Soil microorganisms control plant ectoparasitic nematodes in natural coastal foredunes

Belowground herbivores can exert important controls on the composition of natural plant communities. Until now, relatively few studies have investigated which factors may control the abundance of belowground herbivores. In Dutch coastal foredunes, the root-feeding nematode Tylenchorhynchus ventralis is capable of reducing the performance of the dominant grass Ammophila arenaria (Marram grass). However, field surveys show that populations of this nematode usually are controlled to nondamaging densities, but the control mechanism is unknown. In the present study, we first established that T. ventralis populations are top-down controlled by soil biota. Then, selective removal of soil fauna suggested that soil microorganisms play an important role in controlling T. ventralis. This result was confirmed by an experiment where selective inoculation of microarthropods, nematodes and microbes together with T. ventralis into sterilized dune soil resulted in nematode control when microbes were present. Adding nematodes had some effect, whereas microarthropods did not have a significant effect on T. ventralis. Our results have important implications for the appreciation of herbivore controls in natural soils. Soil food web models assume that herbivorous nematodes are controlled by predaceous invertebrates, whereas many biological control studies focus on managing nematode abundance by soil microorganisms. We propose that soil microorganisms play a more important role than do carnivorous soil invertebrates in the top-down control of herbivorous ectoparasitic nematodes in natural ecosystems. This is opposite to many studies on factors controlling root-feeding insects, which are supposed to be controlled by carnivorous invertebrates, parasitoids, or entomopathogenic nematodes. Our conclusion is that the ectoparasitic nematode T. ventralis is potentially able to limit productivity of the dune grass A. arenaria but that soil organisms, mostly microorganisms, usually prevent the development of growth-reducing population densities.
Introduction
Root herbivores play an important role in shaping the composition of natural plant communities (Brown and Gange 1990). Nematodes and insects represent the vast majority of the belowground herbivores (Brown and Gange 1990; Stanton 1988). Nematodes are more abundant than soil insects, and in some grassland ecosystems, nematodes are the dominant herbivores (Ingham and Detling 1986). Root-feeding nematodes have been estimated to take up as much as one quarter of the net primary production of grassland vegetation (Stanton 1988), and they affect plant quality (Davis et al. 1994; Troelstra et al. 2001), plant diversity, and vegetation succession (de Deyn et al. 2003). Root-feeding nematodes can also indirectly affect plant performance by their influence on bottom-up and top-down control of aboveground invertebrate herbivores (Bezemer et al. 2005). However, in spite of the increasing knowledge on the significant role of belowground herbivores in the control of plant abundance and plant community composition, relatively few studies have investigated which factors control the abundance of the belowground herbivores in natural ecosystems (Strong et al. 1996, 1999).
Herbivore abundance can be influenced by natural enemies (top-down), by the host plant (bottom-up), and by competition with other herbivores (horizontal control). In (semi) natural ecosystems, most studies on the control of root-feeding nematodes have focused on plant quality (Yeates 1987), interspecific competition (Brinkman et al. 2004, 2005), plant community composition (de Deyn et al. 2004), plant succession and soil conditions (Verschoor et al. 2002), and mycorrhizal fungi (de la Peña et al. 2006). Soil food web models assume root-feeding nematodes to be controlled by carnivorous nematodes and microarthropods (Hunt et al. 1987; Neutel et al. 2002). However, most biological control studies in agricultural systems focus on managing nematode abundance by parasitic soil microorganisms (Kerry 2000; Sikora 1992) or mycorrhizal fungi (Hol and Cook 2005), suggesting that root-feeding nematodes are mainly controlled by microorganisms. Therefore, previous studies show little agreement and do not clearly predict how root-feeding nematodes will be controlled in natural ecosystems.
Empirical data for top-down mechanisms are rare for terrestrial ecosystems relative to the many studies in aquatic systems (Walker and Jones 2001). In general, trophic cascades have been argued to be less common on land than in water (Polis and Strong 1996). Nevertheless, there is empirical evidence supporting the existence of trophic cascades in terrestrial plant–predator–prey systems (Schmitz et al. 2004). Tritrophic systems of plants, aboveground insect herbivores, and their natural aboveground enemies are the best-studied terrestrial examples of top-down and bottom-up herbivore controls (Carson and Root 1999; Rosenheim 1998). Below ground, tritrophic interactions may not essentially differ from what is known above ground (Bezemer and van Dam 2005), although rates of dispersal of organisms and chemical compounds will be lower than is mostly the case above ground (Rasmann et al. 2005; van der Putten 2003). Therefore, the challenge is, similar to that above ground (Schmitz et al. 2004), to assess what controls the abundance of root herbivores. This knowledge will enhance our understanding of belowground multitrophic interactions and their influences on plant performance and plant community composition.
In the present study, the role of microarthropods, nematodes, and microorganisms in controlling the abundance of the root-feeding nematode Tylenchorhynchus ventralis (Loof 1963) Fortuner and Luc (synonym Telotylenchus ventralis) was experimentally compared. This nematode is a polyphagous ectoparasite, which means that it is a quite generalistic root feeder that penetrates outer cortical cells with its stylet to collect and ingest cell contents (Yeates et al. 1993). T. ventralis is a root parasite of the dominant coastal foredune grass Ammophila arenaria (Marram grass). In field soil, T. ventralis reaches densities that are 80 times lower than achieved when inoculated into sterilized dune soil (de Rooij van der Goes 1995). Whereas T. ventralis can strongly reduce growth of A. arenaria in sterilized soil, field densities in nonsterilized soil are too low to directly influence plant performance (de Rooij van der Goes 1995). The roots of A. arenaria are parasitized by an array of herbivorous nematodes ranging from ectoparasites to sedentary endoparasites (de Rooij van der Goes et al. 1995). The control mechanisms of root herbivorous nematodes in dunes appear to highly depend on the feeding type of the nematode, and even on the species of nematode. Whereas the sedentary root knot nematode Meloidogyne maritima (Jepson 1987) Karssen, van Aelst and Cook is controlled by competition (Brinkman et al. 2005), the sedentary cyst nematode Heterodera arenaria (Cooper 1955) Robinson, Stone, Hooper and Rowe appears to be controlled by bottom-up processes (van der Stoel et al. 2006). The migratory endoparasitic root lesion nematode Pratylenchus penetrans (Cobb 1917) is controlled by arbuscular mycorrhizal fungi (de la Peña et al. 2006). Thus far, the factors that control the ectoparasitic nematode T. ventralis associated with A. arenaria are unknown.
Previous studies showed bottom-up control of A. arenaria to occur only when the plants were severely growth reduced (de Rooij van der Goes et al. 1995). Alternatively, competition with cyst and root lesion nematodes is a potential factor controlling ectoparasitic nematodes (Eisenback 1993). However, endoparasitic nematodes did not control abundance of T. ventralis (Brinkman et al. 2004). In the present study, the top-down factors that may be involved in the control of T. ventralis populations were investigated in order to determine how belowground trophic interactions might influence plant performance and vegetation composition.
To assess the top-down control of T. ventralis, three experiments were performed. The aim of experiment 1 was to elucidate the potential top-down control of T. ventralis by the dune soil community. In experiment 2, the particular role of microorganisms was investigated by selective elimination of soil fauna (nematodes and microarthropods). In experiment 3, the hypothesis that emerged from experiment 2, that soil microorganisms are the main cause of top-down control of T. ventralis, was tested. Here, we applied Koch’s postulates by collecting microorganisms, nematodes, and microarthropods from dune soil and adding them to sterilized soil inoculated with T. ventralis. New evidence that top-down control by soil microorganisms is the most important factor controlling the abundance of ectoparasitic nematodes in dune soil is presented and discussed.
Materials and methods
Soil
In summer 2003, soil samples were collected from mobile and stable foredunes at Voorne, The Netherlands (Latitude 51°55′N to Longitude 04°05′E). The samples were collected along six transects parallel to the beach and 50 m apart. At each sampling station in the mobile and stable dune, 60 kg of soil was collected from the youngest root zone of A. arenaria. The soil was sieved (0.5-cm mesh size) to remove plant parts and debris and stored in plastic bags at 4°C until used (van der Stoel et al. 2002).
Plants
Seeds of A. arenaria were collected from the same foredune area and stored dry until used. In order to obtain seedlings, the seeds were germinated for 2 weeks on moist glass beads in a climate room at a 16/8 h light/dark regime at a temperature of 25/15°C, respectively. When the first leaf was 2–3 cm long, the seedlings were transplanted to 1.5-l plastic pots filled with 1,500 g of dune soil. In each pot, four seedlings of A. arenaria were planted, and the soil surface was covered with aluminum foil to protect the soil from desiccation. The soil moisture was adjusted to 10% w/w and maintained at this level throughout the experiment by weighing the pots twice a week and resetting their initial weight using demineralized water. Once a week, full-strength Hoagland nutrient solution was added at a weekly rate of 12.5 ml pot−1 for the first 3 weeks and then 25 ml pot−1, subsequently (Brinkman et al. 2004). This nutrient supply rate was effective to compensate for effects of nutrient release as a result of soil sterilization in dune soil (Troelstra et al. 2001; van der Putten et al. 1988). The experiments were carried out in a greenhouse at a day temperature of 21°C ± 2°C (day length 16 h) with additional light (to maintain a minimum of 225 μmol m−2 s−1 PAR with SON-T Agro lamps) and a night temperature of 16°C. These temperatures are comparable with summer conditions in the field and are optimal for both plant and nematode development (Troelstra and Wagenaar unpublished results).
Experiments
Experiment 1: multiplication of T. ventralis in sterilized and nonsterilized dune soil
In this experiment, the effect of soil origin (mobile and stable dunes) and soil organisms on multiplication of the ectoparasitic nematode T. ventralis was tested. Half of the soil was sterilized by gamma irradiation at an average dose of 25 kGray, which eliminates microorganisms and nematodes effectively from dune soil (de Rooij van der Goes et al. 1998). One week after the seedlings of A. arenaria had been transplanted, half the pots were inoculated with 50 T. ventralis pot−1. The noninoculated pots served as controls for effects of T. ventralis on plant biomass production. There were six replicates of each treatment.
Experiment 2: reproduction of T. ventralis in partially sterilized soil
Multiplication of T. ventralis was studied in soils from which microarthropods and nematodes had been selectively removed by stirring the soil for 15 min at 1,500 rpm. This method has proven to effectively kill the soil fauna (de Rooij van der Goes et al. 1998). We confirmed this by inspecting the soil following stirring and found no live nematodes or microarthropods. The experiment was carried out as described above, but now the soil was completely sterilized by gamma irradiation (average 25 kGray), partially sterilized by stirring to remove the soil fauna, or nonstirred in order to have a nonsterilized control soil. Each soil was inoculated with 0, 25, or 250 T. ventralis pot−1 in order to examine any interaction between the effect of type of soil sterilization and nematode inoculation density. There were six replicates of each treatment.
Experiment 3: reinoculation of microorganisms, nematodes, and microarthropods into sterilized soil with T. ventralis
In order to completely apply Koch’s postulates, microarthropods, nematodes, and microorganisms were extracted from the soil of mobile and stable coastal foredunes and inoculated alone and in all factorial combinations into sterilized dune soil. Then, seedling plants of A. arenaria were grown as in the previous experiment, and every pot was inoculated with 50 T. ventralis. All treatments were carried out in six replicates.
The microorganisms were obtained by shaking soil samples of 100 g with demineralized water (1:1 w/w) for 10 min and filtering the supernatant through a 20-μm mesh (Klironomos 2002). Prepared microbial filtrate contained no nematodes, but bacteria and fungi had readily passed through the filter. The pots with microorganisms were inoculated with 10 ml of the filtrate, which was 1/15 of the original soil density. For each pot, nematodes had been extracted from 1,500 g of nonsterile soil by Cobb’s method (Oostenbrink 1960) and added in a suspension of 10 ml pot−1, so that nematode inoculation density corresponded with the density of nematodes in field soil. The nematode community added to the pots was analyzed microscopically (magnification 200×) and consisted of plant parasites (T. ventralis, T. microphasmis, Pratylenchus spp, Paratylenchus spp., Meloidogyne spp., Rotylenchus spp., Criconematidae), bacterivores (Acrobeles spp., Acrobeloides spp., Chiloplacus spp., Cephalobidae, Plectus spp.), omnivores (Aporcelaimellus spp., Microdorylaimus spp.), and carnivores (Choanolaimus spp.).
Microarthropods were collected from nonsterile dune soil by wet sieving through 180-μm mesh and added as 10 ml of suspension pot−1, which corresponded with the field density of microarthropods. Demineralized water was added to all pots in equal amounts.
Assessing the presence of microbial enemies on nematodes in field soil
In order to confirm whether microbial enemies may occur on T. ventralis in the field, we extracted mobile nematodes from 100 cm3 of the field soil from each of the sampling sites using an adaptation of the Tray method (Whitehead and Hemming 1965). Half of the resulting nematode suspension was inspected using an inverted microscope (magnification 200×), and the nematodes were checked for symptoms of infection by bacteria or fungi. Nematodes infected by fungi were picked from the suspension and transferred to a corn-meal agar plate with antibiotics to encourage sporulation (Smith and Onions 1994), making possible identification of fungi that were previously found in a vegetative state. Identification of fungal natural enemies was done by observing mycelia and spore structure morphology and comparing this with the descriptions of Barron (1977). Endospores of the parasitic bacterium Pasteuria spp. were recorded when observed attached to the nematode cuticle. Symptoms of infection by a nonlethal bacterial parasite Microbacterium nematophilum were assessed according to Sulston and Hodgkin (1988).
To detect whether nematode natural enemies may occur as dormant forms in the soil, nematode-baited sprinkle plates were used. Soil (1 g) from each of the samples was sprinkled on water agar (1%) in a 9-cm-diameter Petri dish. A concentrated suspension of an estimated 500 Caenorhabditis elegans synchronized in the young adult stage (Sulston and Hodgkin 1988) was added to the plates. A negative control containing nematodes only in water agar (1%) was used. The plates were sealed, kept at room temperature, and observed after 2 weeks and subsequently at weekly intervals up to 5 weeks (Barron 1977). Identification of fungal natural enemies was done as described above.
Harvest
All three experiments were harvested 12 weeks after inoculation of T. ventralis, allowing this nematode to complete two reproductive cycles (de Rooij van der Goes 1995). The nematodes were extracted from soil by Cobb’s decantation method and from the roots using a mistifier (Oostenbrink 1960). The numbers of T. ventralis were counted using a microscope (magnification 200×) and expressed as numbers 100 g−1 of dry soil. The roots and shoots of A. arenaria were dried for 48 h at 75°C and weighed.
Data analysis
Normal distribution of data and homogeneity of variance were checked by inspection of the residuals after model fit (using the package Statistica 7). To obtain the normal distribution of data and homogeneity of variances, numbers of T. ventralis were log transformed in experiment 1 and square-root transformed in experiment 2. In all three experiments, the soil origin (stable or mobile dune) did not affect significantly (P > 0.05) the measured variables. Therefore, all data from treatments with those two soil origins was pooled, resulting in 12 replicates per treatment. Numbers of T. ventralis of experiment 1 were analyzed using one-way analysis of variance (ANOVA) with main the factor “soil treatment.” Two-way ANOVA with the main factors “soil sterilization” and “nematode inoculation” were performed for root and shoot biomass. Three-way ANOVA with the main factors “stirring,” “sterilization,” and “inoculation density” were performed for analyzing the numbers of T. ventralis shoot and root biomass in experiment 2. Experiment 3 was analyzed by three-way ANOVA with the main factors “invertebrates,” “nematodes,” and “microorganisms.” Treatments were compared by posthoc analysis using Tukey honestly significant difference (HSD) tests (P < 0.05).
Results
Experiment 1
The numbers of T. ventralis at harvest differed significantly between sterilized and nonsterilized soils (F2,33 = 77.9 and P < 0.001). In the nonsterilized soil, addition of T. ventralis resulted in a significant increase of numbers at the end of the experiment compared with nonsterilized, noninoculated soil (Fig. 1). However, there were five times more T. ventralis in the inoculated sterilized soil than in the inoculated nonsterilized soil (Fig. 1; P < 0.05; sterilized soil without T. ventralis added was not included because the nematodes were absent). These results show that multiplication of T. ventralis in nonsterilized soil was significantly enhanced by inoculation but that T. ventralis multiplication was significantly reduced by some factor in the nonsterilized soil that could be excluded by soil sterilization.
Fig. 1Numbers of Tylenchorhynchus ventralis in 100 g of nonsterilized and sterilized dune soil 12 weeks after inoculation with T. ventralis. Error bars indicate standard error, and different letters above the bars indicate significant difference at P < 0.05 (experiment 1)
Soil sterilization influenced shoot biomass more than did T. ventralis inoculation (F1,44 = 117, P < 0.001 for soil sterilization and F1,44 = 4.17, P < 0.05 for inoculation, Fig. 2), and the effect of T. ventralis inoculation depended on soil sterilization (F1,44 = 7.06, P < 0.05). Most shoot biomass was produced in sterilized soil, whereas T. ventralis inoculation significantly reduced shoot biomass (Fig. 2). As expected, the least shoot biomass was produced in nonsterile soil; however, addition of T. ventralis caused no further reduction in growth (Fig. 2). As expected, root biomass was also strongly influenced by soil sterilization (F1,44 = 56.1 and P < 0.001), whereas the effect of T. ventralis addition was greater than for shoot biomass (F1,44 = 16.8 and P < 0.001). As for shoot biomass, the effect of T. ventralis inoculation on root biomass depended on soil sterilization (F1,44 = 9.87 and P < 0.005), which reflects that shoot biomass was significantly reduced by T. ventralis inoculation in the sterilized soil only (Fig. 2).
Fig. 2Shoot and root biomass of Ammophila arenaria in sterilized and nonsterilized soil after 12 weeks from inoculation with Tylenchorhynchus ventralis. Error bars and letters above indicate significant differences at P < 0.05 (experiment 1)
Experiment 2
Significantly greater populations of T. ventralis developed in sterilized than in nonsterlilized soil at both inoculation densities (Tables 1 and 2). At the low-inoculation density, the number of the nematodes in nonsterliized soil was 30 times less than in sterilized soil and 15 times less at the high-inoculation density. There was no significant effect of soil stirring on the numbers of T. ventralis, although there was a trend (P = 0.06) that stirring reduced T. ventralis multiplication.
Table 1Three-way analysis of variance (ANOVA) of the numbers of Tylenchorhynchus ventralis in nonsterilized and sterilized, and stirred and nonstirred dune soil at three inoculation rates (0, 25, 250 pot−1) after 12 weeks from inoculation to Ammophila arenaria. The data has been square-root transformed to achieve normal error distributiondfFPStirring (1)13.4810.06Sterilization (2)1137.41<0.001Inoculation density (3)295.55<0.0011 × 210.8580.361 × 321.2220.302 × 3243.94<0.0011 × 2 × 321.4990.23Error125Table 2 Effects of soil sterilization on numbers of Tylenchorhynchus ventralis in 100 g of soil [±1 standard error (SE)] 12 weeks from inoculation. Letters indicate significant differences at P < 0.05 (experiment 2)NonsterilizedSterilized0 T. ventralis added0.77 ± 0.22a0.08 ± 0.03a25 T. ventralis added12.3 ± 2.62b390 ± 73.03c250 T. ventralis added69.2 ± 16.76b,c1,162 ± 203.7d
As expected, both soil stirring and sterilization influenced shoot biomass (Fig. 3; Table 3). Shoot biomass was greater in sterilized than in nonsterilized soil and in stirred than in nonstirred soil; however, inoculation density of T. ventralis did not influence the shoot biomass (Fig. 3). Root biomass was affected by stirring, soil sterilization, and inoculation density of T. ventralis, whereas effects of inoculation density depended on soil stirring as well as on soil sterilization (Table 3). If no nematodes were inoculated to the pots, soil sterilization almost doubled the root biomass. However, if inoculated with 25 or 250 T. ventralis per pot, the roots in sterilized soil with T. ventralis did not produce more biomass than those in nonsterilized soil (Fig. 3). Root biomass was significantly increased by soil stirring when no or few (25 pot−1) T. ventralis were added to the pots, but there was no increase in root weight at the high inoculation rate.
Fig. 3Effects of soil stirring, soil sterilization, and addition of Tylenchorhynchus ventralis on shoot and root biomass of Ammophila arenaria. Error bars and letters above indicate significant differences at P < 0.05 (experiment 2)Table 3Shoot and root biomass of Ammophila arenaria 12 weeks after inoculation with nematodesdfShoot biomassRoot biomassFPFPStirring (1)114.13<0.0018.69<0.01Sterilization (2)130.49<0.000126.8<0.001Inoculation density (3)21.9980.13910.53<0.0011 × 213.2880.0721.910.1691 × 321.9160.1513.622<0.052 × 322.1450.1216.452<0.011 × 2 × 321.9210.1511.9110.152Error132The results of a factorial analysis of variance (ANOVA) with factors “stirring,” “sterilization,” and “inoculation density” (experiment 2)
Experiment 3
The multiplication of T. ventralis numbers was significantly reduced by adding a mixture of soil nematodes; however, the effect of adding microorganisms was far greater (Fig. 4; Table 4). If microorganisms were present alone or in combination with other soil organisms, the number of T. ventralis was always less than when microorganisms were absent. On average, adding a suspension of nematodes reduced final numbers of T. ventralis by 15%, whereas adding microorganisms reduced numbers of T. ventralis by 55% (Fig. 4). Therefore, the effect of adding nematodes on T. ventralis multiplication was substantially weaker than the effect of microorganisms. Microarthropods did not have a significant effect on the numbers of T. ventralis. Adding microarthropods, nematodes, and microorganisms did not influence shoot or root biomass (P > 0.05; data not shown).
Fig. 4The effects of mixed nematode inoculum and microorganisms on Tylenchorhynchus ventralis multiplication. Error bars and letters above indicate significant differences at P < 0.05 (experiment 3)Table 4The numbers of Tylenchorhynchus ventralis after 12 weeks from inoculationDegrees of freedom (df)F valueP valueMicroarthropods (1)10.00060.981Nematodes (2)15.167<0.05Microorganisms (3)187.84<0.00011 × 211.7360.1911 × 310.6180.4342 × 310.6720.4151 × 2 × 310.0470.828Error88The results of a three-way analysis of variance (ANOVA) with factors “microarthropods,” “nematodes,” and “microorganisms” (experiment 3)
In the suspension of nematodes obtained from the pots to which microorganisms had been added, 47.3% showed signs of infection by culturable microbial enemies (Table 5). The fungal parasite Catenaria spp. was found infecting 16 out of 110 T. ventralis inspected, and an unidentified fungus was detected inside 30 destroyed of 110 T. ventralis checked. Bacterial attachment to the cuticle was also observed for six nematodes, a Paenibacillus-like organism was found on four nematodes, and Pasteuria spp. on two out of 110 nematodes.
Table 5Microbial enemies in or attached to Tylenchorhynchus ventralis in a suspension obtained from microorganism treatment pots in experiment 3Microbial enemy detectedFraction of affected nematodes (%)Unidentified assimilative hyphae27.3Paenibacillus-like3.6Catenaria spp.14.5Pasteuria spp.1.8Total47.3(Healthy)52.7Total nematodes examined100 (n = 110)
Assessing the presence of microbial enemies on nematodes in field soil
The fungal natural enemies Catenaria spp., Harposporium spp., and Myzocytium spp. were found infecting nematodes extracted by the Tray method (Whitehead and Hemming 1965). The bacterium P.penetrans was attached to root knot nematodes (M. maritima). The fungal genera were also detected using nematode-baited sprinkle plates. Some of the nematodes on the plates had a swollen region behind the anus not observed in the original culture or in the negative control. This is a symptom of infection by a nonlethal bacterial parasite, M. nematophilum. An unidentified trapping fungus with nonconstricting rings was also detected in the sprinkle plates but could not be identified, as it did not sporulate. These identifications may not have been exhaustive, but they confirmed the presence of antagonistic microorganism species on T. ventralis as well as in the soil from the field.
Discussion
In coastal foredunes, the root-feeding ectoparasitic nematode T. ventralis would significantly influence the pioneer grass A. arenaria if the density of this nematode was not controlled naturally. Our study strongly suggests that the natural control of T. ventralis in coastal foredune soil is mostly due to soil microorganisms. When inoculated into sterilized soil, numbers of T. ventralis were more than five times greater than when inoculated into nonsterilized soil, whereas selective elimination of soil fauna by stirring did not affect nematode numbers. These results from selective elimination studies were confirmed by isolating microarthropods, nematodes, and microbes and adding these together with T. ventralis to sterilized soil. Inoculation with soil microorganisms reduced T. ventralis more strongly than did inoculation with a nematode community consisting of other plant parasites, bacterivores, omnivores, and carnivores. The negative effect of the nematode community on T. ventralis density might have been due to competition with other root feeders. However, competition between T. ventralis and endoparasitic nematodes (i.e. H. arenaria, M. maritima and P. penetrans) occurred only if numbers of the competitors strongly exceeded present field densities (Brinkman et al. 2004). Therefore, the observed effect of adding nematodes on reducing T. ventralis was due to soil microorganisms cointroduced with the nematode suspension, carnivorous nematodes (Jairajpuri and Bilgrami 1990), or by effects of other non-plant-feeding nematodes. Our results highlight an important discrepancy in thinking about control mechanisms of plant-feeding (also called plant-parasitic) nematodes between biocontrol studies on the one hand and soil food web studies on the other. The majority of studies on the biocontrol of nematodes in agricultural ecosystems mostly focus on parasitic bacteria, such as P. penetrans and fungi (Kerry 2000), for example Arthrobotrys spp. and Pochonia spp. (Stirling and Smith 1998). Biological control practice usually does not consider microarthropods to be relevant for parasitic nematode control (Kerry and Gowen 1995), whereas the role of carnivorous nematodes has been considered (Mankau 1980; Yeates and Wardle 1996) but not successfully used. According to the food web model used by Neutel et al. (2002), root-feeding nematodes in coastal ecosystems are affected by predaceous mites and carnivorous nematodes. The role of microorganisms in nematode control is generally ignored in prominent soil food-web models (de Ruiter et al. 1993; Hunt et al. 1987). In our study system, however, soil microorganisms appeared to play a more important role than soil fauna in the control of plant ectoparasitic nematodes. In dune grasslands, the densities of soil fauna are usually rather low, perhaps too low to control significantly the abundance of nematodes (Petersen and Luxton 1982). Our results, therefore, support the view of biological control studies more than it supports soil food-web models for the control of the ectoparasitic nematode T. ventralis.
Our results suggest that top-down control by natural enemies is more important for ectoparasitic feeding generalists, such as T. ventralis, than are competition (Brinkman et al. 2004) or mycorrhizal fungi (Piśkiewicz and Hol unpublished results), although these control mechanisms have been suggested for the other endoparasitic root-feeding nematode species in the same study system (Brinkman et al. 2005; de la Peña et al. 2006; van der Stoel et al. 2006).
The control of T. ventralis in nonsterilized soil to which T. ventralis was added was not as good as in nonsterile soil, with only the background population of T. ventralis present. Earlier studies have shown that multiplication of T. ventralis is density and time dependent (de Rooij van der Goes 1995). Over time, a low inoculation density of nematodes may result in population increase, whereas a high inoculation density may result in population decline (de Rooij van der Goes 1995). However, we have no information as to how the microbial control shown in the present study may depend on nematode density, and subsequent long-term studies are required to further explore density dependence of nematode top-down control by microbes.
The two screening methods used to detect microbial enemies of nematodes in the soil yielded a diversity of fungal and bacterial antagonists. The Tray method (Whitehead and Hemming 1965) permitted the extraction of mobile stages of nematodes only, and therefore, dead or dying nematodes could not be screened. The nematode-baited sprinkle plate method, although useful for detecting microbial enemies in dormant forms in the soil, produced biased results that reflected the choice of nematode added, the bacterial-feeding nematode C. elegans. The fungal endoparasite Harposporium spp. infects nematodes that ingest its spores and therefore would not be able to infect plant parasites due to the narrow lumen of the stylet (Barron 1977). The bacterium M. nematophilum is thought to be a specialist parasite of C. elegans (Hodgkin et al. 2000). T. ventralis was attacked by a subset of the microbial enemy genera found in the soil, which reflects some specificity in the action of these microbes. Some microorganisms, namely Pasteuria spp. and the Paenibacillus-like organism, have not been detected in the dune soil, although they are widespread in the dune sites. This may be due to their absence or to presence in small numbers that are below the level of detection. The unidentified assimilative hyphae could be the result of attack by generalist trapping fungi. This was a first assessment to confirm the presence of nematode antagonistic microorganisms on T. ventralis, and further studies are needed to isolate culture and inoculate those antagonists in order to evaluate their contribution in root-feeding nematode control.
Soil sterilization always led to increased root and shoot biomass. Previous studies have already shown that soil biota may reduce performance of A. arenaria (van der Putten et al. 1988; van der Stoel et al. 2002). The effect of soil sterilization was greater for root biomass than for shoot biomass. In sterilized soil, enhanced plant growth can be caused by nutrient release as a result of the soil sterilization process (Troelstra et al. 2001). We avoided different nutrient status of the sterilized and nonsterilized treatments by adding nutrient solution (van der Putten et al. 1988). When added to sterilized soil, the root-feeding nematode T. ventralis reduced root and shoot biomass of A. arenaria, and the effect of nematodes on root biomass increased with increasing inoculation density. Addition of T. ventralis to the nonsterile soil did not change root biomass, showing that the contribution of this nematode species to growth reduction of A. arenaria is limited (de Rooij van der Goes 1995). Our study shows that the dune soil not only contains biotic factors that reduce growth of A. arenaria but that there are also (micro) organisms that control population abundance of T. ventralis. In prairie grassland ecosystems, root-feeding nematodes have been assessed to account for reducing 58% of aboveground biomass (Stanton 1988). These estimates are based on elimination trials using soil biocides. However, these studies did not verify the biocide effects by inoculation trials, and they also did not account for natural top-down control of the root-feeding nematodes. Our results suggest that when assessing the effects of root feeders on plant production, interactions of the root feeders with their natural predators needs to be taken into account as well.
We conclude that soil microorganisms contribute to controlling the plant ectoparasitic nematode T. ventralis at a low population density in natural coastal foredunes. When not controlled, T. ventralis would be a key control factor for A. arenaria performance. Addition of other soil fauna, i.e., nematodes and microarthropods, did not influence the abundance of T. ventralis as much as microorganism addition did, which confirms the marginal effects of soil fauna removal on reproduction of T. ventralis. Our results suggest that belowground multitrophic interactions can be crucial for plant performance. Revealing the precise identity of the microorganisms that have negative effects on the T. ventralis population, as well as mechanisms and involvement of the host plant, need further studies.

tylenchorhynchus ventralis
ammophila arenaria
top-down control
multitrophic interactions
root herbivory


Tolerance to coxibs in patients with intolerance to non-steroidal anti-inflammatory drugs (NSAIDs): a systematic structured review of the literature

Adverse events triggered by non-steroidal anti-inflammatory drugs (NSAIDs) are among the most common drug-related intolerance reactions in medicine; they are possibly related to inhibition of cyclooxygenase-1. Coxibs, preferentially inhibiting cyclooxygenase-2, may therefore represent safe alternatives in patients with NSAID intolerance. We reviewed the literature in a systematic and structured manner to identify and evaluate studies on the tolerance of coxibs in patients with NSAID intolerance. We searched MEDLINE (1966–2006), the COCHRANE LIBRARY (4th Issue 2006) and EMBASE (1966–2006) up to December 9, 2006, and analysed all publications included using a predefined evaluation sheet. Symptoms and severity of adverse events to coxibs were analysed based on all articles comprising such information. Subsequently, the probability for adverse events triggered by coxibs was determined on analyses of double-blind prospective trials only. Among 3,304 patients with NSAID intolerance, 119 adverse events occurred under coxib medication. All adverse events, except two, have been allergic/urticarial in nature; none was lethal, but two were graded as life-threatening (grade 4). The two non-allergic adverse events were described as a grade 1 upper respiratory tract haemorrhage, and a grade 1 gastrointestinal symptom, respectively. In 13 double-blind prospective studies comprising a total of 591 patients with NSAID intolerance, only 13 adverse reactions to coxib provocations were observed. The triggering coxibs were rofecoxib (2/286), celecoxib (6/208), etoricoxib (4/56), and valdecoxib (1/41). This review documents the good tolerability of coxibs in patients with NSAID intolerance, for whom access to this class of drugs for short-term treatment of pain and inflammation is advantageous.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used therapeutics in the outpatient management of pain and inflammation in a wide spectrum of diseases. Their primary mode of action is the blockade of prostaglandin synthesis by cyclooxygenases (COX): Constitutively expressed COX-1 is involved in fundamental mechanisms of homeostasis, whereas the inducible COX-2 mediates inflammation. Therapeutic effects of NSAIDs are primarily related to their ability to inhibit COX-2, whereas some of their most frequent adverse effects may be caused by COX-1 inhibition (Fig. 1). In contrast to most “classic” NSAIDs which block both isoforms, the so-called coxibs preferentially inhibit COX-2. This may result in better tolerability, namely reduction of gastrointestinal side effects [29, 85].
Fig. 1Cyclooxygenase isoforms. Non-steroidal anti-inflammatory drugs (NSAIDs) block prostaglandin synthesis by cyclooxygenases (COX). Two isoforms exist of this enzyme: constitutively expressed COX-1 is involved in fundamental mechanisms of homeostasis, whereas the inducible isoform COX-2 mediates inflammation
Respiratory and cutaneous adverse events triggered by NSAIDs are among the most common drug-related intolerance reactions in medicine. Typically, these manifest as asthma attacks or urticaria. Pathogenesis of these symptoms seems to be related to COX-1 inhibition [76]. Therefore, the hypothesis was put forward that coxibs may safely be used in patients with known NSAID intolerance. However, serious intolerance reactions to coxibs have also been observed, thus cautioning too euphoric expectations [71]. We therefore reviewed the relevant literature in a systematic and structured approach for evidence of coxib tolerance in patients with NSAID intolerance.
Methods
Search strategy
We searched MEDLINE (1966–2006), the COCHRANE LIBRARY (4th Issue 2006) and EMBASE (1966–2006) up to December 9, 2006. The following search terms were used: rofecoxib OR celecoxib OR valdecoxib OR parecoxib OR etoricoxib, combined with hypersensitivity OR intolerance. Those coxibs used in veterinary medicine (deracoxib, tiracoxib and cimicoxib) were not included in the literature search. No language or publication restriction was predefined. All publications reporting individual patients with NSAID intolerance and subsequent exposure to a COX-2 Inhibitor were identified and the reference lists of these articles were hand-searched for further publications. If articles could not be retrieved in full text, a copy was requested from the corresponding author and/or journal editor.
Inclusion criteria
Each publication was appraised for inclusion in a stepwise approach (Fig. 2). Only publications describing individual patients and providing a rational medication scheme as well as a sufficiently specific outcome report were included in this review. In a first step, evaluation focused on clinical symptoms and severity of adverse events in patients with NSAID intolerance. Therefore, all articles on this topic were included for this analysis. Subsequently, the probability for adverse events was analysed based exclusively on publications of double-blind prospective trials.
Fig. 2Flow chart documenting the handling data (NSAID non-steroidal anti-inflammatory drugs)
Data extraction and synthesis
All articles were analysed using a predefined evaluation sheet. Uncertainties were resolved by consensus decisions among the investigators. Data synthesis was qualitative and descriptive. The Common Terminology Criteria for Adverse Events version 3.0 (CTCAE) was used to categorize adverse events.
Results
We identified 230 publications on coxibs and/or NSAID intolerance. Hundred and forty-six references not focusing on individual patients with NSAID intolerance were excluded. Unclear medication schemes or outcome reports led to exclusion of two articles. Eighty-four publications were evaluated for severity and type of adverse reactions to coxibs. Thirteen publications on double-blind studies were used to determine the probability of adverse reactions to coxibs.
Severity and type of adverse events to coxibs
A total of 119 adverse events among 3,304 patients exposed to coxibs were identified [1–18, 20–24, 26–28, 30–35, 37–58, 60–75, 77–84, 86–89]. Adverse events were described for rofecoxib (48/1,732), celecoxib (59/1,148), etoricoxib (10/328), and valdecoxib (2/69), but not parecoxib (0/27) (Table 1). All adverse events, except two, have been allergic/urticarial in nature, and none was lethal. One adverse event after administration of rofecoxib and one after celecoxib were reported as life-threatening (grade 4). The two non-allergic adverse events were described as a grade 1 upper respiratory tract haemorrhage, namely blood in nasal discharge after provocation with rofecoxib, and a grade 1 gastrointestinal symptom, namely nausea and abdominal pain.
Table 1Mode and severity of adverse event (CTCAE common terminology criteria for adverse events)PatientsEvents (total)Urticaria (grade)Allergic reactions (grade) Allergic rhinitis (grade)Airway obstruction (grade)Gastroin-testinal symtpms (grade)Pruritus (grade)Hemorrhage, upper respiratory (grade)Rofecoxib1,732489 (1)8 (2)1 (3)25 (3)1 (4)1 (2)2 (1)001 (1)Celecoxib1,1485919 (1)1 (2)1 (2)33 (3)1 (4)01 (1)03 (2)0Etoricoxib328103 (1)5 (2)1 (3)001 (1)00Valdecoxib6921 (1)1 (2)000000Parecoxib2700000000Total3,304119486213131Numbers and grade of adverse events (in parentheses) are indicated
Evaluation of double-blind studies
We identified 13 double-blind studies evaluating tolerability of coxibs in a total of 591 NSAID intolerant patients (Table 2). The coxibs tested in the context of these studies were rofecoxib (n = 286), celecoxib (n = 208), etoricoxib (n = 56) and valdecoxib (n = 41). Among the 591 patients described, 13 exhibited adverse reactions upon provocation with a coxib. The triggering coxibs were rofecoxib (2/286), celecoxib (6/208), etoricoxib (4/56), and valdecoxib (1/41) [13, 28, 48, 60, 69, 74, 75, 77, 78, 83, 84, 87, 88]. One of these reactions was clearly non-allergic, this one being the above-mentioned individual with grade 1 upper respiratory tract haemorrhage.
Table 2Snopsis of double blind studies (NSAID non-steroidal anti-inflammatory drugs)ReferencesSubstance (number of patients)DoseOral provocation with NSAIDFollow-upAdverse events (number of patients/grade)[87]Celecoxib (17)Celecoxib 200 mgYesClinical examinations and forced expiratory volume hourly for 8 h.0[74]Celecoxib (12)/rofecoxib (3)Celecoxib 200 mg/rofecoxib 12.5 mgYesNo information0[75]Rofecoxib (60)Rofecoxib 37.5 mg total doseYesClinical examinations and flow/volume recording hourly0[77]Rofecoxib (12)Rofecoxib 25 mgYesClinical observation every 30 min for 8 h and finally after 24 h.Traces of blood in nasal discharge on days 3–5 of rofecoxib administration (1/1), transient urticaria for 30 min after administration of 5 mg rofecoxib, but not after further administration of higher doses of the drug (1/1)[78]Celecoxib (14)Rofecoxib 10Celecoxib 200–400 mg total dose Rofecoxib 37.5 mg total doseYesForced expiratory volume was recorded each hour for 3 h0[60]Rofecoxib (104)Rofecoxib 25 mg total doseYesClinical control for at least 6 h after each challenge and the next day.0[83]Celecoxib (60)Celecoxib 200 mgYesClinical examinations were conducted hourly0[28]Celecoxib (33)Celecoxib 130 mg total doseYesSpirometry and vital signs at 15- to 30-min intervals after each dose.0[88]Celecoxib (18)/rofecoxib (18)Celecoxib 300 mg total dose/rofecoxib 37.5 mg total dose1YesVital signs and forced expiratory volume every 15 min for 6 h.0[84]Rofecoxib (60)Rofecoxib 50 mgYesClinical examinations and forced expiratory volume hourly.0[13]Valdecoxib (41)Valedcoxib 30 mg total doseYesClinical examination and monitoring of pulse/blood pressare until 16 h past last exposureGeneralised urticaria (1/2)[69, 70]Celecoxib (54)Etoricoxib (56)Celecoxib 200 mgEtoricoxib 120 mgYesVital signs and pulmonary function was monitored at baseline and hourly for 3 h and the skin, nose, thorax were examined at the same intervalsCelecoxib: urticaria (3/1), lip angioedema (1/3), eyelid wheals and angioedema (1/3), urticaria, rhinorrhea, and conjunctival injection (1/1)etoricoxib: urticaria (3/1), tongue, eyelid and lip angioedema (1/3)[48]Rofecoxib (19)Rofecoxib 37.5 mg total doseYesSerial measurement of forced expiratory volume after 30, 60 and 120 min0
Discussion
This systematic review documents the low probability of allergic/pseudo-allergic reactions induced by coxibs in patients with NSAID intolerance. To our knowledge, this is the first comprehensive analysis of data published on this topic. We have searched all three major medical databases available, namely MEDLINE, COCHRANE LIBRARY, and EMBASE using very broad and general search terms. Following identification of relevant publications, these were evaluated by means of a pre-defined evaluation form. The data available are described in the form of a structured review [19].
NSAIDs are among the most commonly prescribed therapeutics in the world. Although generally considered safe, their wide and frequent use results in these drugs being among the most common causes of drug-related intolerance reactions. This may at least in part be due to their non-selective inhibition of both cyclooxygenase isoforms. In line with this hypothesis, NSAIDs characterized by pronounced COX-1 inhibition bear a high risk to trigger asthma attacks in patients with aspirin-sensitive asthma bronchiale, whereas preferentially COX-2 inhibiting NSAIDS are better tolerated by these patients [36]. Our analysis of published studies on this issue further supports this notion, as only 13 of 591 NSAID-intolerant patients showed adverse reactions upon provocation with coxibs in double-blind clinical studies; all of these were grade 3 or milder. Still, relatively selective COX-2 inhibitors have been identified as triggers of serious intolerance reactions [71]. This implies that our current understanding of NSAID-triggered intolerance is still imperfect, and its pathogenesis cannot be reduced to cyclooxygenase-mediated effects alone, but needs to take into account clinically relevant additional NSAID-mediated effects such as secretion of leukotrienes from mast cells and other leukocytes.
It has been suggested that coxibs may confer an elevated risk for acute myocardial infarction and sudden cardiac death, namely after long-term therapy [25]. As a reaction, several coxibs are no longer available despite a recommendation by the participants of an expert meeting organized by the Food and Drug Administration to grant further prescription of rofecoxib, celecoxib and valdecoxib in the US [59]. On the other hand, substantial evidence described here points towards a good tolerability of coxibs in patients with NSAID intolerance. Given the wide use of NSAIDs in the short-term treatment of trivial signs and symptoms, the availability of coxibs for these indications would be advantageous for this relevant subpopulation of patients, since this type of application is unlikely to increase cardiac risk.

coxibs
intolerance
non-steroidal anti-inflammatory drugs
urticaria
adverse drug reaction


CT colonography: optimisation, diagnostic performance and patient acceptability of reduced-laxative regimens using barium-based faecal tagging

To establish the optimum barium-based reduced-laxative tagging regimen prior to CT colonography (CTC). Ninety-five subjects underwent reduced-laxative (13 g senna/18 g magnesium citrate) CTC prior to same-day colonoscopy and were randomised to one of four tagging regimens using 20 ml 40%w/v barium sulphate: regimen A: four doses, B: three doses, C: three doses plus 220 ml 2.1% barium sulphate, or D: three doses plus 15 ml diatriazoate megluamine. Patient experience was assessed immediately after CTC and 1 week later. Two radiologists graded residual stool (1: none/scattered to 4: >50% circumference) and tagging efficacy for stool (1: untagged to 5: 100% tagged) and fluid (1: untagged, 2: layered, 3: tagged), noting the HU of tagged fluid. Preparation was good (76–94% segments graded 1), although best for regimen D (P = 0.02). Across all regimens, stool tagging quality was high (mean 3.7–4.5) and not significantly different among regimens. The HU of layered tagged fluid was higher for regimens C/D than A/B (P = 0.002). Detection of cancer (n = 2), polyps ≥6 mm (n = 21), and ≤5 mm (n = 72) was 100, 81 and 32% respectively, with only four false positives ≥6 mm. Reduced preparation was tolerated better than full endoscopic preparation by 61%. Reduced-laxative CTC with three doses of 20 ml 40% barium sulphate is as effective as more complex regimens, retaining adequate diagnostic accuracy.
Introduction
Full bowel purgation remains a major cause of discomfort prior to any colonic investigation [1–3]. Furthermore, fluid and electrolyte imbalance may occur following aggressive cleansing [4]. A potential advantage of computed tomography colonography (CTC) over colonoscopy is the ability to reduce laxative requirements while maintaining diagnostic accuracy [5–7]. Reduced laxative regimens often incorporate orally ingested contrast agents to “tag” or “label” residual fluid and faecal residue. The ideal tagging regimen remains controversial but must be safe, effective, simple and well tolerated.
In many respects, barium is an ideal tagging agent: it has an established safety profile, is relatively palatable, produces minimal side effects, and is effective for tagging solid residue [8]. Previous work has shown adequate tagging may be achieved using low volumes of 40% w/v barium, although the ideal volume and dosing regimen has not been fully established [9]. Furthermore efficacy for fluid tagging has been questioned [9], with some investigators preferring iodine-based contrast either alone or in combination, claiming a more homogeneous fluid opacification better suited to digital subtraction [10]. We aimed to establish the optimum barium-based reduced-laxative faecal- and fluid-tagging regimen, to assess patient acceptability, and to document diagnostic accuracy compared to an enhanced colonoscopic reference standard.
Materials and methods
Full ethical committee approval was obtained, and all subjects gave written informed consent.
Consecutive patients were recruited from those scheduled to undergo afternoon diagnostic colonoscopy for symptoms suggestive of colorectal neoplasia (change in bowel habit, rectal bleeding, unexplained weight loss or palpable abdominal mass) from one of three institutions. Patients were excluded if below age 50 or if they had a known diagnosis of inflammatory bowel disease. Eligible patients were invited to undergo CTC at 8 am, prior to same day colonoscopy and were randomised via a computer random number generator to one of four reduced-laxative regimens using barium-based faecal tagging [20 ml 40% w/v barium sulphate suspension (Tagitol V, EZEM, Lake Success, NY)] (Fig. 1). For each of the regimens, patients followed a low-residue diet 2 days before the CTC (avoiding fatty food, milk and vegetables). The day prior to CTC, all patients were allowed a low-residue meal kit (Nutraprep, EZEM), and ingested the same reduced-laxative protocol [13 g sachet of senna granules (Reckitt Benckiser Healthcare, Hull, UK) at 7 am and 18 g magnesium citrate (Lo-So prep, EZEM) at 7 pm]. This laxative regimen was defined as “reduced” given the normal laxative preparation prior to colonoscopy at the recruiting institutions includes an additional 18 g of magnesium citrate (see below). Overall fluid intake was restricted to 2.1 l the day prior to CTC for all four regimens.
Fig. 1Details of reduced-laxative tagging regimens. Taken with meals from low residue meal kit (single star). Diluted in 250 mls of water (double stars)
In an attempt to improve fluid tagging, two regimens (C and D) included either an additional 250 ml 2.1% w/v barium sulphate (Readi-Cat Banana Smoothie, EZEM) or 15 ml meglumine amidotrizoate (10 g sodium amidotrizoate/66 g meglumine amidotrizoate per 100 ml; Gastrograffin, Schering) diluted in 250 ml of water, taken 2 h prior to CTC [11]. Patients otherwise took nothing by mouth the day of the CTC.
Study power
The study was powered to detect a 20% difference in tagging quality (see below) across the four regimens. Based on pilot data, the interclass correlation coefficient between colonic segments was calculated to be 0.30, and it was calculated that a sample size of 22 per group was required (alpha 0.05 at 80% power).
CT colonography
Prone and supine CTC was performed with automated CO2 insufflation (Protocol pump, EZEM) [12], using either a 4-detector-row (GE Lightspeed Plus, GE, Milwaukee, WI, USA; 120 kV, 50 mA, 2.5 mm collimation, slice reconstruction 1.25 mm, pitch 1.5, n = 82 patients) or 64-detector-row scanner (Siemens Somatom Sensation 64, SEMS, Germany; 120 kV, 50 mA, 0.6 mm collimation, pitch 0.24, n = 13 patients).
One of three experienced radiologists (each with experience of at least 300 CTC cases with endoscopic validation) evaluated the datasets immediately after the examination using a dedicated workstation with proprietary software (Vitrea 3.8, Vital Images, Minnetonka, MN, USA), and noted the segmental location of any polyps or cancers on a study report sheet, together with lesion size measured using electronic callipers applied to the 2D MPR best showing the maximum diameter. The choice of reporting radiologist for each patient was dependent upon the particular recruiting institution and availability on the day of the scan. All three radiologists used a primary 2D approach with 3D reserved for problem solving.
Colonoscopy
Immediately following CTC patients ingested a further 18 g of magnesium citrate in order to complete the normal endoscopic cleansing regimen prior to afternoon colonoscopy. Colonoscopy was performed as per usual practice by one of five experienced endoscopists, on average 6 h (range 4–8 h) after the CTC scan. Segmental unblinding was used as described previously [13, 14], using the sealed CTC report. In brief, the CTC report was handed to the endoscopy nurse accompanying the patient. CTC findings were revealed to the colonoscopist by the nurse on a segmental basis (caecum to rectum) once examination of each colonic segment was deemed complete during extubation of the colon. If CTC suggested a lesion had been missed, the segment was re-intubated and a second segmental examination performed. There was no time limit imposed on the colonoscopist for this second look. All polyps were photographed, their sizes estimated by direct comparison to adjacent open biopsy forceps, and then excised for histology where possible.
Polyp correlation
Polyps found at CTC were deemed true positive if a corresponding polyp was found in the same or adjacent segment at endoscopy and if the estimated size of the polyp was within 50% of the endoscopic measurement.
Patient experience
Immediately following CTC a questionnaire was administered [15] investigating patient experience of the reduced-laxative tagging regimen (Table 1). Patient responses were compared to a historical cohort of 69 symptomatic patients recruited from the same endoscopy lists during a prior study comparing CTC to colonoscopy [16]. As part of this prior study, patients had undergone full bowel preparation [13 g senna granules and two doses of 18 g magnesium citrate (total 36 g) prior to CTC] and had completed the same questionnaire under similar circumstances.
Table 1Bowel-tolerance questionnaire questions and responses in comparison to historical controls undergoing full bowel preparationVariableResponseReduced preparationa, n (%)Historical controls [full preparation]b, n (%)P valueHow did you find understanding prep sheet?Easy56 (63)48 (70)0.39Fairly easy/difficult33 (37)21 (30)How did you find swallowing medicine?Easy43 (48)44 (64)0.36Fairly easy36 (40)32 (32)Quite difficult/difficult10 (11)3 (4)How did you find coping with special diet?No problem59 (66)42 (61)0.49Bit difficult24 (27)24 (35)Very difficult6 (7)3 (4)How did you feel after medicine?Fine65 (73)48 (70)0.96Unwell/very unwell24 (27)21 (30)Did you have any abdominal pain?None31 (36)25 (36)0.37Mild39 (45)27 (39)Moderate/severe16 (19)17 (25)Did you have any nausea/vomiting?None58 (67)46 (67)0.92Mild22 (26)18 (26)Moderate/severe6 (7)5 (7)Did you experience any faintness or dizziness?None70 (81)54 (78)0.63Mild/moderate/severe16 (29)15 (22)Did you experience any wind?None25 (29)26 (38)0.27Mild41 (48)32 (46)Moderate/severe20 (23)11 (16)Did you experience any soreness?None37 (43)24 (35)0.15Mild37 (43)29 (42)Moderate/severe12 (14)16 (23)Did you experience any incontinence?None68 (80)49 (71)0.2Mild8 (9)13 (19)Moderate/severe9 (11)7 (10)Did you experience any sleep disturbance?None33 (39)41 (59)0.01Mild29 (34)20 (29)Moderate/severe23 (27)8 (12)How many times did you open your bowel after starting the preparation?1–31 (1)N/A3–520 (22)>569 (77)N/A Not applicable (not asked)a13 g senna plus 18 g magnesium citrateb13 g senna plus 36 g magnesium citrate
One week later a follow-up questionnaire (Table 2) was mailed to the current study cohort investigating tolerance of the preparation prior to CT and preference, if any, over the full preparation required for subsequent colonoscopy.
Table 2Questions and responses to follow-up questionnaire pertaining to patient tolerance and preferencesVariableResponsePatient number (%)How did you find taking the low-residue diet?No problem46 (67%)Moderately inconvenient20 (29%)Very inconvenient3 (4%)How did you find drinking the tagging liquid?No problem57 (83%)Moderately inconvenient11 (16%)Very inconvenient1 (1%)How did you tolerate the preparation before CT?Well37 (53%)Fairly well29 (42%)Poorly3 (4%)How did you tolerate the additional preparation prior to colonoscopy, compared to that before the CT?No problem51 (74%)More uncomfortable12 (17%)Much worse6 (9%)How did you find the preparation before CTC compared to the full colonoscopy preparation?Much better18 (26%)Better24 (35%)No better26 (38%)
Grading of bowel preparation
Two experienced radiologists (both with experience of over 700 validated CTC cases) in consensus retrospectively reviewed all CTC examinations, grading the quality of preparation and success of tagging. Observers were unaware of the tagging regimen used and divided the colon into six segments (rectum to caecum) for analysis [17].
Grading of residual stool (irrespective of tagging status) was based on the percentage of total mucosal circumference coated on an axial image. For each colonic segment, the slice with the most residual stool was used to assign the score. Scores were as follows: 1: no residue or scattered residue, 2: coating of <25% or thin circumferential “film” less than 2 mm in depth, 3: coating of ≥25 to 50%, 4: >50% coating.
Grading of residual fluid (irrespective of tagging status) was based on the maximum anteroposterior (AP) diameter of the colonic lumen submerged. For each colonic segment, the slice with the most residual fluid was used to assign the score. Scores were as follows: 1: no fluid, 2: <25% AP diameter, 3: ≥25 to 50% AP diameter, 4: >50% AP diameter.
The quality of tagging for solid residue and fluid was scored using a system adapted from Lefere at al. [9] and assigned for supine and prone positions combined for each colonic segment. Residual stool was divided into that measuring ≤5 mm and ≥6 mm (based on 2D measurement using electronic calipers), and the number of stool balls ≥6 mm was counted for each patient. Readers assessed the percentage of total residual stool volume (for each size category) that had been tagged for each colonic segment. Scores were assigned as follows: 1: all residual stool untagged, 2: 1 to <25% tagged, 3: 25 to <50% tagged, 4: 50 to <75% tagged, 5: 75 to 100% tagged.
Tagging of residual fluid was scored as follows: 1: fluid untagged, 2: layered tagging (a mix of tagging densities in one fluid level with a denser dependent layer and visibly less dense non-dependent layer (Fig. 2), 3: fluid homogeneously tagged (single tagging density). The density of tagged fluid (in HU) was also recorded by taking the average of three regions of interest drawn in the deepest fluid pool for each colonic segment. If fluid was layered (Fig. 2), the minimum and maximum HU was recorded. Finally observers recorded their confidence using a percentage score that, based on the bowel preparation, they would be able to exclude a polyp ≥6 mm in day-to-day clinical practice for each colonic segment.
Fig. 2A 68-year-old female with change in bowel habit. Axial CT colonographic image demonstrating layering of contrast (arrow) within tagged fluid
Statistical analysis
Preliminary data analysis revealed skewed data for most bowel preparation and tagging variables, and thus scores were combined for subsequent analysis. For residue, scores were grouped into either a score of 1, or a score of 2 or more. For residual fluid, scores were grouped into scores of 1/2, or scores of 3/4. Scores for solid residue tagging were grouped into those of ≤4 or 5. Fluid tagging scores were grouped into scores of 1/2, or score of 3. Logistic regression was then applied, adjusting for colonic segment, to compare the four regimens overall, and to compare the distal (rectum, sigmoid, descending) and proximal colon (transverse, ascending and caecum). In addition, the prevalence of layering within tagged fluid was compared on a per-patient basis using the chi-squared test. Tagged fluid attenuation was compared using linear regression following log transformation of the data. Confidence scores for excluding a polyp ≥6 mm were grouped as either <100% or 100%, and compared using logistic regression. For all regression analyses, robust standard errors were employed to account for interdependency between colonic segments and scan position (supine/prone) in the same patient. Results were expressed as the odds of the outcome under consideration compared to regimen A.
Questionnaire responses were compared using Fischer’s exact test.
Overall categorical per-polyp and per-patient data are presented using descriptive statistics. Given the relatively low polyp incidence (and thus low statistical power), comparative statistics for polyp detection were not performed across regimens. False-positive numbers were compared using one-way ANOVA.
Results
A total of 95 patients were recruited (50 female, mean age 64 years, range 50–85 years), with 24, 25, 24 and 22 randomised to regimens A to D respectively. Seventy-seven, 13 and 5 patients were recruited from institutions 1, 2 and 3 respectively. Overall, 67 (71%) had a change in bowel habit, 18 (19%) had rectal bleeding, 7 (7%) had non-specific weight loss, and 3 (3%) had a clinically palpable abdominal mass.
Bowel preparation
The percentage of segments (supine and prone combined) assigned a score of 1 for residual solid residue (i.e. no residue or scattered) was 76% (218/288), 82% (246/300), 81% (232/288) and 94% (247/264), for regimens A to D respectively, and on average there were 2.5, 2.0, 2.8 and 1.1 stool balls ≥6 mm per patient. The improved quality of preparation in regimen D reached statistical significance [odds of score 2–4: 0.19 (95% CI: 0.07, 0.56), P = 0.02]. Across all regimens, the distal colon was significantly better prepared then the proximal colon (P = 0.006). For example the number of segments assigned at least a score of 2 for solid residue in the caecum was 42% (20/48), 48% (24/50), 35% (17/48) and 23% (10/44) for regimens A to D respectively, compared to 25% (12/48), 16% (8/50), 21% (10/48) and 0% (0/44) for the rectum.
The percentage of segments (supine and prone in total) assigned a score of 1 or 2 for residual fluid was 51% (148/288), 59% (178/300), 53% (153/288) and 43% (114/264) for regimens A to D respectively. There was no significance difference between groups either overall or between the proximal and distal colon (P = 0.22–0.37).
Tagging quality
Tagging quality was generally good (mean tagging score 3.7–4.5) (Fig. 3). While there was weak evidence of improved proximal colonic tagging for residue ≤5 mm for regimens C and D (P = 0.08), overall there was no difference in the efficacy of solid residue tagging across regimens (Table 3).
Fig. 3A 54-year-old male with unexplained rectal bleeding. Axial CT colonographic image showing homogeneous tagging of stool ≤5 mm (arrow) and ≥6 mm (arrowhead) in sizeTable 3Efficacy of tagging of solid residue according to size and regimenSolid residue sizeColon segmentsRegimenMean tagging score (SD)Odds ratio (95% CI)aP value≤5 mmAll segmentsA4.3 (1.2)1B4.3 (1.2)0.94 (0.39, 2.32)C4.5 (1.1)1.47 (0.56, 3.87)D4.5 (1.2)1.67 (0.68, 4.14)0.56Distal colonbA1B0.97 (0.37, 2.52)C0.76 (0.26, 2.27)D1.00 (0.38, 2.61)0.97Proximal coloncA1B0.85 (0.26, 2.80)C4.06 (0.74, 22.1)D3.22 (0.95, 10.9)0.08≥6 mmAll segmentsA4.1 (1.6)1B4.3 (1.5)1.82 (0.44, 7.62)C4.1 (1.7)1.56 (0.32, 7.76)D3.7 (1.8)0.43 (0.11, 1.65)0.24Distal colonbA1B1.30 (0.26, 2.52)C3.84 (0.37, 39.2)D0.17 (0.02, 1.27)0.12Proximal coloncA1B3.22 (0.26, 39.8)C0.94 (0.11, 7.87)D0.91 (0.11, 7.54)0.78SD Standard deviationaOdds of tagging score of 5 (best) compared to regimen AbRectum, sigmoid and descending colon combinedcTransverse, ascending colon and caecum combined
The average per-segment fluid tagging score was 2.5 (SD 0.8), 2.3 (SD 1.0), 2.5 (SD 0.9) and 2.4 (SD 1.2) for regimens A to D respectively. The odds of homogeneous tagging (i.e score 3) did not differ across the four regimens either overall or between the proximal and distal colon (P = 0.65–0.95). In total, 4% (6/144), 1% (2/150), 2% (3/144) and 5% (6/132) of segments respectively contained non-tagged fluid (score 1) (Fig. 4).
Fig. 4A 75-year-old female with change in bowel habit. Axial CT colonographic image showing failure of fluid tagging (arrow) (grade 1)
Overall 33% (8/24), 44% (11/25), 38% (9/24) and 36% (8/22) of patients receiving regimens A to D respectively had at least one segment with layering of tagged fluid (P = 0.89) (Fig. 2).
Fluid tagging density
In terms of mean HU of the tagged fluid (excluding the least tagged layer if tagging was layered), there was no significant difference among the four regimens, either overall or between the proximal/distal colon (P = 0.14–0.86). The mean HU of tagged fluid was 522 (SD 283), 504 (SD 395), 430 (SD 268), and 491 (SD 269) for regimens A to D respectively.
However in those segments with layering of contrast, the maximum attenuation (HU) of the least tagged layer was significantly higher for regimens C and D than for regimens A and B (P = 0.002) [77 (SD 105), 119 (SD 139), 155 (SD 118) and 174 (SD 122), for regimens A to D respectively].
Patient experience
A total of 89 of 95 (94%) patients completed the questionnaire (Table 1), although responses were incomplete in 4. Demographic data for the present study cohort (50 female, mean age 64 years, range 50–85 years) were not significantly different from the 69 historical controls (36 female, mean 63 years, range 35–85 years) [16]. Other than sleep disturbance (worse after reduced preparation) (P = 0.01), there was no significant difference in reported tolerance of the reduced and full bowel preparation regimens for any of the factors tested.
Follow-up questionnaire
Sixty-nine (73%) patients returned the follow-up questionnaire (Table 2). Overall 95% tolerated the reduced preparation regimen well or fairly well, and most (83%) found drinking the tagging agents acceptable. Although most [51/69 (74%)] found the additional preparation required for colonoscopy “no problem”, a majority (61%) found the reduced preparation regimen “better” or “much better” than the full preparation required for colonoscopy.
Diagnostic performance
Five patients (one each from regimens A, B, and C and two from regimen D) were excluded from the performance analysis due to a new diagnosis of inflammatory bowel disease (all presenting with rectal bleeding). A further patient (from regimen B) was excluded after refusing colonoscopy. Of the remaining 89 patients, 68 had either normal colonoscopy or diminutive polyps (≤5 mm) only, and 21 had at least one polyp ≥6 mm or cancer. Colonoscopy was incomplete in 10/89 (11%) reaching the transverse colon in 6, sigmoid in 3 and hepatic flexure in 1. Reasons for failure were obstructing stricture (1), severe diverticulosis (1), tortuous colon (5), and pain (3). Only those segments visualised at colonoscopy were included in the assessment of diagnostic performance.
Per polyp analysis
In total there were 9 polyps ≥10 mm (all adenomatous), 12 polyps 6–9 mm (10 adenomatous, 2 hyperplastic) and 72 polyps ≤5 mm (46 were recovered for histology, of which 26 were adenomatous, 15 hyperplastic and 5 normal mucosa). One 6-mm polyp and two 5-mm polyps were found only on re-look endoscopy after segmental unblinding of the CTC report. No CTC-detected polyps were classified as false positives due to segmental or size mismatching with colonoscopy.
Summed across regimens A–D, detection of cancer, polyps ≥10 mm, 6–9 mm and ≤5 mm was 2/2 (100%), 8/9 (89%), 9/12 (75%) and 23/72 (32%) respectively (Fig. 5) (Table 4). In total there were only four false positives ≥6 mm (Figs. 6 and 7), with no significant difference between regimens (P = 0.15).
Fig. 5A 71-year-old male with rectal bleeding. Axial CT colonographic image demonstrating a 30-mm rectal cancer (arrow). Note the adjacent well-tagged residue (arrowheads)Table 4Polyp detection overall and according to tagging regimenRegimenPatient numberDetection cancer, n (%)Detection 10 mm+, n (%)Detection 6–9 mm, n (%)Detection 1–5 mm, n (%)False positive 10 mm+, nFalse positive 6–9 mm, nFalse positive 1–5 mm, nP valueaA232/2 (100%)1/2 (50%)N/A3/10 (30%)0170.15B23N/A3/3 (100%)5/6 (83%)10/30 (33%)01b9C23N/A1/1 (100%)2/2 (100%)6/20 (30%)001D20N/A3/3 (100%)2/4 (50%)4/12 (33%)1c12Overall892/2 (100%)8/9 (89%)9/12 (75%)23/72 (32%)1319N/A Not applicableaComparison of false positives across regimens using one-way ANOVAbIn patient with confirmed 8-mm polypcIn patient with confirmed 10-mm polypFig. 6A 54-year-old male with change in bowel habit. Axial CT colonographic image of a 6-mm filling defect in the rectum reported as a polyp. No lesion was found at colonoscopy with segmental unblinding suggesting the lesion was untagged faecal residueFig. 7Overall number of false positives according to size and regimen (n = 89)
Per patient analysis
Across all regimens sensitivity, specificity, positive and negative predictive values for detection of patients with any lesion ≥6 mm were 96%, 97%, 0.9 and 0.96 respectively (Table 5).
Table 5Per-patient performance overall and according to tagging regimenRegimenPatient numberPolyp ≥6 mm incl. cancer, n (%) [95% confidence limits]Polyp ≥10 mm incl. cancer, n (%) [95% confidence limits]SensitivitySpecificityPPVNPVSensitivitySpecificityPPVNPVA233/4 (75%) [33–100%]18/19 (95%) [85–100%]0.750.953/4 (75%) [33–100%]19/19 (100%) [100–100%]1.00.95B237/7 (100%) [100–100%]16/16 (100%) [100–100%]1.01.03/3 (100%) [100–100%]20/20 (100%) [100–100%]1.01.0C233/3 (100%) [100–100%]20/20 (100%) [100–100%]1.01.01/1 (100%) [100–100%]22/22 (100%) [100–100%]1.01.0D205/7 (71%) [38–100%]12/13 (93%) [78–100%]0.830.863/3 (100%) [100–100%]17/17 (100%) [100–100%]1.01.0Overall8918/21 (96%) [93–100%]66/68 (97%) [93–100%]0.9a0.9610/11 (91%) [74–100%]78/78 (100%) [100–100%]1.0b0.99NPV Negative predictive value, PPV positive predictive valueaPrevalence of abnormality = 0.24bPrevalence of abnormality = 0.12
Reader confidence
The mean segmental observer confidence for excluding a polyp ≥6 mm was 90, 89, 97 and 93% for regimens A to D respectively. There was no significant difference among the regimens, either overall or in the proximal/distal colon (P = 0.15–0.57).
Discussion
In accordance with previous studies [6, 8, 10], we found patients in general preferred reduced preparation to the full purgation required for colonoscopy, although this preference was much less than expected (only 61% preferred the reduced preparation). We defined the laxation used as “reduced” in comparison to the endoscopic preparation used at our institutions which includes an additional 18 g of magnesium citrate. We also used senna rather than bisacodyl as used by Lefere et al. [9]. Both are stimulant laxatives with a similar mode of action and effect at the doses administrated, although senna was preferred as it is widely used at our institutions. However a combination of 13 g of senna and 18 g magnesium citrate clearly produces relatively strong purgation, reflected in the induced symptoms (77% of patients opened their bowels over five times) and marginal patient preference. It would therefore seem reasonable to study reduced laxation further, perhaps omitting the senna and/or reducing the dose of magnesium citrate. It is however important to realise that whereas normally patients took no solids by mouth for 24 h prior to colonoscopy, in the present study they were permitted to eat from a low-residue diet kit, an issue we did not specifically address with our questionnaires, thereby potentially underestimating this benefit.
Recent data have questioned whether CTC is actually better tolerated than conventional colonoscopy [18], but because full bowel purgation is often cited by patients as the worst part of any colonic examination [1, 2], it is assumed that reduced laxative CTC will improve patient compliance (notably in a screening setting). However this assumption has not been proven in prospective trials, and we cannot extrapolate the preferences we found into increased compliance with CTC. Indeed it could be argued the laxative regimen we used would have relative little impact on compliance in a screening setting, given the side-effect profile.
The overall quality of bowel preparation was good, with all four regimens producing at least 76% of segments with either no or scattered residue only. This is similar to data reported by Lefere et al. [9], who combined 16.5 g of magnesium citrate with biscodyl tablets and suppository. Interestingly, regimen D, which included 15 ml meglumine amidotrizoate, resulted in significantly better preparation than the other regimens, possibility due to a “washing effect” of the iodine-based contrast, incorporating solid residue into a more fluid solution [10]. Although we used a small dose, meglumine amidotrizoate is also known to have a laxative effect that may also have added to the superior cleansing.
Tagging of solid residue was in general good, with no significant difference between the regimens tested. This suggests barium-based tagging can be simplified to a 1-day regimen only, when combined with reduced cathartic preparation. Similarly all regimens produced an average tagged fluid density of around 500 HU. Recent phantom data suggest that although submerged polyp conspicuity is optimised at 700 HU, it remains good at 500 HU [19]. We did however demonstrate that fluid tagging could be manipulated by additional oral agents on the morning of CTC. As it is non-miscible with water, barium often produces a layering effect with lower attenuation fluid sitting above denser barium. Although there was no significant difference in layering among the regimens, the addition of morning 2.1% barium sulphate or meglumine amidotrizoate significantly increased the attenuation of the non-dependent fluid layer. It is arguable whether this will have significant impact clinically, not least because fluid moves between supine and prone positions, but it may have implications for subtraction software. Zalis et al. have recently demonstrated suboptimal subtraction when high-density barium is used as the sole tagging agent [10]. It is interesting to speculate whether manipulation of fluid tagging could optimise subtraction. It should also be noted our regimen mildly restricted fluid intake the day prior to CTC, which may have influenced our fluid tagging results.
One major advantage of barium as a tagging agent is its inert nature and safety. Although iodinated contrast medium produces more homogeneous fluid tagging, this is mainly of clinical importance if subtraction software is being used. While the risks of allergy to oral iodinated contrast are minimal, adverse events do occur [20, 21]. Our data suggest good results can be achieved using only barium.
Ultimately, diagnostic performance is the best measure of the success of any reduced-laxative protocol. Reassuringly overall, we prospectively found high diagnostic performance when compared to colonoscopy. Our data confirm again that full bowel preparation is not required to maintain diagnostic accuracy for CTC, assuming adequate reader training [22]. Importantly, there were only four false positive polyps over 6 mm, and across all regimens the positive predictive value for patients with any lesion ≥6 mm and ≥10 mm was 0.9 and 1.0 respectively (Table 5).
Our study does have weaknesses. It could be argued that we used a relatively harsh laxative regime given that good results have been reported without any laxative at all [5, 6]. However the vast majority of CTC worldwide is still performed using full bowel preparation, and in the authors’ opinion, significant changes in practice are most likely to follow gradual reduction of purgation, which allows radiologists to become comfortable with this approach. However we acknowledge that if there was good evidence of acceptable performance data using CTC without prior laxation, this may well rapidly become widely implemented by the CTC community. We compared patient symptomatology with a historical cohort, and although well-matched in demographic terms, we cannot exclude bias completely. We did not instruct colonoscopists to grade the quality of bowel preparation and, in particular, the influence of retained barium on mucosal visibility, although no endoscopy failed due to incomplete bowel preparation. It could be argued that reduced preparation regimens are best suited to increase compliance in asymptomatic screening populations [23]. However for pragmatic reasons we used symptomatic patients, as we do not have access to a large screening population. Finally, although we were able to show high diagnostic performance for reduced-preparation CTC overall, given the relatively low number of polyps, we were unable to meaningfully compare across regimens.
In conclusion, a combination of reduced laxatives and a simple tagging regimen based on 40% barium sulphate the day prior to CTC maintains acceptable diagnostic accuracy. Three doses of 20 ml 40%w/v barium sulphate are as effective as more complex regimens, but fluid tagging can be manipulated by addition of dilute barium or meglumine amidotrizoate on the morning of CTC, the latter also reducing the volume of residual stool.

colonography
computed tomography
cathartics
barium sulfate


The efficacy of anakinra in an adolescent with colchicine-resistant familial Mediterranean fever

Colchicine is the treatment of choice in familial Mediterranean fever (FMF) for the prevention of both attacks and secondary amyloidosis. The overall nonresponder rate is about 5–10%. Anakinra is known to have good effectiveness in a severe autoinflammatory syndrome [chronic infantile neurological cutaneous and articular (CINCA) syndrome] and other recurrent hereditary periodic fevers. Pyrin—the protein involved in FMF—has a role in activating the proinflammatory cytokine interleukin (IL)-1β. We report the effectiveness of the addition of an IL-1-receptor inhibitor (anakinra) to colchicine in controlling the febrile attacks and acute phase response in an adolescent with FMF resistant to colchicine.
Case report
A 15-year-old girl with familial Mediterranean fever (FMF) was admitted to our hospital because of the persistence of recurrent fever attacks (twice a month), accompanied by chest and abdominal pain with headache, despite taking colchicine at a high dose (2 mg/kg per day). She suffered severe discomfort and complained of several school absences.
Acute phase reactants [erythrocyte sedimentation rate (ESR) 110, C-reactive protein (CRP) 43 mg%] and serum interferon-γ (125.4 pg/ml) were high during the crises. There was no proteinuria, but given the severity of the clinical picture, we performed a rectal biopsy to definitely exclude the presence of amyloidosis and it was negative.
Anakinra was started at 50 mg/day (1 mg/kg per day) subcutaneously, without stopping colchicine. Approval was obtained from an Ethics Committee (Burlo Garofolo Health Authority).
In the following 3 months the patient developed only three mild episodes of abdominal pain without fever, which resolved in a few hours. During these episodes acute phase reactants were negative. Moreover, serum interferon-γ was low (23 pg/ml).
After 3 months, anakinra was discontinued to confirm its real efficacy, without stopping colchicine. Following this interruption, the girl complained of eight fever attacks in 3 months accompanied by abdominal pain, vomiting, chest pain, and headache. Anakinra was reintroduced. Since then (15 months of follow-up), the patient has been in good health, without any fever attacks or abdominal or chest pain.
There have been no adverse effects due to anakinra, apart from minor local stinging and erythema at the injection sites, which gradually diminished.
Discussion
Colchicine is the recommended treatment of FMF since it can prevent the febrile attacks and the development of amyloidosis, even though a few patients develop amyloidosis also when on colchicine treatment [3, 5]. However, approximately one third of the patients have a partial remission and about 5–10% are nonresponders [3]; another 2–5% do not tolerate the drug well [3]. In single case reports several drugs (interferon-α, infliximab, thalidomide, etanercept) have been suggested as a possible alternative treatment for these cases [3, 6].
Anakinra is a recombinant form of the human interleukin (IL)-1-receptor antagonist that targets type I IL-1-receptor which is expressed in many tissues. There are some studies and case reports that show a good efficacy of anakinra in a severe autoinflammatory syndrome (CINCA, chronic infantile neurological cutaneous and articular syndrome) [2] and other recurrent hereditary periodic fevers [4]. Despite the elevated costs (approximately 50 € per 100 mg) its use appears justified in some inflammatory diseases that lack alternative treatments.
Since pyrin—the protein involved in FMF—activates the precursor form of IL-1β into its biologically active fragments there is a rationale for testing anakinra in FMF [1, 5]. Moreover, anakinra did appear to be effective in suppressing acute phase reactants in a patient with FMF (a nonresponder to the treatment with colchicine) and amyloidosis [1].
In our case, the addition of anakinra to colchicine had a dramatic therapeutic benefit without relevant side effects. As the febrile attacks recurred after discontinuing anakinra and disappeared again after reintroduction, we believe in the real therapeutic efficacy of this drug. In light of the present evidence [3, 5], colchicine should be continued in order to prevent further developments of amyloidosis.

anakinra
familial mediterranean fever
colchicine resistance