Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2–3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.
Introduction
Poinsettia, Euphorbia pulcherrima Willd. Ex Klotzsch, is a contemporary symbol of Christmas in most parts of the world. Since it was introduced to the United States in 1825 from Mexico, poinsettia has become the primary potted flower produced and sold in North America, Europe, Asia and Australia (Ecke et al. 2004; Williams 2005). Today, Europe and North America represent the largest volume of production and sales, but demand is growing quickly in the Australian region as poinsettia becomes more popular each year (Williams 2005). Global production of poinsettia has exceeded hundreds of millions and is still expanding, indicating its economic and market potential for the floral industry.
Genetic engineering is an important tool for breeding ornamental plants with addition of desirable traits such as novel colour, better quality and resistance to pathogens and insects (Mol et al. 1995; Deroles et al. 2002; Hammond 2006; Hammond et al. 2006). This technology has been successfully utilized in the production of a number of important ornamental crops e.g. blue roses (Yoshikazu 2004), novel carnations (http://www.florigene.com), transgenic gladiolus (Kamo et al. 1997) and improvement of chrysanthemums (Teixeira da Silva 2004). To date, transgenic ornamentals of over 30 genera have been produced by different transformation approaches (Hammond 2006; Hammond et al. 2006). However, there are only a few reports describing genetic transformation of poinsettia: one was the US patent 7119262 (Smith et al. 1997) using the biolistic transformation approach, while the other two were electrophoresis-based transformation attempts (Vik et al. 2001; Clarke et al. 2006). Biolistic transformation requires the use of a gene gun device (Sanford et al. 1987) and tends to generate transformants with a high transgene copy number, complex transgene loci and unpredictable silencing of the transgene (Herrera-Estrella et al. 2004). Electrophoresis of DNA into meristems on a living plant was described as a simple method to generate transformants by avoiding tedious tissue culture work and was utilized in producing transgenic orchid (Griesbach 1994). However, no stable transgenic poinsettia was ever produced using electrophoresis, regardless of the strong transient expressions that were detected in both studies (Vik et al. 2001; Clarke et al. 2006). Thus, Agrobacterium-mediated transformation for poinsettia was developed in the present study.
Poinsettia mosaic virus (PnMV) is a single-stranded, positive-sense RNA virus (Bradel et al. 2000) that belongs to the family Tymoviridae (Dreher et al. 2005). Infection of poinsettia plants with PnMV results in mosaic symptoms during parts of the growing season (Fulton and Fulton 1980), which in turn decreases the commercial value of this ornamental plant. Thus, growers are interested in the potential benefits of growing PnMV-free poinsettias. PnMV-free poinsettia plants can be obtained by heat treatment or in vitro culture of apical meristems, which are time-consuming and cost-ineffective methods. An additional problem is that PnMV-free poinsettia tends to be rapidly reinfected, although no vector is known (Blystad and Fløistad 2002; Siepen et al. 2005). There is therefore a need for a new and effective alternative approach, like Agrobacterium-mediated transformation, which can overcome these difficulties.
RNA silencing is a mechanism by which transcription or translation of a gene is suppressed. It is known to occur in plants, fungi and animals (Fire et al. 1998; Waterhouse et al. 1998; Baulcombe 2005). It is triggered by double stranded RNA (dsRNA) molecules (Meister and Tuschl 2004), which are subsequently recognized and cleaved by the host-encoded endoribonuclease dicer (Bernstein et al. 2001) into small interfering RNA (siRNA) molecules of 21–26 nucleotides in length (Hamilton and Baulcombe 1999). These siRNAs, in conjunction with the RNA-induced silencing complex (RISC), target RNA molecules with homologous sequences for sequence-specific degradation (Hammond et al. 2000; Bernstein et al. 2001; Tabara et al. 2002). In plants, RNA silencing can be achieved by genetic transformation with gene constructs that express highly transcribed sense, anti-sense or self complementary hairpin RNA (hpRNA) containing sequences homologous to the target gene (Smith et al. 2000; Wesley et al. 2001; Helliwell and Waterhouse 2003). RNA silencing has been efficiently used to generate resistance against plant viruses in many plant species including ornamentals (Metzlaff et al. 1997; Tenllado et al. 2004; Bucher et al. 2006; Hammond et al. 2006).
In this study, we report the development of an A. tumefaciens-mediated transformation method for poinsettia, which has never previously been described for this plant species. This method could facilitate the improvement of poinsettia by introducing new traits into existing commercial poinsettia cultivars in order to meet market demands. Using this approach, in combination with RNA silencing technology, transgenic PnMV resistant poinsettia plants carrying PnMV-derived hpRNA constructs were produced for the first time.
Materials and methods
Plant materials
Euphorbia pulcherrima, poinsettia cv. Millenium, plants were kindly supplied by the J. Kristiansen nursery, Grimstad, Norway, in the year 2000. The original stock plants were subjected to heat therapy to eliminate PnMV (Fløistad and Blystad, unpublished). PnMV-free cv. Millenium cuttings were grown in the greenhouse under a photoperiod of 16 h light and 8 h dark with a temperature of 22°C. Internode stem explants from 8 to 10-week-old cv. Millenium plants were used for somatic embryogenesis and A. tumefaciens-mediated transformation.
Somatic embryogenesis of poinsettia cv. Millenium
Internode stem explants 5–15 mm long from cv. Millenium plants were excised and used in the establishment of somatic embryogenesis for poinsettia prior to transformation. In Experiments 1 and 2, the explants were surface sterilized for 10 min in 3% NaOCl and then rinsed three times with sterilized deionized and autoclaved H2O for 5, 10 and 15 min according to Preil (1994). Because of a large number of infections encountered in Experiments 1 and 2, the stem explants were sterilized in Experiment 3 using 70% ethanol for 1 min followed by 5 min with 1% NaOCl and 3 rinsings with sterile deionized and autoclaved water for 3, 10 and 20 min, a modification of the protocol of Preil (1994). The modified protocol was repeated twice before being utilized in Experiment 3 and also in subsequent transformation experiments. After sterilization, stem segments of ca. 1–1.5 mm thickness were placed on callus induction (CI) medium [MS medium (Murashige and Skoog 1962) supplemented with 0.2 mg l−1 BAP and 0.2 mg l−1 CPA and 30 g l−1 sucrose, Table 1] for 8–10 days. Embryogenic calli were then transferred to somatic embryo induction (SEI) medium (MS medium contains 0.3 mg l−1 NAA and 0.15 mg l−1 2iP and 30 g l−1 sucrose, Table 1) for somatic embryogenesis. The somatic embryos emerged after an average of 12 weeks were then transferred to somatic embryo maturation (SEM) medium (MS basal medium containing 0.05 mg l−1 BAP and 30 g l−1 sucrose, Table 1). Shoots and plantlets derived from somatic embryos were subsequently cultured on root induction (RI) medium consisting of 1/2 strength MS, 2 mg l−1 IAA and 20 g l−1 sucrose or on hormone free 1/2 strength MS (HFMS) medium supplemented with 20 g l−1 sucrose for root induction (Table 1). Plants with well-developed roots were transferred to soil and grown in the greenhouse at 22°C. The above culture conditions and media compositions for somatic embryogenesis of cv. Millenium were developed according to Preil (1994) with a number of modifications described above. Detailed information regarding media compositions is summarized in Table 1.
Table 1Media names and compositions modified according to Preil (1994)MediumMSSucrose (%)CPA (mg l−1)BAP (mg l−1)NAA (mg l−1)2iP (mg l−1)IAA (mg l−1)CIFull strength30.20.2–––SEIFull strength3––0.30.15–SEMFull strength3–0.05–––RI1/2 strength2––––2HFMS1/2 strength2–––––CI Callus induction medium, SEI somatic embryo induction medium, SEM somatic embryo maturation medium, RI root induction medium, HFMS hormone free MS medium for root induction
Light conditions were 23 μE m−2 s−1 for callus and somatic embryos and 30 μE m−2 s−1 for plantlets in RI and HFMS media with a 16 h photoperiod. Temperature was constant at 24°C. A light microscope connected to a digital camera was used to follow the development of the somatic embryos.
Agrobacterium strain and hairpin (hp) RNA constructs
The disarmed A. tumefaciens strain LBA4404 (Hoekema et al. 1983; Invitrogen, California, USA) was utilized throughout the study. Three hpRNA constructs, named as pCP, pR2 and pR3, were generated. Construct pCP targeted the viral coat protein (CP), whereas constructs pR2 and pR3 targeted two distinct regions within the viral RNA-dependant RNA-polymerase (RdRp) (Fig. 1a). Briefly, constructs pCP, pR2 and pR3 were generated by amplifying the corresponding fragments from the viral genome and introducing the appropriate restriction sites. The following restriction sites were introduced: XhoI·KpnI (for sense orientation) and ClaI·XbaI (for antisense orientation) for R2 and R3 fragments, and XhoI·EcoRI (sense) and ClaI·XbaI (antisense) for CP fragment. The primers used are presented in Table 2. Each amplified fragment was 500 bp long. Subsequently, the fragments were inserted into pHANNIBAL vector (kindly provided by CSIRO Plant Industry, Canberra, Australia) in sense and antisense orientations, interrupted by an intron pyruvate orthophosphate dikinase (pdk) gene (Fig. 1b) as described by Helliwell and Waterhouse (2003). The three expression cassettes were cloned into pART27 (Gleave 1992, kindly provided by CSIRO Plant Industry, Canberra, Australia), a binary plasmid vector, at Not1 site under the control of CaMV 35S promoter and with ocs terminator (Helliwell and Waterhouse 2003), generating hpRNA constructs pCP, pR2 and pR3 (Fig. 1b). For selection, the neomycin phosphotransferase II (nptII) gene conferring kanamycin resistance was used under the control of nopaline synthase promoter (Nos-P) and with Nos terminator (NosT, Fig. 1b) in the binary plasmid expression vector pART27. Sequence analysis was utilized to verify the junctions and orientations in the expression vectors pCP, pR2 and pR3. All three plasmid vectors were introduced into A. tumefaciens strain LBA 4404 by electroporation according to the manufacturer’s instructions (Invitrogen, California, USA).
Fig. 1Schematic representation of PnMV genome (a) and hairpin (hp) RNA constructs used in the current study (b). The locations of the primers used to generate the constructs as well as for screening purposes are presented over the PnMV genome. Sequences of the primers are presented in Table 2. The location of the probe used to detect siRNA is presented below the PnMV genome (see “Materials and methods”). CP coat protein region, R2 and R3 RNA-dependent RNA-polymerase (RdRp) regions of the PnMV genomeTable 2Primers used in this studyPrimer nameRestriction sitesSequenceaLocationR2F-XhKXhoI, KpnIctcgagggtaccTTTAGCAAAACGCAGCACAAAATCA4,371–4,395R2R-CXbClaI, XbaIcatcgattctagaTCTCCAGACACCATGATTGGGTG4,848–4,870R3F-XhKXhoI, KpnIctcgagcggtaccTTCGCTTTAAAACAGAAAGCACCA4,939–4,962R3R-CXbClaI, XbaIcatcgatgtctagaGCCTCGTAGCTTGGTTGGGTT5,418–5,438CPF-XhEXhoI, EcoRIctcgaggaattcAACCACGTCGACTCCACTCCAT5,478–5,499CPR-CXbClaI, XbaIatcgattctagaAGCTTGCCGCTCACCAGCAC5,958–5,977aPrimer sequences are presented in the 5′–3′ orientation. Sequences complementary to the viral genome are presented in uppercase letters whereas non-complementary restriction sites included in each primer are presented in lowercase letters. The primers used for screening purposes are identical to the ones presented but do not include the non-complementary restriction sites
Transformation of poinsettia stem explants
A. tumefaciens strain LBA4404, harbouring the plasmid pCP, pR2 or pR3, was grown overnight in 15 ml liquid LB medium supplemented with 50 mg l−1 kanamycin (Sigma-Aldrich, St Louis, USA) at 28°C with shaking at 200 rpm until an OD600 of 0.6–0.8 was reached. The bacterium suspension was collected at 2,700 rpm for 10 min, washed twice with MS basal medium supplemented with 2% sucrose (MS-2), and resuspended in 10 ml MS-2.
The internode stem explants taken from 8 to 10-week-old poinsettia plants derived from cuttings were disinfected as described above, excised into stem segments with 1–1.5 mm thickness and inoculated with Agrobacterium suspension for 5 min with gentle shaking. After the infection, stem segments were blotted briefly with sterile filter paper and placed on CI medium at 24°C in the dark for 72 h without selection. After co-cultivation, the explants were blotted gently on sterile filter paper and transferred to the CI medium with selection and light conditions as described above.
Selection and regeneration of transgenic plants
After 8–10 days on CI medium supplemented with 500 mg l−1 claforan (Aventis Pharma Ltd, Norway) and 10 mg l−1 kanamycin, the explants were transferred to SEI medium supplemented with 400 mg l−1 claforan and 25 mg l−1 kanamycin for somatic embryogenesis. The protocol for somatic embryogenesis is detailed above. The somatic embryos obtained after about 12 weeks were then transferred to the SEM medium supplemented with 400 mg l−1 claforan and 10 mg l−1 kanamycin. Shoots and plantlets derived from somatic embryos were subsequently cultured on RI medium or on hormone free 1/2 strength MS (HFMS) medium (Table 1) for root induction and were subsequently transferred to soil and grown in the greenhouse at 22°C.
Screening of transgenic poinsettia plants
Screening of transgenic poinsettia plants was carried out by PCR. The primer pairs used to amplify the CP, R2 and R3 fragments were the same as those for vector constructions, but did not include any additional restriction site (Table 2). HotStarTaq PCR kit purchased from Qiagen (Valencia, California) was used in PCR screening of transformants. Twenty micro litres of reaction mixture containing 2× HotStarTaq Mastermix, 0.4 μM of each primer, 0.1 μg template DNA and H2O were subjected to PCR amplification under the following conditions: 15 min at 95°C (1 cycle), 30 s at 95°C, 30 s at 55°C, 1 min at 72°C (35 cycles) and a final extension 10 min at 72°C (1 cycle) according to the manufacturer’s instructions. All the PCR amplification was performed using Applied Biosystems 96 Thermal Cycler (Applied Biosystems). PCR products were analysed by electrophoresis on 0.8% (W/V) agarose gels.
Southern blot analysis
To confirm the stable integration of transgenes into the poinsettia genome and to evaluate transgene copy number, Southern blot analysis was performed, basically as described by Sambrook et al. (1989). Total genomic DNA was isolated from young leaves of control plant and the putative transformants using a modified CTAB protocol as described by Rogers and Bendich (1988). Ten micrograms of genomic DNA was digested with the restriction enzyme HindIII for 4 h and separated on a 1% (W/V) TBE agarose gel overnight at 37 V followed by transfer onto Gene Screen Transfer membrane (NEN™ Life Science Products Inc., Boston, MA, USA). The HindIII site is not present in the pCP, pR2 and pR3 vectors. The HindIII site from the pHANNIBAL vector was removed during the vector construction. Membranes were hybridized overnight with 32P-labelled probe of 1.5 kb in size targeting the CP, R2 and R3 regions (Fig. 1a). The probe was generated by amplifying a 1.5 kb fragment that included the R2, R3 and CP regions used to generate the hairpin RNA constructs pR2, pR3 and pCP. This allowed us to use one probe instead of three probes for southern blot analyses of pR2, pR3 and pCP transformants.
Northern blot hybridization of low molecular weight RNA
Total RNA was extracted using the Trizol reagent (Invitrogen, USA) as recommended by the manufacturer. Low molecular weight (LMW) RNA was separated from high molecular weight RNA by precipitation with 1 volume of 4 M LiCl4 at 4°C overnight. After centrifugation, the LMW RNA-containing supernatant was transferred to a new tube and precipitated with 1 volume of isopropanol. LMW RNA concentrations were measured using a GeneQuant II spectrophotometer (Amersham Biosciences, UK) and RNA quality was verified by agarose gel electrophoresis. LMW RNA (approximately 30 μg) was mixed with 1 volume of Tris-borate–EDTA–urea sample buffer (Biorad, USA) and heated at 100°C for 5 min. Subsequently, LMW RNA was separated on a 15% polyacrylamid tris-borate–EDTA–urea gel and transferred to a Hybond-N membrane (Amersham Biosciences, UK) overnight. The membranes were hybridized with a digoxigenin (DIG)-labelled (Roche Diagnostics, Germany) RNA probe (Fig. 1a), previously cleaved by alkaline hydrolysis to generated fragments of approximately 50 bp as described by Hamilton and Baulcombe (1999). After hybridization the membranes were washed at room temperature, 2 × 15 min in 5× SSC + 0.5% SDS and 2 × 15 min 1× SSC + 0.5% SDS and then exposed to films.
Virus inoculation and detection
Sap from poinsettia and N. benthamiana plants systemically infected with PnMV was used as an inoculum. Approximately 0.5 g of infected leaf tissue was ground with 4 ml of inoculation buffer [0.03 M sodium phosphate buffer (pH 8)] and mechanically inoculated to leaves of three to four-leaves stage plants lightly dusted with carborundum. Plants were grown in the greenhouse at 22–24°C.
Detection of PnMV infection by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was carried out at 6 and 10 weeks post-inoculation basically as described by Clark and Adams (1977). The uppermost fully expanded leaves of each plant were collected in polyvinyl bags (Bioreba, USA). Extraction buffer [100 mM phosphate buffer (pH 7.4), 20 mM Tris, 137 mM NaCl, 3 mM KCl, 2% PVP 24 kD, 0.05% Tween 20, and 0.02% NaN3] was added to the bags (1/10 w/v) and the samples were macerated. A measure of 100 μl of the homogenates were transferred to wells of microtitre plates previously coated with 100 μl of PnMV IgG (Agdia, USA) diluted 1/1,000 in coating buffer [50 mM carbonate–bicarbonate buffer (pH 9.6) and 0.02% NaN3], followed by incubation at +4°C overnight. Microtitre plates were washed with washing buffer [10 mM phosphate buffer (pH 7.4), 140 mM NaCl, 3 mM KCl, and 0.05% Tween 20] and 100 μl of alkaline phosphatase conjugated PnMV IgG (Agdia, USA) diluted 1/1,000 in extraction buffer was added to each well. Microtitre plates were incubated at +4°C overnight. Subsequently, they were washed with washing buffer and 100 μl of substrate buffer [1 M diethanolamine (pH 9.8) and 0.02% NaN3] containing p-nitrophenyl phosphate (0.5 mg ml−1) was added. Absorbance values were recorded at 405 nm with an Expert plus microtitre plate reader using the Kim software (ASYS HITECH, Austria). Values were considered positive when the A450 value was twofold higher than that of the negative control.
Results
Establishment of somatic embryogenesis for poinsettia cv. Millenium
An efficient protocol for somatic embryogenesis of poinsettia cv. Millenium was developed. Stem explants of cv. Millenium responded well to the induction media and a large number of somatic embryos were effectively induced (Table 3; Fig. 2a–d). In Experiments 1 and 2, somatic embryos could only be induced from 21 to 37% of the explants due to infection, whereas more than 75% of the explants produced somatic embryos in Experiment 3 after the modification of the disinfection protocol. The average numbers of somatic embryos on each embryogenic callus varied from 5.3 ± 2.4 and 5.8 ± 3.1 for Experiments 1 and 2, respectively, to 7.2 ± 4.1 for Experiment 3 (Table 3). The highest induction of somatic embryos was found in Experiment 3 with 18 somatic embryos on a single callus. When globular stage somatic embryos (Fig. 2a, arrowed) reached the mature stage (Fig. 2b), they became loosely attached to the calli. Shoots derived from somatic embryos were subsequently cultured on RI medium or HFMS medium (Fig. 2c) for root induction and were transferred into the greenhouse when the roots were well established (Fig. 2d). The three experiments conducted and the efficiency of somatic embryogenesis are summarized in Table 3.
Table 3Experiments and efficiency of somatic embryogenesisExperimentNo. of explants% of embryogenic explantsRange of SE/embryogenic explantMean SE/embryogenic explantaNo. of regenerated plantletsb1200212–105.3 ± 2.4702200372–145.8 ± 3.11353350753–187.2 ± 4.1768SE somatic embryosaValues are presented with their corresponding standard deviationbRegenerated plantlets after 14 weeksFig. 2Somatic embryogenesis in poinsettia cv. Millenium: a embryogenic structure and globular stage somatic embryos (arrows) that appeared on the callus (bar 1 mm), b cotyledonary stage of somatic embryos (bar 1 mm), c plantlets deriving from somatic embryos on RIM medium; and d regenerated plants established in the greenhouse
The RI and HFMS media used for root induction did not show any clear differences in the percentage (85 and 87% for RI and HFMS, respectively) of plants that developed roots 3 weeks after being transferred to the rooting media.
Development of the A. tumefaciens-mediated transformation method
Prior to the production of transgenic poinsettia plants, a number of optimizations were carried out to reveal the most suitable conditions for inoculation and co-cultivation, concentration of antibiotic for eliminating A. tumefaciens after co-cultivation, and kanamycin selection without inhibiting somatic embryogenesis and regeneration of plants. Results showed that the optimal inoculation time was 5 min with gentle shaking, while the most suitable co-cultivation time was 72 h (Table 4). Using this combination, 26 out of the 80 (33%) explants tested PCR positive after inoculation, co-cultivation and 2 weeks on claforan (Table 4). Those explants were followed and showed normal embryogenesis and regeneration as compared to the control (data not shown). Therefore, these conditions were utilized throughout all the transformation experiments. When inoculation time exceeded 20 min followed by a co-cultivation period of 6 days, only 4% of the explants survived due to the overgrowth of Agrobacterium on the surfaces of the explants (Table 4).
Table 4Number of PCR positive explants that survived 2 weeks after Agrobacterium inoculation and co-cultivation treatments. A total of 80 explants were used in each treatmentCo-cultivation time (days)Inoculation time 5 min10 min20 min29 (11%)13 (16%)21 (26%)326 (33%)21 (26%)20 (25%)610 (13%)8 (10%)3 (4%)
For elimination of Agrobacterium growth after co-cultivation, the optimized concentration of claforan was 500 mg l−1 for the CI medium, whereas 400 mg l−1 claforan was an adequate concentration for the SEI and SEM media. For selection of transformed cells, 25 mg l−1 kanamycin was found optimal for the SEI medium, while 10 mg l−1 kanamycin was optimal for the CI and SEM media.
Production of transgenic poinsettia plants, molecular analyses and resistance assays
To produce transgenic poinsettia plants, 5 transformation experiments including 868 stem segment explants were carried out using the established inoculation, co-cultivation and selection conditions described above. Of those explants, 228 (26.3%) somatic embryo-derived plants were obtained. The results are summarised in Table 5.
Table 5Summary of five Agrobacterium-mediated transformation experiments on poinsettia cv. Millenium with pCP, pR2 and pR3 constructsExperimentaNo. of explantsNo. of regenerated plantsNo. of transformantsbTransformation efficiency (%) c1 (CP)1855431.62 (R3)1326932.33 (R3)1726663.54 (R2)2542131.25 (R2)1251832.4aCP, R3, and R2 represent transformation experiments with constructs pCP, pR2 and pR3, respectivelybTransformants verified by PCR and Southern blot analysiscNumber of transformants/total number of explants transformed
PCR analysis was conducted for screening the putative transformants. All 228 regenerated plants were analysed by PCR and 18 transformants were revealed. Of these, three possessed pCP constructs, while six and nine contained pR2 and pR3 constructs, respectively (Fig. 3a–c). Transformation frequency varied from 1.2 to 3.5% with an average transformation frequency of 2.1% (Table 5).
Fig. 3PCR analysis. PCR positive transformants detected with primer pairs for (a) CP, (b) R2 and (c) R3 fragments respectively. Lane A1 1 kb marker; lane A2 non-transformed plant; lanesA3–5 are independent CP-transgenic lines 11-1, 11-2 and 3-1; laneA6 plasmid control. Lane B1 1 kb marker; lane B2 non-transformed plant; lanes B3-8 are R2-transgenic lines 72-1, 72-2, 75-2, 79-1, 84-2, and 84-4; lane B9 plasmid control. Lane C1 1 kb marker; lane C2 non-transformed plant, lanes C3-11 are R3 transgenic lines 18-1, 30A, 38-1, 40A, 40B, 41-2, 41-5, 56-2 and 62-1; lane C12, plasmid control. The 500 bp band of the 100 bp marker is depicted with an arrow
Southern blot analysis was performed on selected PCR positive plants with satisfactory characteristics for studying transgene integration and estimating the transgene copy number. Results of Southern blot analysis confirmed the stable integration of transgene and both single- and multiple-copy transgene integration into the poinsettia genome were detected among the transformants (Fig. 4). Of the eight transformants analysed, six showed single copy integration of transgene (lanes 2–6 and 9 in Fig. 4) with fragment size greater than the length of intact T-DNA (ca 7.5 kb). However, two pR3 transformants (lanes 7–8 in Fig. 4) showed multiple-copy transgene integration with both large (intact T-DNA) and small fragments (partial, non intact T-DNA).
Fig. 4Southern blot analysis of selected PCR positive transformants carrying pCP, pR2 and pR3 constructs. The HindIII-digested total genomic DNA was probed with a 1.5 kb probe homologous to the region of CP, R2 and R3 fragments (Fig. 1) allowing us to analyse all the three different types of transformants (CP, R2 and R3 transformants) at the same time. Lane1 plasmid control; lanes2-4 CP transformants 11-1, 11-2 and 3-1; lanes5-6 R2 transformants 72-2, 79-1; lanes 7-9 R3 transformants 38-1, 40B, 18-1; lane10 negative control
Furthermore, Northern blot analysis of low molecular weight RNA was carried out on the transgenic plants harbouring constructs pCP, pR2 and pR3, prior to inoculation. Transgene-derived siRNA molecules of 21–26 nucleotides length were detected in transgenic lines pCP 11-1, pCP 3-1, pR2 72-1, pR2 72-2 and pR3 18-1 (Fig. 5). This data indicated that the transgene had been transcribed and converted into siRNAs (Fig. 5). Due to limitations in the amount of LMW RNA from transgenic pR3 lines 40B, 41-2, 62-1 and 41-5, these lines were not included in the experiment.
Fig. 5Northern blot analysis of low molecular weight (LMW) RNA to detect small interfering RNAs (siRNA) in non-inoculated transgenic plants and non-inoculated control. aLane 1 control; lanes 2–3 CP transgenic lines 11-1, 3-1; lanes 4–5 R2 transgenic lines 72-1,72-2; lane 6 R3 transformant, 18-1. Ribosomal RNA is presented in (b) to indicate presence of LMW RNA in the gel. Lane 1 control; lanes 2 and 3 PCP transformed plants; lanes 4 and 5 PR2 transformed plants; lane 6 PR3 transformed plant
Transgenic plants and non-transformed controls were mechanically inoculated with PnMV at a three to four leaves stage. The upper non-inoculated leaves were tested by DAS-ELISA at 6 and 10 weeks post-inoculation. Results showed that the control plants were systemically infected with PnMV, whereas the tested transgenic lines carrying pCP and pR2 constructs remained virus free (Table 6). However, of the five pR3-containing transgenic lines studied, two lines remained virus free, while the remaining three lines were systemically infected (Table 6).
Table 6Immunological detection of PnMV on non-transformed and transformed poinsettia plants inoculated with PnMVConstructTransgenic plantaELISAb6 wpi10 wpiR272-10.10.172-20.10.2R318-10.10.240B0.82.441-20.52.662-10.10.241-50.72.8CP11-10.10.23-10.10.1Control +10.82.820.82.7Control −10.10.220.20.230.20.240.10.1wpi Weeks post inoculationaOne plant per transgenic line was challenged in this assaybELISA absorbance values (405 nm) after 60 min of incubation at room temperature are presented. Samples with twice the value of the highest negative control sample were deemed positive
Discussion
Significance of genetic engineering in control of virus diseases in ornamentals
The application of genetic engineering in the floriculture industry has clearly become instrumental and rewarding in meeting the demand for novel desirable traits. A number of genetic engineering approaches have been developed for ornamental plants (Griesbach 1994; Kamo et al. 1995; Deroles et al. 1997; Zuker et al. 1998; Kamo et al. 2000; Kishimoto et al. 2002; Kim et al. 2004; Teixeira da Silva 2004; Hammond 2006 and the current study) to facilitate the improvement of ornamental crops with better quality and enhanced resistance against diseases and pests. Effective resistance against viruses conferred by transgenes has shown significant potential because no chemical can be applied for control of virus diseases. Regardless of the importance, only a few reports have been published demonstrating the virus resistance obtained in ornamentals through genetic engineering (Kamo et al. 1997; review by Hammond et al. 2006). Furthermore, many of the ornamentals were transformed only with marker genes like gus or selectable antibiotic or herbicide resistant markers (Deroles et al. 2002). Using the Agrobacterium-mediated transformation protocol developed in the current study, we have obtained PnMV resistant transgenic poinsettia lines expressing viral sequences targeting the CP and RdRp regions of the PnMV genome. To our knowledge, no PnMV resistance has previously been obtained using classical or molecular breeding approaches, demonstrating the potential of the transgenic lines produced in the present study for the poinsettia industry.
Somatic embryogenesis and Agrobacterium-mediated transformation in poinsettia cv. Millenium
One of the reasons that transformation of ornamental plants has lagged somewhat behind that of major crops is the lack of efficient regeneration systems, as these are key components leading to the successful development of transformation protocols. Moreover, both transformation and regeneration can be cultivar- or even genotype-dependent. Therefore, we developed an efficient somatic embryogenesis system for poinsettia cv. Millenium prior to transformation, although a number of somatic embryogenesis systems were reported previously for old cultivars such as Angelika, Franzi and Diamond (Preil and Beck 1991; Preil 1994; Osternack et al. 1999). A recent study on somatic embryogenesis of cv. Freedom Red and Freedom White demonstrated the influence of cultivar and even plant genotype on somatic embryogenesis in poinsettia (Castellanos et al. 2006), indicating the necessity of developing an effective regeneration system for the individual cultivar prior to the development of transformation systems.
Poinsettia, like other Euphorbia species, produces latex, a milky solution exuded from cut surfaces. When explants were excised from greenhouse grown poinsettia plants, the latex was released from the cutting points, making surface sterilization difficult. In Experiments 1 and 2, a very low percentage of explants produced somatic embryos due to a severe infection found in the callus induction culture (Table 3). This is likely to have been the result of a combination of latex and insufficient sterilization based on the sterilization procedure described for cv. Angelika, Franzi and Diamond (Preil 1994), which might not be optimal for cv. Millenium (Table 3). A modified protocol was thus used for surface sterilization in Experiment 3 as well as in the five transformation experiments, and the efficiency of somatic embryogenesis was greatly improved (see Table 3), demonstrating the significance of optimization of sterilization and regeneration protocols for the individual cultivar.
In addition to the availability of an efficient regeneration system, successful A. tumefaciens-mediated transformation depends on several factors, e.g. inoculation and co-cultivation times with A. tumefaciens, antibiotic selection and regeneration. The optimized infection conditions and co-cultivation times in our study (Table 4) are consistent with the range of published reports on ornamental plants, where 5–30 min infection time and 1–6 days co-cultivation have been reported (Aida et al. 1999; Kishimoto et al. 2002; Cui and Ezura 2003; Kim et al. 2004; Teixeira da Silva 2004). However, in our study, 6 days co-cultivation led to A. tumefaciens overgrowth and explant death, thus differing from the results for Begonia, in which 6 days co-cultivation is required (Kishimoto et al. 2002). These data indicate that co-cultivation times vary among different plant species.
Antibiotic selection is another key factor influencing the establishment of a transformation protocol. To eliminate Agrobacterium after co-cultivation, several antibiotics such as cefotaxim (claforan), carbenecillin, and timentin have been used in previous studies. The influence of the type of antibiotic and/or concentration on each transformation system varies greatly (Ishida et al. 1996; Nauerbey et al. 1997; Sunikumar and Rathore 2001). In the present study, optimised concentrations of claforan in the CI, SEI and SEM media effectively eliminated Agrobacterium. For selecting transformed cells, the nptII gene was used. Although selection media containing 50 and 100 mg l−1 kanamycin have been documented in the production of transgenic plants (Kishimoto et al. 2002; Kim et al. 2004; Wu et al. 2005), those concentrations severely inhibited the development of embryogenic calli and somatic embryos in poinsettia. In our study the optimal concentrations of kanamycin were 10 and 25 mg l−1. In the case of Agrobacterium-mediated transformation of Platanus acerifolia, kanamycin at 20 mg l−1 was found effective for the inhibition of regeneration in non-transformed shoots (Li et al. 2007), suggesting the variability of antibiotic selection in each transformation system and the necessity of optimization of antibiotic selection for each plant species.
Although transgenic PnMV resistant poinsettias were produced using the Agrobacterium-mediated transformation protocol developed in the current study, the transformation frequency was fairly low (Table 5), suggesting that further improvement is required. This will be carried out in the near future.
Southern blot analysis confirmed the integration of transgenes into the poinsettia genome and detected both single- and multiple-copy integration of the transgenes among the transformants (Fig. 4). The majority (six out of eight) of the transformants showed single copy transgene integration with fragment sizes greater than that of the intact T-DNA (ca 7.5 kb), indicating that the intact T-DNA was integrated into the poinsettia genome. However, the bands with sizes around 4–6 kb, smaller than that of intact T-DNA, were also found in the transgenic lines with multiple insertions (lanes 7 and 8 in Fig. 4).
According to Afolabi et al. (2004) and Zhu et al. (2006), insertion of non-intact T-DNAs were found in more than 70% of the transgenic rice lines causing 14–21% of the loci to contain only part of the T-DNA, due to T-DNA truncation. Moreover, T-DNA rearrangement is well documented in many plant species including both dicots and monocots (Deroles and Gardner 1988; Puchta et al. 1992; Azhakanandam et al. 2000; Yin and Wang 2000; Rai et al. 2007). Almost 50% of the 27 transgenic rice lines studied showed rearrangement of T-DNA inserts according to Rai et al. (2007). Consequently, such T-DNA truncations and/or rearrangements could also possibly occur in our study causing partial T-DNA insertion. Since Agrobacetrium-mediated transformation of poinsettia has never been described before, there is no available information regarding T-DNA transfer and integration into the poinsettia genome.
The hp RNA constructs and PnMV resistance in transgenic poinsettia cv. Millenium
A number of effective approaches have been utilized to control viral diseases (Goldbach et al. 2003). Among these, coat protein-mediated and RNA silencing-based resistance have been shown to be efficient transgenic approaches to engineer virus resistance in plants (Powell-Abel et al. 1986; Barker et al. 1998; Waterhouse et al. 1998; Smith et al. 2000; Domínguez et al. 2002; Lu et al. 2003). Furthermore, according to previous studies, hpRNA-induced RNA silencing is more efficient than sense- or antisense-mediated silencing (Chuang and Meyerowitz 2000).
In the current study, the establishment of Agrobacterium-mediated transformation enabled us to transform poinsettia plants with intron-containing hpRNA constructs homologous to the PnMV genome. PnMV resistant transgenic poinsettia lines showing no systemic infection were identified after mechanical inoculation as determined using the DAS-ELISA assay (Table 6). The presence of transgene-derived siRNA molecules in non-inoculated transgenic poinsettia plants carrying pCP, pR2 and pR3 constructs, and the absence of these in the non-transformed control plants, indicates that the transgenes were expressed and subsequently cleaved. This data indicates that RNA silencing underlies the PnMV resistance observed in our study (Fig. 5). As mentioned previously, LMW Northern analysis included only one pR3 line (i.e.18-1) due to the limitations in the amount of RNA available for the remaining lines. Therefore, the underlying reason for lack of resistance in some of the pR3-containing transformants (Table 6) needs to be clarified by further studies.
Poinsettia is a non-food, non-feed and vegetatively propagated ornamental plant. Pollen mediated transgene outflow is restricted, a significant advantage over sexually propagated plants. The combination of genetic engineering and RNA silencing technologies for the generation of virus-resistant transgenic ornamentals is therefore appealing, especially in an industry in which quality is so important. Transgenic vegetatively propagated ornamentals like PnMV resistant transgenic poinsettia are likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.
In summary, we have developed an A. tumefaciens-mediated transformation method for poinsettia. Using this approach, transgenic PnMV resistant transgenic lines expressing PnMV-derived hpRNA constructs were produced. Phytoplasma, the poinsettia branch-inducing factor for good branching (“free-branching”) and for a compact growth habit (Lee et al. 1997), is currently being reintroduced into the transgenic poinsettias. Candidate transgenic lines with PnMV resistance and desired ornamental values will be selected for further study with the future goal of possible commercialization. The methodology developed here could facilitate the future engineering of poinsettia to meet customers’ expectations for new colours and other desirable traits.

agrobacterium tumefaciens
transformation
euphorbia pulcherrima
poinsettia mosaic virus
somatic embryogenesis


Quality of life and functional status in patients with cancer of the oral cavity and oropharynx: pretreatment values of a prospective study

We assessed the pretreatment health-related quality of life (HRQOL) and functional status of patients with advanced oral and oropharyngeal cancer. Eighty patients were investigated. HRQOL was assessed by EORTC QLQ-C30/QLQ-H&N35 questionnaires. Functional status assessment comprised speech and oral function tests. The results revealed a wide range of HRQOL and functional deficits before treatment. HRQOL appeared to be related to some extent to tumor site (patients with oral tumors reported more pain compared to patients with oropharyngeal tumors) and tumor classification (patients with T3–T4 tumors reported more trouble opening the mouth and felt more ill compared to patients with T2 tumors). Comorbidity appeared to have a major impact. Patients with comorbidity had significantly worse scores on several scales/items on both the EORTC questionnaires. Functional deficits were related to tumor site, classification and comorbidity. Patients with oral cavity tumors (versus oropharyngeal tumors), patients with T3–T4 tumors (versus T2 tumors), and patients with comorbidity (versus without comorbidity) scored significantly worse on several speech and oral function tests. Impaired speech and oral function appeared to be clearly related to global quality of life (QLQ-C30) and self-reported speech (QLQ-H&N35). Many patients with advanced oral and oropharyngeal cancer have compromised HRQOL and functional status before the start of treatment. In addition to tumor site and tumor classification, comorbidity appears to have a major impact on HRQOL and functional status. Knowledge of pretreatment HRQOL and functional status levels is useful for better understanding the impact of treatment on these outcomes over time.
Introduction
The most important outcome for cancer patients is overall survival. However, the disease and its treatment often have a major impact on health-related quality of life (HRQOL) and functional status in patients with head and neck cancer [1]. Therefore, HRQOL and functional status are important aspects to consider in treatment evaluation. To interpret outcome following treatment, it is necessary to assess HRQOL and functional status following diagnosis, but before the start of treatment [2].
Studies on pretreatment HRQOL [2–5] and functional status [6, 7] often include heterogeneous groups of head and neck cancer patients. However, there are substantial differences between patient groups that are related to tumor site and stage [7]. Patients with advanced oral or pharyngeal cancer, for example, often have the poorest HRQOL and functional status [5, 8]. Pretreatment HRQOL among patients with oral or oropharyngeal cancer is often compromised, although it tends to be better compared to HRQOL following treatment [9–11]. There is also evidence that functional status, including oral function, speech and swallowing abilities is significantly deteriorated before treatment [12–15]. Comorbidity is another important factor that varies substantially among subpopulations of patients, and can have a significant influence on the choice of initial treatment, the care that patients receive, and on treatment outcomes [16]. Therefore, comorbidity should be taken into consideration evaluating the HRQOL of patients, both at time of diagnosis, and over the course of treatment [17–19].
The primary objective of the present study is to assess pretreatment HRQOL and functional status in relation to tumor site, tumor classification and comorbidity, in a well-defined group of patients with advanced oral and oropharyngeal cancer.
Patients and methods
Patients
Between January 1998 and December 2001, 92 consecutive patients diagnosed with stage II-IV oral or oropharyngeal squamous cell carcinomas were asked to participate in the study. The planned treatment was composite resection with microvascular soft tissue transfer (i.e., radial forearm free flap) for the reconstruction of surgical defects, and radiotherapy on indication. Exclusion criteria were age greater than 75 years, serious cognitive impairment and lack of basic fluency in the Dutch language. Twelve patients declined to participate, resulting in a final sample of 80 patients (response rate = 87%). All patients were treated at the Department of Otolaryngology/Head and Neck Surgery of the VU University Medical Center, Amsterdam, The Netherlands.
Data collection and study measures
All data were collected prior to the start of treatment, including sociodemographics (age and gender), disease stage, comorbidity, HRQOL, and functional status. Comorbidity was assessed dichotomously (yes or no) by review of medical records and on the basis of self-report, and was noted if one or more relevant medical ailments accompanied the primary medical illness. The comorbid conditions were cardiovascular, respiratory, gastro-intestinal, renal, endocrine, neurological, and immunological disorders, previous malignancy and considerable weight loss or alcohol abuse. For example, cardiovascular problems such as a myocardial infarct or hypertension, respiratory problems such as restrictive lung disease or COPD, or endocrine disorders such as diabetes mellitus with insulin usage were defined as relevant comorbid conditions.
HRQOL was assessed by means of the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire (version 2.0) [20] and the EORTC head and neck cancer module QLQ-H&N35 [21]. The QLQ-C30 is composed of multi-item scales and single items assessing five areas of functioning (physical, role, emotional, cognitive, and social), fatigue, pain, emesis, dyspnea, insomnia, appetite loss, constipation, and diarrhea. Overall quality of life and the perceived financial impact of the disease and treatment are also assessed. The head and neck cancer-specific QLQ-H&N35 module comprises seven symptom scales: pain, swallowing, senses, speech, social eating, social contact, and sexuality. There are 11 additional, single items covering problems with teeth, opening the mouth wide, dry mouth, sticky saliva, cough, feeling ill, weight loss, weight gain, use of nutritional supplements, feeding tubes, and painkillers. The scores of both the QLQ-C30 and of the QLQ-H&N35 are linearly transformed to a scale of 0–100, with a higher score indicating a higher (i.e., more positive) level of functioning or global HRQOL, or a higher (i.e., more negative) level of symptoms or problems.
The QLQ-C30 data of the patients were compared with published normative data from the general population of Norway [22]. Comparisons were made with the normative subsample that corresponded closely to the age and gender distribution of the patient sample (i.e., age range of 50–59 years and 48% female). The QLQ-H&N35 results were compared with reference values derived from a randomly selected sample of the Swedish general population [8].
Speech and oral functional status
Speech analyses were performed according to a standardized speech assessment protocol. Speech recordings of a read aloud text were performed in a sound-treated room and digitized using Cool Edit PRO 1.2 (Adobe Systems Incorporated, San Jose, CA, USA) with 22 kHz sample frequency and 16 bit resolution. Recording level was adjusted for each speaker to optimize signal-to-noise ratio. All recordings were made with a mouth-to-microphone distance of 30 cm. A computer program was developed to perform blinded randomized speech evaluation and to score overall intelligibility, and quality of articulation and nasal resonance. Overall intelligibility was assessed on a 10-point scale ranging from poor to excellent by two trained speech therapists. Scores below six were defined as insufficient intelligibility (according to the Dutch educational system). To obtain more insight into the cause of decreased intelligibility, evaluation of the quality of articulation and nasal resonance was performed by the same two speech therapists on a 4-point scale ranging from deviant (score 1–3) to normal (score 4). Speech rate was measured by calculating words per minute on a read aloud standardized text.
Oral function was evaluated by a trained investigator (blinded for the clinical data regarding tumor site and stage) according to a protocol described by Teichgraeber et al. [23]. All assessments were based on 5-point scales ranging from poor to excellent (transformed scores ranging from 0–100). The oral function evaluation included three tests; (1) tongue mobility (mean score of tongue straight out, elevation of tongue tip, elevation of the base of the tongue, tongue deviation left and right, symmetry left and right, and tongue withdrawal), (2) lip mobility (mean score of general movement, spreading and rounding, symmetry left/right, lip closure without speech, and lip closure during speaking), and (3) diadochokinesis (mean score of repetitive motion ability concerning tongue movement left/right, up/down, and in/out, and repeating the syllables /ta/ /cha/, and /ka/). Tongue and lip strength were quantified by a calibrated digital voltameter. To measure tongue strength the patient was asked to push the tongue against a metallic disc with the lips positioned around a cylinder, and to resist the force. To measure lip strength, the patient was asked to keep his lips around a button with a string attached to it on which the examiner pulled. Scores ranged from 0 (no strength) to 0.5 mV (normal strength).
Statistical analyses
Chi-square tests were used to assess associations between the independent variables tumor site, classification and comorbid condition. Student’s t-tests were performed to test for differences between the patient sample and the normative samples. These tests were based on the mean scores of the study and normative samples and the standard deviations of the study sample only, because no standard deviations of normative samples were available. Student’s t-tests (HRQOL) and Mann–Whitney tests (functional status) were performed to determine the impact of tumor site (oral versus oropharyngeal), tumor classification (T2 versus T3–T4) and comorbidity (yes or no). Spearman correlation coefficients (r) were calculated to investigate the association between self-reported HRQOL (i.e., the QLQ-C30 global quality of life scale and the QLQ-H&N35 speech scale) and speech and oral functional status. Statistical significance was defined as a P-value less than or equal to 0.05.
Results
Sample description
Patient characteristics are shown in Table 1. The patients’ age ranged from 23 to 74 years (mean 58 years). Forty-one percent of the sample was female. One patient was operated on for a recurrent tumor after prior transoral excision (1 year earlier) in which the rTNM stage was used, and one patient had undergone previous radiotherapy (3 years earlier) for a neck node of an unknown primary. Four patients had a synchronous second primary tumor, and in these cases the stage of the largest tumor was used. An equal percentage (50%) of patients had a tumor originating on the left or right side, and in the majority of patients (73%) the tumor did not extend over the median line. All 80 patients completed the EORTC questionnaires, speech rate tests (word count) and oral function tests (mobility and strength). In one patient speech recording could not be done due to logistical problems, and in three patients speech recordings were inadequate for interpretation due to technical problems. Speech quality analyses could thus be performed on 76 patients. Presence of comorbid conditions and tumor classification were significantly associated. Significantly more patients with comorbidity were diagnosed with a larger tumor (χ2 = 10.37, P < 0.01). No other significant associations were observed between tumor site, tumor classification, age, gender and comorbidity.
Table 1Characteristics of 80 patients included in this studyAge (years) Range23–74  Mean58n (%)Gender Male47 (59) Female33 (41)General condition Comorbidity48 (60) No comorbidity32 (40)Tumour site Oral cavity38 (47) Oropharynx42 (53)T 2 35 (44) 3 42 (52) 4 3 (4)N 024 (30) 116 (19) 2a2 (3) 2b30 (38) 2c6 (7) 3 2 (3)
Health-related quality of life
Compared to the general population, the patients with oral or oropharyngeal cancer scored significantly worse on 5 (out of 15) scales or single items regarding the QLQ-C30: role functioning (P = 0.000), emotional functioning (P = 0.000), pain (P = 0.026), insomnia (P = 0.016) and appetite loss (P = 0.003). Conversely, patients scored significantly better regarding social functioning (P = 0.009), fatigue (P = 0.030), emesis (P = 0.001), and diarrhea (P = 0.001). Regarding the QLQ-H&N35, the patients scored significantly worse on 7 (out of 10) scales or single items compared to the general population: pain (P = 0.000), swallowing (P = 0.000), senses (P = 0.050), social eating (P = 0.000), teeth (P = 0.009), opening the mouth wide (P = 0.000) and sticky saliva (P = 0.005). Conversely, the patients scored significantly better on two items: coughing (P = 0.002) and feeling ill (P = 0.025).
Regarding tumor site, the QLQ-C30 results were comparable between oral cavity and oropharynx cancer patients. Only the pain score was significantly worse (P = 0.027) for patients with oral cavity tumors as compared to patients with oropharynx tumors. The QLQ-H&N35 also revealed that patients with oral cavity tumors had significantly more pain (P = 0.002); furthermore, problems with their teeth appeared to be different (P = 0.001).
No statistically significant differences were observed for any of the QLQ-C30 scores as a function of tumor classification (T2 versus T3–T4). Regarding the QLQ-H&N35, patients with T3–T4 tumors scored significantly worse on opening the mouth (P = 0.009) and reported feeling more ill (P = 0.035) compared to patients with T2 tumors.
Regarding comorbidity (Table 2), patients with one or more comorbid conditions scored significantly worse on QLQ-C30 physical functioning (P = 0.002), global quality of life (P = 0.016), fatigue (P = 0.014), pain (P = 0.024), constipation (P = 0.042) and diarrhea (P = 0.017) compared to patients without comorbidity. Additionally, based on the QLQ-H&N35 data, patients with comorbidity reported significantly more pain (P = 0.007), trouble with social eating (P = 0.036), teeth problems (P = 0.034), problems with opening the mouth (P = 0.013) and feeling ill (P = 0.003). The use of pain medication was significantly higher among patients with than those without comorbidity (75 versus 50%; P = 0.025).
Table 2EORTC QLQ-C30 and QLQ-H&N35 scores for comorbidityComorbidityNoYesMean (SD) n = 32Mean (SD) n = 48PEORTC QLQ-C30 Physical functioning95.0 (12.4)81.7 (21.4)0.002 Role functioning85.4 (18.3)79.2 (24.9)0.228 Cognitive functioning89.6 (18.3)87.2 (19.2)0.574 Emotional functioning69.5 (23.4)72.2 (24.6)0.626 Social functioning93.2 (13.3)88.5 (21.5)0.275 Global quality of life82.6 (19.6)71.5 (19.7)0.016 Fatigue13.9 (20.0)26.9 (24.1)0.014 Emesis1.6 (4.9)1.4 (4.7)0.874 Pain22.4 (25.3)37.8 (31.8)0.024 Dyspnea6.3 (19.7)10.4 (18.4)0.338 Insomnia36.5 (36.3)31.3 (39.1)0.550 Appetite loss7.3 (20.3)18.7 (31.4)0.072 Constipation5.2 (20.9)18.7 (32.9)0.042 Diarrhea1.0 (5.9)8.3 (16.1)0.017 Financial impact2.1 (8.2)7.6 (20.9)0.157EORTC QLQ-H&N35 Pain27.1 (17.6)41.5 (25.5)0.007 Swallowing14.6 (22.6)24.1 (22.6)0.068 Senses 6.3 (14.5)8.7 (21.5)0.577 Speech 9.4 (14.1)13.0 (17.5)0.336 Social eating14.6 (25.3)27.4 (27.1)0.036 Social contact3.1 (7.0)3.7 (6.1)0.674 Sexuality16.1 (24.9)16.3 (27.6)0.981 Teeth13.5 (26.6)30.6 (38.8)0.034 Opening mouth7.3 (20.3)25.7 (37.2)0.013 Dry mouth15.6 (26.8)22.2 (28.6)0.303 Sticky saliva12.5 (29.0)21.5 (31.1)0.196 Coughing6.3 (13.2)14.6 (21.6)0.055 Feeling ill0 (0)9.7 (18.1)0.003% Yes% YesPain medication50.075.00.022
Speech and oral functional status
Abnormal scores were observed in 17% of the patients regarding overall intelligibility, in 25% regarding nasality and in 37% regarding articulation. Functional results in relation to tumor site, classification and comorbidity are shown in Table 3. Regarding tumor site, patients with oral cavity tumors scored significantly worse on intelligibility (P = 0.015), articulation (P = 0.039), nasality (P = 0.040), tongue and lip mobility (P = 0.000, 0.009), diadochokinesis (P = 0.004), and tongue strength (P = 0.001) compared to patients with oropharyngeal tumors. Regarding tumor stage, patients with T3–T4 tumors scored significantly worse on intelligibility (P = 0.011), articulation (P = 0.003), tongue mobility (P = 0.001), diadochokinesis (P = 0.012), and tongue and lip strength (P = 0.021, 0.018) than patients with T2 tumors. Patients with comorbidity scored significantly worse on articulation (P = 0.043), diadochokinesis (P = 0.026), and tongue strength (P = 0.045) as compared to patients without comorbidity.
Table 3Functional status tests for tumour site, tumour stage and comorbidityTumour siteTumour stageComorbidityOral cavityOropharynxT2T3–T4NoYesMean (SD) n = 37Mean (SD) n = 39PMean (SD) n = 32Mean (SD) n = 44PMean (SD) n = 29Mean (SD) n = 47PSpeech Intelligibility  (0–10)6.0 (1.3)6.7 (1.0)0.0156.8 (1.0)6.0 (1.2)0.0116.6 (1.0)6.2 (1.3)0.132 Articulation  (0–4)3.4 (0.8)3.7 (0.6)0.0393.8 (0.4)3.3 (0.8)0.0033.8 (0.4)3.4 (0.8)0.043 Nasality  (0–4)3.6 (0.6)3.8 (0.4)0.0403.8 (0.4)3.7 (0.6)0.9113.7 (0.4)3.7 (0.5)0.771n = 38n = 42n = 35n = 45n = 32n = 48 Rate  Words per minute183 (31.6)182 (38.5)0.988189 (33.8)178 (35.8)0.201189 (31.4)179 (38.1)0.233n = 38n = 42n = 35n = 45n = 32n = 48Oral functions Mobility  Tongue (0–100)82.3 (20.0)96.4 (5.4)0.00096.0 (5.8)84.8 (19.3)0.00193.9 (9.9)89.9 (14.4)0.209  Lip (0–100)87.4 (5.7)99.7 (0.9)0.00999.4 (2.0)98.0 (5.1)0.14099.3 (2.2)98.5 (3.9)0.239  Diadoch (0–100)87.4 (20.7)97.2 (6.1)0.00497.5 (5.0)88.7 (19.6)0.01297.4 (5.1)91.5 (15.4)0.026 Strength  Tongue (0–0.5)0.2 (0.2)0.4 (0.2)0.0010.4 (0.2)0.3 (0.2)0.0210.4 (0.2)0.2 (0.2)0.045  Lip (0–0.5)0.3 (0.1)0.4 (0.2)0.3280.4 (0.2)0.3 (0.1)0.0180.4 (0.2)0.3 (0.1)0.370
Correlations between self-report and observer rated data
Significant correlations (P < 0.01) were observed between self-reported global quality of life scale (QLQ-C30) and observer ratings of intelligibility (r = 0.41) and articulation (r = 0.36) (Table 4). Additionally, statistically significant (P < 0.05) but relatively low correlations were found between self-reported speech (QLQ-H&N35) and observer ratings of intelligibility (r = −0.28), articulation (r = −0.24), nasality (r = −0.27) and diadochokinesis (r = −0.28).
Table 4Correlations between functional status tests and QLQ-C30 global quality of life scale/QLQ-H&N35 speech problemsScalesQLQ-C30 global quality of lifeQLQ-H&N35 speech problemsCorrelation coefficient (r) n = 76PCorrelation coefficient (r) n = 76PSpeech Intelligibility0.4110.000−0.2850.013 Articulation0.3550.002−0.2420.035 Nasality0.1520.190−0.2700.019n = 80n = 80 Rate0.1290.267−0.1420.221n = 80n = 80Oral functions Mobility  Tongue0.0780.490−0.1880.095  Lip0.1570.164−0.1760.119  Diadochokinesis0.1090.337−0.2800.012 Strength  Tongue0.1880.095−0.1750.121  Lip0.0560.624−0.0490.665
Discussion
Pretreatment HRQOL and functional status were investigated in a well-defined sample of patients with advanced oral and oropharyngeal cancer. Deteriorated HRQOL and functional status before treatment have been reported in earlier studies, albeit for less well defined patient groups [9–15]. The results of the present study indicate a wide range of HRQOL and functional deficits in patients with advanced oral and oropharyngeal cancer before treatment. Impaired speech and oral function, as assessed objectively, was associated significantly with self-reported global HRQOL and speech problems, which is in accordance with the conclusions of Rogers et al. and Karnell et al. [14, 24].
Compared to the general population, patients scored significantly worse on 5 of 15 scales or items of the EORTC QLQ-C30 but, conversely, they scored significantly better on four others. Regarding the QLQ-H&N35, patients scored significantly worse on most scales or items but significantly better on coughing and feeling ill. The significantly better scores for patients compared to the reference groups on some scales or items may be explained by the fact that people from the general population may have (other) chronic conditions as well. Alonso et al. [25] reported that, 55% of the general population has one chronic health condition, and 30% has more than one chronic condition. They also found that comorbidity can have a substantial impact on HRQOL, and stated that the presence of comorbidity limits the ability to attribute HRQOL deficits to one specific disease (e.g., to head and neck cancer).
In head and neck cancer patients, comorbidity has proven to be an important factor associated with complications and mortality rates [26–29]. Studies on the impact of comorbidity on functional status in head and neck cancer patients are lacking. Studies that have examined the impact of comorbid status on HRQOL are scarce, have yielded conflicting results, and have examined the post-treatment period only. Pourel et al. [17] found no significant association between comorbidity and HRQOL in 113 long-term survivors 2 years after treatment for oropharyngeal carcinomas. Similarly, Taylor et al. [30] found no impact of comorbidity on work-related disability among 384 patients after treatment for head and neck cancer. However, Terrel et al. [19] in a study on HRQOL of 570 head and neck cancer patients after treatment, reported a clear effect of comorbidity in patients with two or more comorbid conditions. In our study on patients with oral or oropharyngeal carcinomas before treatment, comorbidity was present in 60% of the patients, and proved to have a major impact on HRQOL and functional status. Patients with comorbidity had significantly worse scores on several general and head and neck specific quality of life aspects and on speech and oral function tests compared to patients without comorbidity. Unfortunately, the cause of comorbidity was not systematically noted in the present study; future studies may provide more insight into the relation between the cause of comorbidity and HRQOL.
The impact of tumor site and classification on HRQOL appeared to be limited, with patients with oral cavity tumors reporting more pain, and patients with T3–T4 tumors reporting more trouble opening the mouth and feeling more ill. With regard to tumor site, comparisons with earlier studies are difficult because most studies included patients with oral cavity tumors only or reported on heterogeneous samples not stratified by (sub)sites [4, 9–11]. Regarding tumor classification, other studies have reported worse HRQOL for patients with higher tumor classifications and stages before treatment [2, 14].
Tumor site and classification were found to have a clear impact on functional status. Patients with oral cavity tumors (versus oropharyngeal tumors) and patients with T3–T4 tumors (versus T2 tumors) had worse speech and oral function scores, which is in accordance with the results of earlier studies [13, 14].
In conclusion, we observed compromised HRQOL and functional deficits among patients with advanced oral and oropharyngeal cancer before the start of treatment. In addition to the impact of tumor site and classification, comorbidity proved to have a major impact on HRQOL and functional status. Prospective studies are needed to obtain insight into the relation between pretreatment HRQOL and functional status and outcome after treatment, and the relationship between changes in HRQOL and functioning over time and tumor site, tumor classification, and comorbid conditions.

quality of life
functional status
speech
head and neck cancer
microvascular reconstruction


Antithrombin III in patients admitted to intensive care units: a multicenter observational study

Introduction The administration of antithrombin III (ATIII) is useful in patients with congenital deficiency, but evidence for the other therapeutic indications of this drug is still uncertain. In Italy, the use of ATIII is very common in intensive care units (ICUs). For this reason we undertook an observational study to determine the pattern of use of ATIII in ICUs and to assess the outcome of patients given this treatment.
Introduction
Antithrombin III (ATIII) is a recognized treatment for patients with congenital ATIII deficiency [1,2,3,4,5] (see also the approval of this indication by the Food and Drug Administration); in contrast, the evidence supporting its use for other clinical indications is uncertain [6,7,8,9,10].
In Italian hospitals this drug is widely used in patients admitted to intensive care units (ICUs), who are generally given ATIII for the treatment of sepsis or disseminated intravascular coagulation (DIC). The approval of ATIII by the Italian Ministry of Health was granted nearly 10 years ago (before the profound reform of the Drug Regulatory Agency made by the Italian Ministry of Health in 1993) and has remained unchanged since then. This approval of ATIII was rather generic and included 'congenital deficiency of ATIII and all clinical conditions that can cause an acquired deficiency of ATIII'.
Three small randomized studies [7,8,9] and one large international trial [10] assessed the effectiveness of ATIII in sepsis, but none of these trials found a significant benefit in terms of reduced morbidity or mortality. As regards congenital deficiency, the effectiveness of ATIII is fairly well documented [1,2,3,4,5], but these patients are rare. The other clinical indications (such as acute thrombosis or thromboembolism, prevention of DIC in hepatic coma, and treatment of bleeding episodes in cirrhosis) are supported by a small series of very preliminary studies (see, for example, the Drugdex databank, CD-ROM Drugdex, volume 110; Micromedex, Englewood, Colorado, USA).
To achieve a better definition of the current use of ATIII in Italian hospitals and to generate naturalistic data (based on routine practice) about the outcome of this treatment, we undertook a multicenter observational study.
Methods
Design of the study and aims
The study was based on a multicenter observational design. From 20 May to 20 July 2001 all consecutive patients admitted to ICUs in 20 Italian hospitals and treated with ATIII were enrolled in the study. The study had the following aims: (1) surveying the use of ATIII in patients admitted to ICUs; (2) determining the outcome of patients treated with ATIII; and (3) comparing the results obtained from our observational study with those previously found in the randomized controlled trials (RCTs).
A meta-analysis was also conducted to summarize the information deriving from four RCTs [7,8,9,10] that studied the effectiveness of ATIII in sepsis.
Data collection
The following information was recorded from each patient enrolled in the study: (1) demographic characteristics (age, sex, weight); (2) congenital deficiency (y/n); (3) baseline ATIII level; (4) ward of first admission in the hospital; (5) clinical indication for using ATIII (sepsis or DIC or any other clinical condition); (6) daily dose and duration of treatment with ATIII; (7) outcome of hospitalization (alive or dead); and (8) concurrent administration of antibiotics and/or heparin.
Analysis
The information collected from each patient was analyzed by standard descriptive statistics. In the subgroup of patients with sepsis, the in-hospital mortality rate observed in our study was compared with that previously reported by the four RCTs. All rates were presented together with their 95% confidence interval (CI), which was calculated by using Equations 1.26 and 1.27 of Fleiss [11].
Results
The overall number of patients who were admitted to ICUs during the study period was 1648. Of these patients, 216 (13%) were enrolled in our study. The characteristics of these 216 patients are presented in Table 1.
The clinical indication for using ATIII was sepsis (n = 56), DIC (n = 50), or other (n = 101). Table 1 also reports separate information for the subgroup of 56 patients treated for sepsis.
The duration of ATIII therapy did not differ at levels of statistical significance between patients treated for different clinical indications (P = 0.57 according to an analysis of variance). The daily dose of ATIII showed a difference between sepsis and other indications (Table 1).
Table 2 reports the outcome of hospitalization according to clinical indication. With regard to the use of ATIII in patients with sepsis, Figure 1 shows the percentage mortality rate (with 95% CI) observed in our study, together with the rates found in four previous studies [7,8,9,10].
Subgroup analyses within the patient cohort of our study did not identify any relationship between mortality and patient characteristics. The administration of heparin, which Warren et al. [10] found to have some implications for outcome, did not influence mortality in our patient series: mortality was 19.6% in the 107 patients who received heparin, compared with 30.5% in the 95 patients who did not receive this drug (P = 0.10) by Fisher's exact test; mortality was 28.6% in the 28 patients with sepsis who received heparin, compared with 42.3% in the 26 patients with sepsis who did not receive this drug (P = 0.39).
Discussion
The main scientific value of our observational and prospective study lies in its naturalistic design; the population of patients that we studied was in fact drawn from the everyday practice of more than 20 hospitals and was intentionally free from specific exclusion criteria.
In interpreting our outcome data, one disadvantage is that the group treated with ATIII was not compared with any reference group observed prospectively within our research; neither did we include any retrospective control group not treated with the drug. However, historical retrospective controls would have raised profound problems of matching the retrospective data with the prospective ones. A prospective enrollment of controls not treated with ATIII was not feasible because the therapeutic policy of the ICUs involved in our study was to administer ATIII to virtually all patients with a diagnosis of sepsis or DIC.
Regardless of our statistical indexes, a 'first-look' comparison between the data on sepsis produced by the previous RCTs (including four treatment groups and four control groups) and those observed in our naturalistic study indicates a complete overlap of the various survival rates and of their respective 95% CIs. This qualitative impression (Figure 1) is in agreement with the meta-analysis shown in Figure 2 (see Appendix2 for details of its methodology).
This meta-analysis gave the following results: summary odds ratio 0.98; 95% CI 0.83–1.15, P = 0.80; χ2 for heterogeneity 1.86; 3 degrees of freedom; P = 0.60. In this meta-analysis, the large-scale trial by Warren et al. [10] outweighed the other three small RCTs in that Warren's trial included 93% of the overall cohort of the four RCTs. In the light of the above data, there seems to be no clinical benefit in administering ATIII to critical patients with sepsis; in this context, one crucial point is that the most recent large-scale trial gave very clear results and was negative. The other clinical indications reported in our patients' series were more difficult to interpret because of the nearly complete lack of previous controlled studies exploring these therapeutic issues.
There has been a lively debate in the literature on the relative merits of observational studies and RCTs in providing useful evidence of clinical effectiveness [12,13,14]. Although the great majority of researchers stick to the concept that RCTs are the gold standard, common sense suggests that having information both from RCTs and from observational studies is better than having information from RCTs only. In this framework, our study advances knowledge about the use of ATIII in critical patients.
In conclusion, our findings based on an observational prospective study and on an updated meta-analysis of the previous RCTs do not support the use of this drug in ICU patients with sepsis.
Key messages
Antithrombin III (ATIII) is a recognized treatment for patients with congenital ATIII deficiency; in contrast, the evidence supporting its use for other clinical indications is uncertain.
In Italian hospitals this drug is widely used in patients admitted to intensive care units (ICUs), who are generally given ATIII for the treatment of sepsis or disseminated intravascular coagulation.
Three small randomized studies and one large international trial have assessed the effectiveness of ATIII in sepsis, but none of these trials has found a significant benefit in terms of reduced morbidity or mortality.
Our findings, based on an observational prospective study and on an updated meta-analysis of the previous randomized controlled trials, do not support the use of this drug in ICU patients without congenital deficiency.
Competing interests
In 2001 our research group received a grant from Eli-Lilly (Italy) to conduct an original study on factors influencing length of stay in critical patients with sepsis. In Italy, anti-thrombin III is marketed by Aventis-Behring and by Baxter.
Appendix 1: Gruppo di Studio sull'antitrombina III (The Antithrombin Study Group)
The Antithrombin Study Group includes the study coordinators (A Messori, F Vacca, M Vaiani, S Trippoli, Laboratorio di Farmacoeconomia, c/o Azienda Ospedaliera Careggi, Firenze) and a total of 51 participants. The names and addresses of the participants involved in the project were the following (all located in Italy): R Banfi, M Cecchi, E Cini, D Dupuis, T Falai, R Fornaini, A Ipponi, ML Migliaccio, F Pelagotti, L Rabatti, I Ruffino, R Silvano, E Tendi (Firenze, four hospitals); P Becagli, M Monciatti (Empoli); B Bozzone, R Casullo, F Cattel, S Pardossi, R Passera, S Stecca, U Tagliaferro (Torino, two hospitals); P Di Bartolomeo, T Faggiano, M Lattarulo (Bari); N Caboni, A Cannas (Cagliari); A Plescia, M Sorci (Rimini); L Bonistalli, M Puliti (Prato); B Ciammitti, M Costantini, F Mammini (Terni); L De Cicco, G Mazzaferro (Napoli); P Marrone, R Tetamo (Palermo); P Beneduce, MG Celeste, P Fiorani, S Galeassi, G Guaglianone, A Pecere, L Ragni (Roma, two hospitals); SM Germinario (Andria); O Basadonna, L Todesco (Camposampiero, Padova); R Calle-gari, M Pegoraro (Asolo); E Lamura (Ancona).
Appendix 2: Methodology of the meta-analysis
A MedLine search (PubMed, ) was performed to cover the period from January 1980 to November 2001. The search was limited to the studies published in English and was based on four index terms combined with the following Boolean syntax: "antithrombin III" AND (sepsis OR septic shock OR "disseminated intravascular coagulation"). This search was supplemented by examining the Drugdex databank (CD-ROM Drugdex, volume 110; Micromedex, Englewood, Colorado, USA).
Eligible studies were included if they met the following criteria: patients were admitted to an ICU; randomized design; diagnosis of sepsis, septic shock or DIC; assessment of survival. The odds ratio was used as the main index to assess the treatment effect within each trial and to generate the overall results of the meta-analysis. The calculation of the summary odds ratios was based on a random-effect model [15,16]. Heterogeneity was assessed as described previously [17].
Abbreviations
ATIII = antithrombin III; CI = confidence interval; DIC = disseminated intravascular coagulation; ICU, intensive care unit; RCT = randomized controlled trial.

antithrombin iii
sepsis
disseminated intravascular coagulation
septic shock


Colonic irrigation for defecation disorders after dynamic graciloplasty

Background and aims Dynamic graciloplasty (DGP) improves anal continence and quality of life for most patients. However, in some patients, DGP fails and fecal incontinence is unsolved or only partially improved. Constipation is also a significant problem after DGP, occurring in 13–90%. Colonic irrigation can be considered as an additional or salvage treatment for defecation disorders after unsuccessful or partially successful DGP. In this study, the effectiveness of colonic irrigation for the treatment of persistent fecal incontinence and/or constipation after DGP is investigated.
Introduction
Dynamic graciloplasty (DGP) is a proven effective treatment for fecal incontinence. Success rates vary from 42–92% [1–5]. Comparison of results is difficult because the outcome of this method seems to be influenced by the etiology of fecal incontinence, pre-existing stoma, length of follow-up, method of stimulation, and the surgeon’s experience [1, 6]. DGP significantly improves quality of life and anal continence for most patients [2]. Quality of life and patient satisfaction scores correlate significantly with continence scores [7, 8]. However, in some patients, fecal incontinence is unsolved or only partially improved after DGP.
Morbidity rates are high after DGP: complications associated with the technique can often be prevented or treated, but other complications like reduced sensitivity of the rectum by destruction of sensory nerves can be very hard to treat and often result in DGP failure [4, 8, 9]. Morbidity rates are lower and success rates higher in the hands of surgeons experienced in the technique [3, 10].
Constipation is a significant problem after graciloplasty, occurring in 13–90% of the patients after DGP [6–8, 11, 12]. Constipation due to technical failure, like a gracilis wrap that is too tight, can be treated by revisional surgery. Other causes of constipation can first be treated with dietary measures and medication. In case of pelvic floor dysfunction, biofeedback training can be started [9]. When this first line treatment is unsuccessful, retrograde colonic irrigation can be considered. Colonic irrigation can also be used as an additional therapy or salvage therapy for persistent fecal incontinence after failed or partially failed surgery [13]. Not much is known about the success rate of retrograde colonic irrigation, as there is only a limited number of published studies [13, 14].
Antegrade irrigation through a colostomy or appendico-cecostomy is another technique for colonic irrigation, with a reasonable success rate of 64–85% [15–17].
In this study, the effectiveness of colonic irrigation for the treatment of persistent fecal incontinence and/or constipation after DGP was investigated.
Materials and methods
Patients with DGP and postoperative defecation disorders were selected for colonic irrigation between January 1999 and June 2003. The patients were offered colonic irrigation as additional or salvage therapy, as a colostomy was the final option for these patients. The inclusion criteria were invalidating fecal incontinence and/or constipation after DGP and a signed informed consent; exclusion criteria were patients not willing to perform the irrigation because of either embarrassment or wish for colostomy or patients physically and/or mentally not capable of performing irrigation.
All patients visited the outpatient clinic of the University Hospital Maastricht. Relevant physical and medical history were collected. The patients could be divided in six groups according to the etiology of fecal incontinence before DGP: congenital, trauma (rupture/anal surgery), pudendopathy (PNTML > 2.6 ms), spinal cord lesion, cancer (abdominal perineal resection in rectumcarcinoma), and prolapse. The patients were asked to fill out a questionnaire. This questionnaire contained questions concerning the method of irrigation, the time needed for irrigation, the amount of water used, the frequency of irrigation, added substances to the irrigation water, and side effects of colonic irrigation. The endpoint was defined that irrigation had to be successful and satisfying for the patients. Successful irrigation was defined as reaching continence for feces (pseudo-continence) and/or complete resolution of straining, feeling of incomplete evacuation, bloating abdomen, and abdominal pain during irrigation.
The patient was considered satisfied when he or she indicated that the colonic irrigation rendered a major improvement of the quality of life. The improvement of quality of life was measured by a visual analog scale/specific questions within the questionnaire. The irrigation was performed in most patients as retrograde irrigation; only four patients performed antegrade irrigation through an appendico-cecostomy or a colostomy. Patients with fecal incontinence can gain continence as a result of colonic irrigation. This is called pseudo-continence because these patients are only continent, as there is no fecal filling of the rectum and distal colon. The definition of resolved constipation was no straining and no feeling of incomplete evacuation after defecation. Defecation occurred during irrigation in most of these patients.
The Biotrol® Irrimatic pump (Braun®; Fig. 1) or the irrigation bag (Braun®; Fig. 2) were used for colonic irrigation. The Irrimatic pump is an irrigation pump using a flexible tube with a cone-shaped end. The tube is introduced either in the anal canal in case of retrograde irrigation or placed in an appendico-cecostomy or colostomy in case of antegrade irrigation. The pump can hold a maximum of 2 l. The patients were instructed to start the irrigation daily with 500 ml of water. Thereafter, the frequency and the amount of water were adjusted until a satisfactory result was achieved. Water used for irrigation was at body temperature; too cold water can cause abdominal cramps or collapse and too hot water can cause burns. Soap or laxatives could be added if necessary. The pump works on a storage battery, and the speed of water ejection can be regulated. The working mechanism of the irrigation bag is similar, except for the water ejection, which is induced by gravity.
Fig. 1Biotrol® Irrimatic pump (Braun®)Fig. 2Irrigation bag (Braun®)
Data analysis was performed by using SPSS 14.0 (SPSS release 14.0, SPSS, Chicago, USA), with the Mann–Whitney U test. P ≤ 0.05 was considered statistically significant. Data are given as the mean values with the standard deviation.
Results
The questionnaire was sent to 74 patients. Fourteen patients did not wish to participate in the study for various reasons, and 14 patients did not return the questionnaire. Forty-six patients (62%) could be included for analysis of which 37 were female (80%), and these patients had an overall mean age of 59.3 ± 12.4 years. Complications of the DGP occurred in 23 (50%) patients of which 10 patients had two or more complications. Twenty-two patients suffered from constipation, 35 patients were still incontinent for feces, and seven patients had pain in the IPG pocket, leg, or anus.
The medical history besides the DGP is mentioned in Table 1. On average, the patients started the irrigation 21.39 ± 38.77 months after the DGP. Eight patients already used irrigation before DGP.
Table 1Medical historyMedical historyNumber of patients (percentage of total)Anal rupture during delivery13 (28%)Anal repair14 (30%)Prolapse operation19 (41%)Hysterectomy17 (37%)Abdomino-perineal resection for rectum carcinoma8 (17%)Sigmoid resection for sigmoiditis2 (9%)Cholecystectomy8 (17%)Anus atresia1 (2%)
Twenty-four (52%) patients used irrigation as an additional therapy for fecal incontinence in partially failing DGP. Eleven (24%) patients used irrigation for constipation and 11 (24%) patients for both. Most patients (91%) used the irrigation pump. Three patients added soap and one patient an enema to the water to achieve a better result. These were patients with constipation or a combination of fecal incontinence and constipation.
Irrigation was usually performed in the morning (70%) and sometimes in the evening (16%). The frequency of irrigation was 0.90 ± 0.40 times per day. The amount of water used for irrigation was 2.27 ± 1.75 l with duration of 39 ± 23 min. Seventy-four percent of the patients with fecal incontinence irrigated in the morning, 17% in the evening, and 9% twice a day. For the patients with constipation, these numbers are, respectively, 45, 18, and 36%. Ninety percent of the patients with a combination of defecation disorders used the colonic irrigation in the morning.
There was no significant difference in the frequency of irrigation between patients with fecal incontinence and patients with constipation (0.92 vs 0.98/day; P = 0.108). The amount of water used for irrigation was higher in patients with fecal incontinence compared to patients with constipation, but this difference was not significant (2.31 vs 1.91 l; P = 0.484). There was no significant difference in the time needed for irrigation between patients with fecal incontinence and those with constipation (40.8 vs 32.9 min; P = 0.154).
Overall, 81% of the patients were satisfied with the irrigation, 80% of the patients with fecal incontinence, 90% of the patients with constipation, and 72% of the patients with a combined defecation disorder. Thirty-seven percent of the patients with fecal incontinence reached (pseudo-) continence, and in 30% of the patients, the constipation completely resolved (Fig. 3). In 29% of the patients with a combined defecation disorder, the constipation resolved and they were (pseudo-) continent. Overall, satisfaction was related with symptomatic improvement. Only one patient with good symptomatic improvement was not satisfied because he felt that the irrigation was time-consuming and not practical.
Fig. 3Success of rectal irrigation for defecation disorders
Four patients, two with fecal incontinence, one with constipation, and one with both, performed antegrade irrigation through a (3) colostomy or an (1) appendico-cecostomy with good results, and all indicated that the irrigation improved their quality of life.
The patients could be divided in six groups according to the underlying cause of fecal incontinence before DGP (Table 2). When the different etiologies before DGP were compared with the result during irrigation, the patients with a rupture or anal surgery had the worst results of irrigation. The patients with a spinal cord lesion seemed to have the best results.
Table 2Success of rectal irrigation in different etiologies for fecal incontinence before DGPEtiologyNumber of casesSuccess (%) during irrigationCongenital11 (100%)Trauma143 (21%)Pudendopathy155 (33%)Spinal cord lesion53 (60%)Abdomino-perineal resection for rectum carcinoma83 (37%)Prolapse20 (0%)Total4515 (33%)
Side effects of the irrigation were reported in 61% of the patients: leakage of water after irrigation (43%), abdominal cramps (17%), and distended abdomen (17%). Twenty-four (53%) patients changed the irrigation method during their course to improve the results. Seven (16%) patients stopped the rectal irrigation: in five patients, the result was unsatisfactory; two other patients did not need the irrigation anymore because the complaints resolved completely. Finally, two patients received a permanent stoma.
Discussion
Colonic irrigation can be used effectively to treat defecation disorders when other conservative treatments fail or in addition to unsuccessful or partially successful surgical treatment [18].
Colonic irrigation is usually forgotten as an alternative conservative treatment for defecation disorders, although many health care takers are familiar with colonic irrigation, particularly, for its use as perioperative colonic cleansing [19, 20].
Retrograde colonic irrigation is performed through the anorectum or via a colostomy. Only a few publications addressed to retrograde colonic irrigation are found in literature. Briel et al. [13] found a success rate of 38% of retrograde colonic irrigation for fecal incontinence and a significant improvement in quality of life. Recently, another study reported a success rate of 41% in patients with fecal incontinence and 65% in patients with constipation [18]. The success rate is based on patient satisfaction. Although patient satisfaction is the primary goal of treatment, it is a subjective measure. In future research, this should be combined with objective measures such as validated quality of life questionnaires in a prospective study design.
Antegrade colonic irrigation is especially known for the treatment of evacuation disorders in small children and can be performed through an appendico-cecostomy (MACE) or a cecostomy button [16, 21–23]. Alternative enteral access is a sigmoid tube or transverse colonic conduit for patients with a left colonic evacuation disorder [24–26]. The most common problems of MACE are stoma stenosis and leakage [16, 17]. The results in our hospital of antegrade irrigation by an appendico-cecostomy were described earlier [23]. In this study, four patients performed antegrade irrigation via a colostomy or a MACE with good results. O’Bichere et al. performed an experimental study investigating the effect of retrograde vs antegrade colonic irrigation in pigs. That study demonstrates that colonic emptying is more efficient with antegrade irrigation compared to retrograde irrigation [27]. Although a reasonable success rate can be achieved with antegrade irrigation (64–85%) [15–17], retrograde colonic irrigation is preferred above antegrade irrigation as initial treatment because of its non-invasive nature and benign complications.
The amount of water used for irrigation was higher in patients with fecal incontinence compared to patients with constipation. This can be explained by the difficulty of retaining water in the rectum in case of fecal incontinence. Theoretically, the amount of water retained in the rectum is higher in patients with constipation. The grade of colonic emptying by irrigation in constipated patients by means of scintigraphy was investigated by Christensen et al. [14]. The effect of retrograde colonic washout was significantly better in idiopathic fecal incontinence compared to idiopathic constipation, and its effect correlated with the extent to which the irrigation fluid had entered the colorectum.
Surgical treatment of fecal incontinence gives an overall success of 50–84%, depending on the etiology of fecal incontinence and type of surgical procedure [28–32]. Success percentages in DGP vary from 42–92% [1–5]. The success rate of 80% of colonic irrigation is comparable to the success rate of surgical treatment for fecal incontinence. Sixty-one percent of the patients that received the questionnaire completed it. Even when we assume that the patients who did not complete the questionnaire were dissatisfied with the treatment (bad case scenario), the success rate in the present study would still be 50%. It is important to realize that this is a selected group of patients with persistent incontinence and/or constipation after DGP. Regarding the fecal incontinence, this group of patients represents the 8–58% (assuming 42–92% success after DGP) with insufficient results of DGP [1–5].
Regarding the different underlying causes for fecal incontinence before DGP, the patients with a trauma to the anal sphincter have the lowest success rate and the patients with a spinal cord lesion have the highest success rate. It is not possible to draw conclusions about the statistical significance of these results because of the limited group size.
Previous studies already reported an increased incidence of constipation after DGP [3, 6, 8, 11, 12].
In this study, we included 22 patients that were constipated after DGP, of which 11 patients had constipation in combination with persistent fecal incontinence. It is important to realize that many patients are incontinent for feces after periods of severe evacuation problems. These evacuation problems are present again after treatment of the fecal incontinence. Its etiology is not well understood, but is likely to be multifactorial [33, 34]. This is known after all surgical procedures for fecal incontinence [35]. Constipation can be due to technical problems such as a tight graciloplasty, which can be solved by revision of the DGP. It is also possible that nerve tissue around the anus sustains damage during the operation, causing pudendopathy which cannot be treated by revisional surgery. Previous surgery in the pelvis, like an abdomino-perineal resection (eight patients in this study), is known to have a very high rate of obstructed defecation, probably due to reduced rectal sensitivity and pelvic floor dysfunction [36]. Constipation related to physical impairment, like a disturbed sensation and/or motility because of a congenital cause or degeneration, is difficult to treat [9]. These patients are usually served well with colonic irrigation.
Surgical treatment of constipation is troublesome and only indicated in a selected group of patients with intractable constipation. The bowel frequency usually increases, but abdominal pain and bloating persists in most patients [37]. In this study, 90% of the patients were satisfied with the irrigation, and in 30%, the constipation resolved with colonic irrigation.
The final step of treatment after the colonic irrigation in the patients of this study would be a colostomy or ileostomy. In the study of Norton et al., the majority of the patients with fecal incontinence managed by a colostomy reported to be positive about the stoma. However, a few could not adapt to the stoma and disliked it intensely [38]. Harris et al. investigated patients with fecal incontinence or constipation managed with a stoma. Many of these patients reported that lifestyle restrictions were imposed by the stoma, and almost half of the patients felt stigmatized. Up to a third of these patients had significant depression, especially the younger women [39]. Cultural issues also have an important impact on the acceptance of a stoma [40]. Keeping these disadvantages of a definitive stoma in mind, colonic irrigation offers an attractive alternative, which is worthwhile trying before proceeding to a definitive stoma.
Conclusion
Colonic irrigation is an effective alternative for the treatment of persistent fecal incontinence after DGP and/or recurrent or onset constipation additional to unsuccessful or (partially) successful DGP. Colonic irrigation is an undervalued and often forgotten treatment option, which deserves its rightful place among the other treatment modalities.

colonic irrigation
dynamic graciloplasty
fecal incontinence
constipation