A genome search for primary vesicoureteral reflux shows further evidence for genetic heterogeneity

Vesicoureteral reflux (VUR) is the most common disease of the urinary tract in children. In order to identify gene(s) involved in this complex disorder, we performed a genome-wide search in a selected sample of 31 patients with primary VUR from eight families originating from southern Italy. Sixteen additional families with 41 patients were included in a second stage. Nonparametric, affected-only linkage analysis identified four genomic areas on chromosomes 1, 3, and 4 (p < 0.05); the best result corresponded to the D3S3681-D3S1569 interval on chromosome 3 (nonparametric linkage score, NPL = 2.75, p = 0.008). This region was then saturated with 26 additional markers, tested in the complete group of 72 patients from 24 families (NPL = 2.01, p = 0.01). We identified a genomic area on 3q22.2–23, where 26 patients from six multiplex families shared overlapping haplotypes. However, we did not find evidence for a common ancestral haplotype. The region on chromosome 1 was delimited to 1p36.2–34.3 (D1S228-D1S255, max. NPL = 1.70, p = 0.03), after additional fine typing. Furthermore, on chromosome 22q11.22–12.3, patients from a single family showed excess allele sharing (NPL = 3.35, p = 0.015). Only the chromosome 3q region has been previously reported in the single genome-wide screening available for primary VUR. Our results suggest the presence of several novel loci for primary VUR, giving further evidence for the genetic heterogeneity of this disorder.
Introduction
Vesicoureteral reflux (VUR) (OMIM 193000) is the most common disease of the urinary tract in children and affects 1–2% of the Caucasian population [1]. VUR may be associated with both acquired postinfectious and congenital parenchymal damage, currently known as reflux nephropathy (RN) [2, 3]. The most serious consequence of RN is chronic renal insufficiency (CRI), leading to end-stage renal failure (ESRF), dialysis, and/or renal transplantation: 25.4% of children affected with CRI have RN [4]. As a consequence, the impact of VUR on public health is considerable and, despite medical and surgical interventions for the past decades, the incidence of VUR-related renal failure has not decreased [5].
The reflux may occur isolated or in association with other congenital abnormalities of kidney/urinary tract (CAKUT) or as part of syndromic entities, such as renal-coloboma or branchio-oto-renal syndromes [6–8]. The exact etiology of primary VUR is not known, but it is probably related to an abnormal morphogenesis of the ureteral bud, leading to a defect of the ureterovesical junction [9]. The initial evidence suggesting a genetic origin of primary VUR came from twin studies, showing an 80–100% concordance for VUR in monozygotic twins vs. a 35–50% concordance in dizygotic twins [10, 11]. Subsequent evidence included familial clustering of VUR [12], ethnic differences between affected and nonaffected individuals [13], and an increased risk (30–50%) of developing VUR in first-degree relatives of an index case [14–16]. From family studies, a range of inheritance patterns was reported, including autosomal dominant with incomplete penetrance [17–20], autosomal recessive [21], polygenic [22], and even X-linked [23].
Previous studies suggest a urinary tract malformation locus on chromosome 6p [24, 25]. Studies of humans with chromosomal abnormalities also suggest candidate loci or genes on chromosomes 10q26 [26], 19q13 (USF2 gene) [27], and 13q33–34 [28]. Because mutations in PAX2 on 10q24 cause renal-coloboma syndrome, a rare autosomal dominant disease with kidney anomalies that include VUR, this gene was also proposed as a candidate [6, 7]. However, none of these loci or genes has been shown causally related to primary VUR [19, 20]. Recently, Lu et al. [29] showed that mutations in the ROBO2 gene contribute to the pathogenesis of VUR/CAKUT in a small proportion of families. In the only genome-wide linkage study reported to date, Feather et al. [18] demonstrated linkage to chromosome 1p13 for primary VUR under a model of autosomal dominant inheritance with reduced penetrance.
Here, we describe the results of the second genome-wide scan for primary VUR. Differently from previous studies and aiming to collect a homogeneous sample set, our patients were ascertained in a single geographic region. Our results suggest the presence of several novel loci for primary VUR, giving further evidence for the genetic heterogeneity of this disorder.
Methods
Patients and families
Fifty-one pedigrees with multiple patients with VUR coming from Campania (southern Italy) were enrolled in the study (Fig. 1). All families were ascertained through an index case, with VUR documented by voiding cystourethrography (VCUG) in males and direct radionuclide cystography (RNC) in females and family members. Three pediatric nephrologists and one radiologist assessed the patients. RN was diagnosed by DMSA scintigraphy (dimercaptosuccinic acid labeled with Technetium-99 m) and defined as focal defects of radionuclide uptake and/or by one-kidney differential uptake below 43% [30]. VUR grading was made according to the International Grading System of Vesicoureteral Reflux [31].
Fig. 1Selection and distribution of families and patients included in the study. VUR vesicoureteral reflux
Additional family members were considered as “affected” based on the presence of reflux documented by VCUG/RNC and/or the diagnosis of RN, or the detection of ESRF/renal replacement in absence of other known causes. As VUR may spontaneously disappear during childhood and adolescence [32], the finding of scintigraphic signs of RN in relatives of VUR patients strongly suggests the previous occurrence of reflux [33]. Individuals with renal symptoms indicative of VUR, such as previous urinary tract infections and/or hypertension and/or proteinuria, not supported by additional findings were classified as “diagnosis unknown”. Patients with secondary VUR, i.e., neurogenic bladder and posterior urethral valves, or other urinary tract abnormalities, i.e., ureterocele and obstructive hydronephrosis, were excluded. The study focused on primary familial VUR. Eight families with 31 patients with VUR were selected for study phase 1 (genome scan) according to the following criteria: diagnosis of primary VUR in absence of any other malformation, two or more affected individuals per family, and a pattern of inheritance compatible with an autosomal dominant model. The second sample (follow-up) consisted of five affected relative pairs (parent–child trios, ten patients) and 11 small families (31 patients) fulfilling the same criteria (Fig. 1).
Informed consent from patients and family members (parents for their children) and approval from the Ethic Committee at Second University of Naples were obtained previously.
Laboratory analysis
Genomic DNA was isolated from peripheral blood leukocytes by standard techniques and was sent from the Paediatrics Department of Second University of Naples to the Department of Clinical Genetics, Erasmus Medical Centre in Rotterdam. A systematic genome scan was performed using the ABI Prism MD-10 set (Applied Biosystems) consisting of 382 short-tandem-repeat polymorphisms markers (STRPs), average spaced 10 cM. Additional markers for further characterization of candidate regions were selected from the gender-average Marshfield genetic map. Information about marker order and distances were obtained from the National Center for Biotechnology Information (NCBI) physical map and Marshfield integrated genetic map. Polymerase chain reaction (PCR) products were resolved on an ABI3100 automated sequencer, and genotypes were analyzed using the GeneMapper software v.2.0 (Applied Biosystems).
Linkage analysis
One thousand simulations were performed (SLINK, MSIM) [34] to investigate the statistical power of our sample set. An autosomal dominant mode of inheritance was assumed with a penetrance of 70%, a mutant allele frequency of 1%, and a phenocopy rate of 1%. The calculations were done under the assumption of genetic heterogeneity. Assuming 35% of unlinked families, a maximum total log of odds (LOD) score of 5.4 was obtained with a low average LOD of 1.58 [standard deviation (SD) = 1.26], after analyzing all eight pedigrees selected previously. Increasing the percentage of unlinked families to 50%, a maximum LOD score of 4.94 was observed (average 0.99, SD = 1.04). Families 1, 4, and 12 had the largest contribution to the total LOD score (LOD = 0.86, 1.04, and 1.62, respectively), followed by families 5, 7, and 13 (LOD = 0.57). Due to their small size, families 2 and 11 had less contribution to the final LOD (LOD = 0.29). Simulations were calculated in the replication group (11 families) after exclusion of five parent–child trios, giving a maximum LOD of 4.57 (average 1.49, SD = 1.07) and a maximum LOD of 4.85 (average 1.14, SD = 1.08), under assumption of 35% and 50% unlinked families, respectively. Family 25 with seven patients was the main contributor to the total LOD score (LOD = 1.47). Finally, simulations were performed in all 19 families (genome scan group and replication group) reaching a maximum LOD of 9.19 (average of 2.76, SD = 1.78) and a maximum LOD of 7.36 (average of 1.71, SD = 1.34), under assumption of 35% and 50% unlinked families, respectively.
Mega2 [35] was used to process the genetic data, whereas the accuracy of allele segregation within the families was confirmed with Pedcheck. The program GENEHUNTER v.2.1 [36] was used to compute multipoint parametric and nonparametric (or model-free) linkage analysis. As VUR may often disappear with age, all individuals in whom VUR, RN, or both were not clinically proven were classified (and analyzed) as “diagnosis unknown”. Thus, a conservative “affected-only” analysis was performed based on these criteria. Due to the uncertain pattern of inheritance, nonparametric analysis was computed. The rationale of a nonparametric analysis is that, among affected relatives, excess sharing of haplotypes identical by descent would be expected, irrespective of the mode of inheritance. The nonparametric linkage statistics examining all individuals simultaneously (NPL-all), is reported.
A parametric analysis was performed as well. An autosomal dominant mode of inheritance with reduced penetrance, as described above (power calculations), was used. Marker allele frequencies were calculated using all spouses (unrelated individuals) coming from the same geographic area. Due to the genetic heterogeneity of the disease, the genome scan data was analyzed, maximizing the heterogeneity LOD score (HLOD) with respect to the proportion of linked families (α). Nonparametric (NPL) and parametric (LOD) scores were calculated for each of the families, and then total NPL and HLOD scores were obtained.
Results
Patients
Fifty-one pedigrees with multiple patients with VUR, originating from the same region of southern Italy (Campania), were available for the study. All persons in the study were ascertained through an index case documented by VCUG/RNC. A total of 143 patients were detected. According to the phenotype, three groups of families were identified: primary VUR; VUR associated with additional abnormalities of kidney/urinary tract such as duplicated collecting system, renal agenesis, or hypospadia; and VUR occurring in syndromes, such as renal-coloboma, branchio-oto-renal syndromes, and reflux associated with congenital ichthyosis (Fig. 1).
A total of 78 primary VUR patients belonging to 25 pedigrees were identified (33 based on a positive VCUG/RNC, 18 based on detection of RN, 21 diagnosed as having both reflux and RN, and six with ESRF). During the course of the study, a new patient affected with RN was detected in family 1 and included in the second stage. Twenty-eight patients were males and 51 were females (ratio M/F = 0.57). The median age at diagnosis was 3.5 years (range 1 month–68 years). Among the 79 patients, 19 were treated surgically, and four out of six who developed ESRF underwent renal transplantation.
The most informative pedigrees are shown in Fig. 2a,b. The pedigrees showed patients in several generations, and male-to-male transmission was observed in some families, supporting an autosomal dominant mode of inheritance. As reported before for primary VUR, we observed several obligate carriers in our families, i.e., families 4, 6, 12, and 13. Family 6 is especially interesting: when assessing the parents (asymptomatic) of the four symptomatic children, both individuals were found to have RN. No consanguinity was reported, but history of ESRF in both branches was described. We are currently recruiting clinical information of those patients (family 6, second generation, Fig. 2). Due to this bilinear transmission, this family was excluded from the linkage analysis.
Fig. 2A selection of pedigrees enrolled in the study is shown. Squares indicate males and circles indicate females. Family members with unknown phenotype are in grey, whereas those unaffected (normal voiding cystourethrography or direct radionuclide cystography before age of 5 years and unrelated spouses) are in white. An asterisk highlights the individuals genotyped in the study. A number following the asterisk indicates the patients/obligate carriers reported in Fig. 3. Individuals with urinary tract infections by history and “obligate carriers” are considered as unknown in all analyses. a Nine multiplex pedigrees included in the genome-wide scan are shown. An additional five parent–child trios are not displayed. Family 6 was excluded from the linkage analysis due to the bilineal inheritance. b All 11 multiplex pedigrees included in the fine-typing stage are shown. VUR vesicoureteral reflux, RN reflux nephropathy, ESRF end-stage renal failure, UTIs urinary tract infections
Genome search
We performed a systematic genome-wide scan using 382 microsatellite markers. Results from both parametric and nonparametric linkage analysis excluded most of the genome (data not shown). Furthermore, negative scores were found on both VUR loci, 1p13 [18] (NPL = −0.86, p = 0.86) and 13q33–34 [28] (NPL = −0.21, p = 0.58) previously reported.
We used a nominal p value of  < 0.05 to decide whether a region was promising for further study. Table 1 summarizes the genome-wide scan results, showing all regions that yielded a total NPL corresponding to p < 0.05. These regions were located on chromosome 1 (D1S468-D1S255, D1S213-D1S2785), chromosome 3 (D3S3681-D3S1569), and chromosome 4 (D4S402-D4S1597). Furthermore, on chromosome 22q11.22–12.3, patients from a single family showed excess allele sharing (D22S539-D22S280). The best evidence of linkage was observed on chromosome 3p12.3–3q24, which yielded the highest NPL score (2.75, p = 0.008, HLOD = 1.52, α = 0.76) and on chromosome 1p36.32–1p34.3 (NPL = 2.22, p = 0.02, HLOD = 1.13, α = 0.62). Recently, mutations in ROBO2 were described in two VUR/CAKUT patients [29]. ROBO2, which is an ideal functional candidate gene for VUR/CAKUT, maps on chromosome 3p12.3 at the border of our region. Thus, we retrospectively sequenced all gene exons in our index patients, and no mutations were found.
Table 1Summary of the genome-wide scan results. Total nonparametric linkage (NPL) scores with p values <0.05PedigreesD1S468-D1S255D1S213-D1S2785D3S3681-D3S1569D4S402-D4S15971p36.32–1p34.31q41–1q433p12.3–3q244q26–4q32.34–65 cM242–266 cM109–158 cM117–169 cM1−0.80 (−1.09)0.59 (0.19)2.48 (0.87)−0.37 (0.07)21.34 (0.27)−0.42 (−0.76)1.34 (0.28)0.44 (0.27)41.14 (0.72)0.26 (−0.36)1.04 (−0.31)3.05 (1.3)51.9 (0.55)1.78 (0.55)1.89 (0.58)1.69 (0.57)71.59 (0.54)1.07 (0.42)1.75 (0.58)−0.33 (−1.37)111.34 (0.27)1.34 (0.27)1.34 (0.28)0.44 (0.28)121.47 (0.19)1.58 (0.35)0.65 (0.10)−0.04 (−1.66)131.25 (0.54)1.97 (0.54)1.25 (0.57)1.25 (0.57)Total NPL (p)2.22 (p = 0.02)2.35 (p = 0.02)2.75 (p = 0.008)2.15 (p = 0.02)Total HLOD (α)1.13 (0.62)0.61 (0.57)1.52 (0.76)1.31 (0.51)NPL and (LOD) scores per family are shown at each locus. Total NPL (p value) and HLOD (α) obtained from the analysis of all eight pedigrees are displayed. HLOD was obtained maximizing the “heterogeneity” LOD score with respect to the proportion of linked families (α). Negative NPL and LOD scores indicate no linkage or inconclusive resultNPL nonparametric LOD score, HLOD heterogeneity LOD score, α proportion of “linked” families
The NPL score of 2.15 (p = 0.02) on chromosome 4q26–32.3 was mainly due to family 4 (NPL = 3.05, p = 0.062, LOD = 1.3). Yet, allele sharing among the patients of this family was observed also on chromosome 2p23.2–2p15 (D2S165-D2S337, NPL = 3.05, p = 0.062, LOD = 1.3). The only region in which patients from family 12 (Fig. 2) showed excess allele sharing was on chromosome 22q11.22–12.3 (NPL = 3.65, p = 0.015, LOD = 1.49). We then closely inspected the haplotypes from the positive regions in all genotyped families. Families 1, 2, 5, 7, 11, and 13 displayed haplotype sharing among each family’s patients on chromosome 3p12.3–3q24, supporting the linkage to this area. Thus, in six out of eight informative families, we observed allele/haplotype sharing in an overlapping area on chromosome 3q. We noted also on chromosome 1p haplotype sharing in four out of eight multiplex families (families 2, 5, 7, and 11) extending from D1S2667 until D1S255. Therefore, we decided to explore these genomic areas.
Refinement of the chromosome 3 locus
The chromosome 3q region between D3S3681 and D3S1569 was saturated with 26 additional microsatellites markers (average spaced approximately 2 cM). A maximum HLOD = 2.69 (α = 0.95) was reached with an NPL = 2.96, p = 0.001. In a second stage, the fine mapping was extended with 16 additional families with 41 patients. The expansion of pedigrees introduced additional unlinked families, which lowered the maximum HLOD to 1.24 (α = 0.36), giving a total NPL = 2.01 (p = 0.01) (Table 2). Pedigree 25 was the main contributor to the total score, with an NPL = 4.63, p = 0.03, (LOD = 1.45). We then performed a detailed haplotype analysis in all 24 families. Patients from families 2, 5, 7, 13, 20, and 25 shared a common region delimited by markers D3S3641 and D3S1764, containing 36 genes according with the NCBI map, built 36.2. Instead, families 1 and 11 were sharing a more centromeric area not overlapping with the other families (Fig. 3). We did not observe a common “ancestral” haplotype shared across the families.
Fig. 3Haplotype analysis in families supporting the chromosome 3 region after fine typing. A selection of microsatellite markers is shown. The shared genomic region is shown in grey; recombinants are shown in white blocksTable 2Summary of fine-typing results in chromosome 1 and 3 (total NPL scores with p values <0.05)PedigreesD1S468-D1S255D3S3681-D3S15691p36.32–1p34.33p12.3–3q244–65 cM109–158 cM1−0.79 (−2.19)1.43 (0.74)21.34 (0.28)1.34 (0.28)41.59 (1.17)0.46 (−0.43)51.89 (0.56)1.79 (0.58)71.6 (0.57)1.75 (0.57)111.34 (0.28)1.34 (0.28)121.51 (0.05)0.81(−0.73)131.26 (0.57)1.98 (0.57)141.41 (0.27)0.004 (0.001)15−0.24 (−0.07)1.41 (0.28)16−0.07 (−0.02)0.0004 (−0.008)171.41 (0.28)1.41 (0.28)18−0.37 (−0.32)−0.57 (−0.48)190.9 (0.28)−0.81 (−0.9)20−0.37 (−0.29)1.73 (0.48)21−0.48 (−0.45)−0.58(−0.48)221.37 (0.27)−0.57 (−0.48)232.43 (0.86)−1.41 (−0.96)250.22 (0.28)4.63 (1.45)Total NPL (p)1.70 (p = 0.03)2.01 (p = 0.01)Total HLOD (α)1.65 (0.55)1.24 (0.36)NPL and (LOD) scores per family are shown at each locus. Total NPL (p value) and HLOD (α) obtained from the analysis of all eight pedigrees are displayed. HLOD was obtained maximizing the “heterogeneity” LOD score with respect to the proportion of linked families (α). Negative NPL and LOD scores indicate no linkage or inconclusive resultNPL nonparametric LOD score, HLOD heterogeneity LOD score, α proportion of “linked” families
Refinement of chromosome 1 locus
The 1p36.22-p34.3 region between D1S468 (4.2 cM) and D1S255 (65.5 cM) was saturated with seven extra microsatellite markers (total 12, spaced 4–5 cM) in the complete group of 24 families (Table 2). We observed a maximum HLOD of 1.65 (α = 0.55) and NPL of 1.7 (p = 0.03). The haplotype analysis showed allele sharing in families 2, 5, 7, 11, 19, and 23 between D1S228 (29.8 cM) and D1S255 (65.5 cM). Patients from families 13, 14, and 22 displayed haplotype sharing, but it was limited to the upper or lower part of the region, thus showing no overlapping with the rest of the families.
Candidate genes
There were several interesting genes map on the chromosome 3q region. The PPP2R3A gene encodes the protein phosphatase 2A, one of four major protein phosphatases identified in eukaryotic cells, implicated in the regulation of most major metabolic pathways. It has been shown to be expressed ubiquitously in 15-day-old kidneys, regulating the mitogenic activity in the early embryonic kidney [37]. The FNDC6 gene encodes a type of fibronectin, expressed mainly in fetal kidney [38], implicated in the development of renal basement membrane [39]. RBP1 and RBP2 genes  (Retinol-binding-proteins type 1 and 2) are involved in the metabolism of vitamin A, which in its active form of retinoic acid plays a critical role during kidney development, even connecting ureters to bladder [40]. The AGTR1 gene encodes the angiotensin receptor II type 1 (AT1), shown to be related to gross abnormalities in renal morphogenesis in mutant mice (hydronephrosis) as well as a poor proliferation of ureteral smooth cells [41].
Discussion
This study reports the second genome-wide search for primary VUR. Our results suggest the existence of several loci mapping to chromosomes 1, 3, 4, and 22, further supporting the hypothesis that primary VUR is genetically heterogeneous.
We, as others, encountered several pitfalls when studying VUR. We observed clinical variability among and within families and the presence of obligate carriers (individuals who carry and transmit the disease allele but do not manifest any disease sign or symptom). Although the appropriate clinical investigations were performed in several apparently healthy individuals (carriers), no evidence of disease was found, indicating a reduced or age-dependent penetrance. As most of these individuals were recruited during their adulthood, we could not exclude an earlier disease condition that evolved to a spontaneous resolution. To overcome these problems, we first performed a careful clinical evaluation of patients and available relatives. All individuals older than 5 years of age with insufficient or no evidence of VUR were classified as diagnosis unknown, despite the consequent loss of power for the statistical analysis.
We performed the first genome search in VUR that includes families with the same ethnic origin. In order to strengthen the genetic homogeneity of the patients, all families included in this study originated from the same geographic area in the southern part of Italy. Yet, our results strongly support that primary VUR is a (highly) genetic heterogeneous condition. Whereas a large number of our patients and families supported the chromosome 3q locus, one relatively large pedigree (family 12) showed evidence of linkage on chromosome 22. Six of our families supported a locus on chromosome 3q22.2–23 (149.8–153.2 cM). This region is fully overlapping with one of the loci reported by Feather et al. [18]. In their study, the interval on chromosome 3q (from GATA128C02 to D3S1763, 112–176 cM) showed a high α (0.98) with an NPL = 3, p = 0.008. This region was supported by one of their largest pedigrees (with seven patients) that was clearly not linked to the chromosome 1p13 locus. It is interesting that two independent studies have found the same genomic region in distinct groups of patients.
Besides the number of patients included in our genome scan, the results of the statistical analysis are quite modest and do not reach the criteria suggested by Lander and Kruglyak [42] to declare significant linkage. Therefore, some of the identified loci may represent false positive hits and should be interpreted with caution.
The complex disease etiology of primary VUR has shown to be difficult to disentangle. Genetic heterogeneity and lack of knowledge of the true genetic model for VUR are probably the main difficulties in the identification of the genetic etiology of VUR. Although genetic studies in VUR are still in an early phase, we can presume that primary VUR is likely a complex disorder, with a number of not fully penetrant genes causing most of the familiar cases. Finally, primary VUR could be caused by simultaneous gene-environment interactions.
In conclusion, our results show further evidence for the genetic heterogeneity in primary VUR. We will next focus on the refinement of the identified genomic regions and the sequence analysis of the candidate genes according to their tissutal expression and biological function. Replication of the results in additional families will be essential, first to confirm and eventually to evaluate the contribution of these loci to the pathogenesis of primary, nonsyndromic VUR.

vur
complex disorder
primary familial vesicoureteral reflux
genetic linkage


Calcium scoring using 64-slice MDCT, dual source CT and EBT: a comparative phantom study

Purpose Assessment of calcium scoring (Ca-scoring) on a 64-slice multi-detector computed tomography (MDCT) scanner, a dual-source computed tomography (DSCT) scanner and an electron beam tomography (EBT) scanner with a moving cardiac phantom as a function of heart rate, slice thickness and calcium density. Methods and materials Three artificial arteries with inserted calcifications of different sizes and densities were scanned at rest (0 beats per minute) and at 50–110 beats per minute (bpm) with an interval of 10 bpm using 64-slice MDCT, DSCT and EBT. Images were reconstructed with a slice thickness of 0.6 and 3.0 mm. Agatston score, volume score and equivalent mass score were determined for each artery. A cardiac motion susceptibility (CMS) index was introduced to assess the susceptibility of Ca-scoring to heart rate. In addition, a difference (Δ) index was introduced to assess the difference of absolute Ca-scoring on MDCT and DSCT with EBT. Results Ca-score is relatively constant up to 60 bpm and starts to decrease or increase above 70 bpm, depending on scoring method, calcification density and slice thickness. EBT showed the least susceptibility to cardiac motion with the smallest average CMS-index (2.5). The average CMS-index of 64-slice MDCT (9.0) is approximately 2.5 times the average CMS-index of DSCT (3.6). The use of a smaller slice thickness decreases the CMS-index for both CT-modalities. The Δ-index for DSCT at 0.6 mm (53.2) is approximately 30% lower than the Δ-index for 64-slice MDCT at 0.6 mm (72.0). The Δ-indexes at 3.0 mm are approximately equal for both modalities (96.9 and 102.0 for 64-slice MDCT and DSCT respectively). Conclusion Ca-scoring is influenced by heart rate, slice thickness and modality used. Ca-scoring on DSCT is approximately 50% less susceptible to cardiac motion as 64-slice MDCT. DSCT offers a better approximation of absolute calcium score on EBT than 64-slice MDCT when using a smaller slice thickness. A smaller slice thickness reduces the susceptibility to cardiac motion and reduces the difference between CT-data and EBT-data. The best approximation of EBT on CT is found for DSCT with a slice thickness of 0.6 mm.
Introduction
The presence of calcium in coronary arteries is known to be a strong indicator for coronary artery disease (CAD) [1]. It has been shown that quantification of coronary calcium enables the assessment of cardiac event risk stratification [1]. In 1990, Agatston et al. described a method which determines the amount of coronary calcium from tomographic images [2]. This method, known as the Agatston score (AS), depends on the area and the maximum CT density of the calcification detected by electron beam tomography (EBT). Since then, EBT is generally accepted as the gold standard for determining the amount of coronary calcium. Alternative scoring methods have been proposed, such as volume scoring (VS), depending on the volume of the calcification, and equivalent mass (EM) scoring, which depends on the volume and the average density of the calcification [3–5].
Calcium scoring (Ca-scoring) on EBT is known to be less susceptible to cardiac motion compared to other CT-modalities, because of its relatively high temporal resolution. However, since the appearance of multi-detector computed tomography (MDCT), scanners of this type are also widely used for Ca-scoring as an alternative to EBT. Although the temporal resolution of MDCT is lower than EBT, the spatial resolution is much higher (0.4 vs. 1.0 mm), enabling the detection of smaller lesions. Whereas Ca-scoring on EBT can only be used in sequential scanning mode, MDCT facilitates Ca-scoring in sequential and spiral mode. Spiral mode scanning has shown to decrease the variability of Ca-scoring when compared to sequential mode scanning [6]. With the development of dual source computed tomography (DSCT) in 2006, CT is finally approaching the temporal resolution of EBT combined with a high spatial resolution [7].
In order to use Ca-scoring as a useful diagnostic test, it must be demonstrated as accurate, clinically relevant and reproducible. Monitoring of coronary atherosclerosis by repeated scans is advocated by Callister et al. to test the response to lipid-lowering pharmacologic therapy [8] and Budoff et al. [9] showed that statin therapy induced a 61% reduction in coronary calcium progression rate. Therefore a highly reproducible scan-method independent of in-vivo conditions to test the accuracy of Ca-scoring is desirable. In this study a cardiac phantom was used to investigate the influence of cardiac motion on the absolute Ca-score for different kinds of scanners. To our knowledge no previous study has systematically investigated the influence of the heart rate on the absolute Ca-score using EBT, 64-slice MDCT and DSCT.
The purpose of this study was therefore to assess Ca-scoring on 64-slice MDCT and DSCT versus EBT on a moving cardiac phantom as a function of heart rate, slice thickness and calcification density using 3 different Ca-scoring methods.
Methods and materials
Cardiac phantom
A moving cardiac phantom (QRM, Möhrendorf, Germany) was used to simulate the movement of the coronary arteries (Fig. 1, left) [7, 10]. The phantom consists of a robot arm which performs a pre-programmed motion (Fig. 2). The robot arm moves in a water container inside a thorax phantom (QRM, Möhrendorf, Germany) [11]. Different inserts can be attached to the robot arm. The motion curves used in this study were based on velocity curves for the LAD given in literature in order to simulate the human coronary motion as realistically as possible [12]. Three different artificial arteries were investigated which were custom built by QRM. The artificial arteries were made of hydroxyapatite (HA) with a diameter of 4 mm and a length of 55 mm (Fig. 1). Each artery contained three artificial calcifications with a length of 10 mm, a spacing of 5 mm and a thickness of 0.5, 1.0 and 2.0 mm, respectively. The density of the calcifications was different in each artery, one with high density calcifications (HDC), one with medium density calcifications (MDC) and one with low density calcifications (LDC). The concentration and density of the calcifications in the three artificial atereries is given in Table 1. The artificial artery had a density of 50 Houndsfield Units (HU), simulating human blood.
Fig. 1Left: the cardiac phantom. Right: schematic figure of the artificial artery, the dimensions are given in millimetersFig. 2Motion curve of the phantom at 70 bpm. The curve is defined by the time-deflection points T1–T8 and the reconstruction intervals of the DSCT and 64-slice MDCT are indicated by the grey areas. Other heart rates are obtained by a time scaling of the data points. For higher heart rates (>90 bpm) the data point T5 was omitted to reflect the relative larger diminishing of the diastolic phaseTable 1The three artificial coronary arteries high, medium and low density calcification (HDC, MDC and LDC) with the properties of the inserted calcifications as specified by the manufacturerArtificial arteryConcentration (mgHA/cm3)Density (g/cm3)HDC7961.58MDC4011.30LDC1971.16
Data acquisition
The phantom was positioned at an angle of 45 degrees relative to the center axis of the scanner. Every scan was repeated five times with a small random translational (approximately 2 mm) and small random rotational repositioning (approximately 2 degrees) of the phantom after each scan. The ECG signal from the phantom was recorded during scanning to enable synchronization with the scanner. The scan parameters on the 64-slice MDCT (Somatom Sensation 64, Siemens, Forchheim, Germany) were: tube voltage 120 kV, tube current 250 mAs effective, collimation 64 × 0.6 mm and rotation time 330 ms. A DSCT (Somatom Definition, Siemens, Forchheim, Germany) was used with similar scan parameters: tube voltage 120 kV, tube current 100 mAs/rot (equivalent to the tube current of 64-slice MDCT), collimation of 64 × 0.6 mm and rotation time 330 ms. A spiral scanning mode was used on both scanners for a better reproducibility. A standard hospital calcium scoring protocol was used on the EBT-scanner (e− Speed, GE Imatron, San Francisco, USA). This protocol uses a sequential mode with a tube voltage of 130 kV, a tube current of 44 mAs, a collimation of 3.0 mm and a scan speed of 50 ms.
A standard calcium scoring kernel (B35f) was used for reconstruction of the CT-data. Images were retrospectively reconstructed with a slice thickness of 0.6 mm (increment 0.4 mm) and 3.0 mm (increment 3.0 mm) for both CT scanners. The phases with minimal motion were selected from the motion curves of the coronary arteries (Fig. 2) and used for reconstruction of the raw data (Table 2). The data from the EBT-scanner were reconstructed with a slice thickness of 3.0 mm (increment 3.0 mm) at 40% of the RR-interval with a standard calcium kernel according to the standard calcium scoring protocol used in our hospital.
Table 2Phases used for reconstruction of the images in percentage of the beat time at different heart rates used in beats per minute (bpm)Heart rates (bpm)506070809010011064-Slice MDCT-phase (%)76746058565351DSCT-phase (%)83827069696766
Ca-scoring was performed on the reconstructed image sets with commercially available software (Syngo CaScore, Siemens, Forchheim, Germany). Three different scoring methods were used: Agatston scoring, volume scoring and equivalent mass scoring. A standard scoring threshold of 130 HU was used during the procedure. Detailed descriptions of these scoring methods can be found extensively elsewhere [4, 11, 13–15]. The three calcifications of the arteries could not be detected individually at higher heart rates (at heart rates larger than 60 bpm for 64-slice MDCT and larger than 90 bpm for DSCT) combined with thin slices in some of the scans. Therefore the Ca-score of the total artery was used instead of the Ca-scores of the individual calcifications.
Data analysis
Two root mean square measures were used to analyze the scoring results. The first measure quantifies the susceptibility of the calcium score to cardiac motion. The second measure quantifies the deviation of the calcium score from the reference value.
We defined a cardiac motion susceptibility (CMS) index in order to assess the susceptibility to cardiac motion of the Ca-scoring methods: in which x0 is the Ca-scoring result at 0 bpm, xi is the scoring result at heart rate i and N is the total number of heart rates used. In the equation for the CMS-index a factor 1/x0 is introduced to make the index independent of the absolute score which enables comparison of Ca-scores obtained at different slice thicknesses and with different scoring methods as a function of cardiac motion. A small CMS-index is equivalent to a low susceptibility of Ca-scores to cardiac motion.
A second measure was introduced to compare the calcium score results of the two CT scanners to the results of the EBT scanner. The deviation of the calcium score on CT versus the reference value on EBT is defined using a Δ-index: in which yi is the EBT-score at heart rate i, zi is the CT-score at heart rate i and yav is the average EBT-score over all heart rates. The normalization factor yav was inserted to make the Δ-index independent of the absolute score and to enable comparison of Δ-indexes obtained with different Ca-scoring methods and slice thicknesses. A low Δ-index is equivalent to a small difference between Ca-scores on CT and EBT. The delta-index, as defined in Eq. 2, is used to quantify the difference in Agatston and volume scores on CT and EBT. For these scoring methods EBT provides the reference value. For the equivalent mass score, however, the reference value is given by the physical mass. The use of a phantom enables the possibility of calculating the true amount of calcium. Therefore the equivalent mass scoring results have been compared to the true values instead of the EBT-values, thus yi is the true value and zi is the CT/EBT-score at heart rate i.
Noise levels were measured using a standard Region of Interest (ROI) technique. The ROI was placed in a section of a slice containing only water. The standard deviation of the measured HU-values within the selected ROI was considered to be a measure for the noise level.
All measurements are considered to be normally distributed. Mean and standard deviation (sd) are given for each measurement.
Results
The Ca-scoring results of the different arteries obtained with 64-slice MDCT, DSCT and EBT are shown in Fig. 3 as a function of slice thickness and heart rate using the three different scoring methods. The scoring results are relatively constant at low heart rates (50–60 bpm). At heart rates higher than 60 bpm, however, the scores deviate from the values at lower heart rates and an increase or decrease of scoring results is observed depending on modality, slice thickness, calcification density and scoring method.
Fig. 3Calcium scores as a function of heart rate in beats per minute using 64-slice MDCT at 0.6 mm (dotted line with circles), 64-slice MDCT at 3.0 mm (solid line with triangles), DSCT at 0.6 mm (dotted line with circles), DSCT at 3.0 mm (solid line with triangles), EBT (solid line with squares). Agatston score (AS), volume score (VS) and equivalent mass (EM) score from top to bottom; artificial arteries high density calcification (HDC), medium density calcification (MDC) and low density calcification (LDC) from left to right. The thick dotted black lines in the figures in the bottom row represent the physical amount of calcium
The results show a general underestimation of the Ca-score for Ca-scoring obtained at 3.0 mm slice thickness when comparing CT-data and EBT at all heart rates except for the Agatston and volume score of the high density calcifications at 70 and 80 bpm. In general, the Ca-scores obtained with 0.6 mm slice thickness on CT are overestimated compared to the EBT-data or are similar to the EBT-data at all heart rates.
The scores obtained with EBT (squares) increased at heart rates above 90 bpm for the artery containing the high density calcifications (Fig. 3, left column), whereas the artery containing the medium density calcifications remained relatively constant throughout the whole range of heart rates (Fig. 3, middle column). The artery containing the low density calcifications showed decreased scoring results at higher heart rates (Fig. 3, right column).
The 64-slice MDCT with a slice thickness of 3.0 mm (solid lines with triangles) showed increased Ca-scores for the Agatston score at 70–90 bpm and for the volumes score at all heart rates for the high density calcification, whereas the equivalent mass score showed a slight decrease. The medium and low density calcification also showed a decrease in scoring results at higher heart rates.
The 64-slice MDCT with a slice thickness of 0.6 mm (dotted lines with circles) showed highly increased Ca-scores above 70 bpm for the high density calcification for all scoring methods. This is also seen for the medium density calcification for the volume score, whereas the equivelnt mass and Agatston score showed a peak in Ca-scores at 80 bpm. The low density calcification showed diminished results at higher heart rates for all scoring methods.
The Ca-scores of the medium and low density calcification obtained with DSCT with a slice thickness of 3.0 mm (solid lines with triangles) were decreased at elevated heart rates. The results of the high density calcification were relatively constant over the whole range of heart rates.
Finally DSCT at 0.6 mm (dotted lines with circles) showed increased results for Agatston and volume score of the high density calcification. The Agatston score of the medium density calcification showed a small decrease and relatively constant results were observed for the equivalent mass score of the high density calcification and volume and equivalent mass score of the medium density calcification. Diminishing results with increasing heart rate were observed for all methods for the low density calcification.
The influence of cardiac motion on the Ca-score (CMS-index) using the different scoring methods is calculated using Eq. 1 and is summarized in Fig. 4a–c for all scanners and slice thicknesses. Looking at the results of the Agatston score, the average CMS-index for EBT was approximately similar to the CMS-index of DSCT at 0.6 mm, which was for its part approximately 60% smaller than the CMS-index of 64-slice MDCT at 0.6 mm. The CMS-index of DSCT at 3.0 mm was approximately 50% higher than the CMS-index at 0.6 mm. The CMS-index of 64-slice MDCT at 3.0 mm was approximately twice as large as the index of DSCT at 3.0 mm (Fig. 4a). The results of the susceptibility to cardiac motion using volume and equivalent mass score were similar to the results obtained with the Agatston score, except for the relatively small CMS-index for 64-slice MDCT at 0.6 mm using for equivalent mass score. The absolute CMS-indexes using equivalent mass were approximately 10% lower compared to the other two methods. The CMS-indexes averaged over scoring method, slice thickness and calcification density were 2.5 for EBT, 3.6 for DSCT and 9.0 for 64-slice MDCT.
Fig. 4Cardiac motion susceptibility-index (see text) determined with Agatston score (AS) (a), volume score (VS) (b) and equivalent mass (EM) (c) score for the high, medium, low density lesions and the average using EBT (with slice thickness of 3.0 mm), 64-slice MDCT (with slice thickness of 3.0 and 0.6 mm) and DSCT (with slice thickness of 3.0 and 0.6 mm). A small CMS-index represents a low susceptibility to cardiac motion. The standard deviations of the CSM-index are indicated by error bars. 64S = 64-slice MDCT; DS = Dual Source CT
The difference between the scoring results using Agatston and volume score of 64-slice MDCT and DSCT compared to EBT are calculated using Eq. 2 and are shown in Fig. 5a–b. For Agatston score (Fig. 5a), the best Δ-index was observed for DSCT with a slice thickness of 0.6 mm (35.9 ± 10.0 averaged over all densities). A Δ-index approximately twice as large was observed for 64-slice MDCT at 0.6 mm (65.7 ± 9.0 averaged over all densities). Both CT-modalities at 3.0 mm had a Δ-index approximately two times the Δ-index of DSCT at 0.6 mm (91.0 ± 10.1 and 88.4 ± 9.1 for DSCT and 64-slice MDCT respectively averaged over all densities). Comparable results were observed for the volume score measurement (Fig. 5b), although the Δ-indexes for the measurements at 0.6 mm were higher with the highest Δ-index for 64-slice MDCT at 0.6 mm.
Fig. 5Δ-index (see text) determined with Agatston score (AS) (a), volume score (VS) (b) and equivalent mass (EM) (c) score for the high, medium, low density lesions and the average using 64-slice MDCT and DSCT, both with slice thicknesses of 3.0 and 0.6 mm. For Agatston and volume score, EBT has been used as a reference value (a and b, respectively), whereas for equivalent mass score the physical mass has been used as a reference value (c). The equivalent mass measurement includes the EBT as well. A small Δ-index represents a good correspondence with the EBT results. 64S = 64-slice MDCT; DS = Dual Source CT
A Δ-index was calculated for all scanners comparing the equivalent mass results to the theoretical true values. The results are shown in Fig. 5c. The smallest Δ-index was observed for 64-slice MDCT (55.9 ± 6.8) followed by higher indexes for DSCT (68.3 ± 8.3) and EBT (71.3 ± 7.9) both with a slice thickness of 0.6 mm, however the indexes of 64-slice MDCT and DSCT and the indexes of EBT and DSCT are within each margins of error shown by the error bars. Both CT-modalities at 3.0 mm showed Δ-indexes approximately twice as large compared to the results at 0.6 mm (140.1 ± 7.8 and 131.1 ± 8.5 for DSCT and 64-slice MDCT respectively averaged over all densities).
The Δ-indexes were 53.2 for DSCT and 72.0 for 64-slice MDCT both with a slice thickness of 0.6 mm averaged over the scoring methods and densities. The Δ-indexes at 3.0 mm were 102.0 for DSCT and 96.9 for 64-slice MDCT averaged over the scoring methods and densities.
Noise levels were as follows: 64-slice MDCT showed 36.1 ± 2.9 HU and 13.2 ± 1.2 HU for 0.6 and 3.0 mm slice thickness, respectively. DSCT showed 43.0 ± 1.6 HU and 16.1 ± 1.0 HU for 0.6 and 3.0 mm slice thickness, respectively. EBT with a slice thickness of 3.0 mm showed a noise level of 20.5 ± 0.8 HU. The noise did not vary at different heart rates.
Discussion
An assessment was made of Ca-scoring on 64-slice multi-detector computed tomography and dual-source computed tomography versus electron beam tomography on a moving cardiac phantom as a function of heart rate, slice thickness and calcification density using 3 different Ca-scoring methods. From the results it can be concluded that the Agatston, volume and equivalent mass scores depend on heart rate, slice thickness and the CT-system used. Furthermore DSCT is approximately 50% less susceptible to cardiac motion as 64-slice MDCT in Ca-scoring.
It has been shown in previous studies that the amount of calcium in coronary arteries is generally underestimated in MDCT with respect to the gold standard EBT. Stanford et al. showed an underestimation of coronary calcium with 4-slice MDCT compared to EBT [16] and the same effect was reported by Horiguchi et al. using 16-slice MDCT [14, 17]. Our results showed underestimation as well, but only for 3.0 mm slice thickness, whereas 0.6 mm showed an overestimation at all heart rates.
Surprisingly the Agatston scores of the medium density calcification at rest using 3.0 mm slices are different for the 64-MDCT and DSCT, while similar scores are expected (approximately 165 for 64-slice MDCT and 135 for DSCT). The same effect is observed for heart rates of 50 and 60 bpm. A possible explanation for this phenomenon lies within the scoring algorithm of the Agatston score. For each calcification the maximum HU value within the calcification is obtained. Based on this maximum value the area of the calcification is multiplied by a weighting factor. For a maximum of more than 400 HU this factor is 4, for a maximum between 300 HU and 400 HU this factor is 3 [2]. The medium density calcification has a CT density of 400 HU. The difference in scoring results can be explained by a small difference in HU between the two scanners. Where the maximum CT density within the medium density calcification could be over 400 HU using the 64-slice MDCT, the maximum CT might have been below 400 using the DSCT. If this explanation is applied, the score obtained using 64-slice MDCT is more similar to the score obtained using DSCT (165*3/4 = 124). Although the difference in HU is very small, the weighting factor of the Agatston algorithm can cause a large difference in scoring result.
At heart rates above 70 bpm Agatston, volume and equivalent mass score differ from the results at rest and at low heart rates. This difference depends on the density of the calcification as can be seen from Fig. 3: calcifications with a high density show elevated scoring results, whereas low density calcifications are associated with diminished scoring results. A decrease of Agatston and equivalent mass score on increased heart rates using a calcification of 400 HU has also been reported by Ulzheimer et al. using a 4-slice MDCT in accordance with our results [11]. We considered the influence of image blurring on the Ca-score as a function of the calcification density in Fig. 6 for a possible explanation for this effect. Two calcifications of identical size are shown by black lines, one with a high density (X) and one with a low density (Y). The corresponding apparent images at a relatively low and high heart rate are given by the solid grey and dotted grey line, respectively. In addition, the default Ca-scoring threshold of 130 HU is shown by the dotted black line. At the level of the threshold the apparent width at high heart rates is larger than the apparent width at low heart rates for the high density object. The reverse effect is observed for the low density object; at high heart rates the apparent size is reduced compared to the apparent size at low heart rates. From this analysis it can be concluded that at high heart rates the apparent volume of high density objects is increased and the apparent volume of low density objects is decreased. With this model we can explain the increase of calcium score on increasing heart rate for high density calcifications, and a decrease of calcium score on increasing heart rate for low density calcifications, as observed in Fig. 3. Decreasing scoring results with increasing movement have previously been reported on 4-slice CT [15].
Fig. 6Theoretical estimated CT profiles for two objects (black) with high (X) and low density (Y) exhibiting a relatively low (solid grey) and high (dotted grey) movement. The dotted black line represents the standard Ca-scoring threshold of 130 HU
The susceptibility of calcium score on heart rate has been assessed by the CMS-index using the 3 different scoring methods available. The results show that the CMS-index of EBT is the lowest for all methods. Therefore it can be concluded that EBT is the least susceptible to cardiac motion. The CMS-index of DSCT is approximately half the CMS-index of 64-slice MDCT, showing a reduction of 50% of the influence of cardiac motion on Ca-scoring on DSCT with respect to 64-slice MDCT. These results can be explained with the improved temporal resolution of DSCT compared to 64-slice MDCT (83 vs. 165 ms). A reduction of the slice thickness also results in a lower CMS-index. Therefore we conclude that the use of a small slice thickness reduces the susceptibility to cardiac motion for both 64-slice MDCT and DSCT.
The difference between CT-data and EBT-data has been assessed by the Δ-index using the Agatston and volume score, the equivalent mass results have been compared to the physical amount of calcium. The results show the lowest Δ-index for DSCT with a slice thickness of 0.6 mm for Agatston and volume score. The CT modalities at 0.6 mm and EBT showed similar Δ-indexes for the approximation to the physical mass. A reduction of the Δ-index was observed comparing the two CT-modalities at 0.6 mm and 3.0 mm. The best resemblance between EBT and CT was observed for DSCT with a slice thickness of 0.6 mm.
The use of a smaller slice thickness has some disadvantages although it was beneficial to the scoring results in this phantom study. The noise measurements showed increased noise levels for the 0.6 mm slices compared to 3.0 mm slices. It is expected that for patient scanning the noise levels at 0.6 mm are too high to guarantee a reliable outcome of the Ca-scoring. To overcome these increased noise levels the tube current can be increased. However, this increases the patient dose as well. Although dose-reduction techniques have been investigated leading to dose-reductions up to 57% [18–21], a good balance between patient dose and accuracy of calcium scoring needs to be found.
Limitations
The EBT-data acquisition of this study was performed with a standard hospital protocol using a tube voltage of 130 kV, whereas CT scanning was performed with a tube voltage of 120 kV. Although higher energies tend to show less density, Nelson et al. reported very small differences between EBT at 130 kV and CT at 120 kV [22]. Therefore we expect that the influence of the difference in tube voltage can be neglected.
The pre-programmed movement of the calcified coronary arteries was 1-dimensional in contrast with the in vivo situation where the motion of human coronary arteries is 3-dimensional and the direction and orientation of the human coronary arteries can vary. In our study the movement of the calcified coronary arteries was in the (x,z) plane with a 45o angle relative to the z-direction of the scanner. We expect that movement more perpendicular to the z-direction of the scanner will cause more blurring in the (x,y) plane and reduce blurring in the z-direction. In addition, we expect that movement more parallel to the z-direction of the scanner will be more subject to partial volume effects when using thick slices. Thin slices will be less subject to the PVE due to the isotropic resolution of 0.6 mm. The motion of the robot arm was programmed according to patient data [11] and therefore we expect that our analysis shows a good correspondence with a clinical situation, but a clinical validation is advocated.
The coronary artery we used for our simulation, the LAD, exhibits lesser motion than the LCX and especially the RCA, which exhibits very large motion swings especially in systole. In our study we, however, wanted to show the influence of motion on the coronary calcium score independent of a specific major coronary artery. We therefore have used motion curves with velocities similar to the LAD to simulate the motion, because if a dependency of calcium score on coronary motion could be proven for the lowest velocity of the LAD, we expect an even stronger dependence for the higher velocities of the LCX and RCA. In our study we have shown that for higher heart rates the under- or overestimation of the calcium score increases as a function of calcification density, independently of the absolute velocity of the artery, but depending on the relative heart rate difference from 0 bpm. Because this motion dependent effect is pronounced visible for the relative low velocity of the LAD, we expect that the results can also be applied to the vaster moving other major arteries.
Conclusion
The results of Ca-scoring are influenced by heart rate, slice thickness and modality used. DSCT is approximately 50% less susceptible to cardiac motion than 64-slice MDCT using a robot phantom. Susceptibility is further reduced with a smaller slice thickness. DSCT gives a better approximation of the absolute calcium score on EBT than results obtained with 64-slice MDCT when using a smaller slice thickness (0.6 mm). The two modalities show similar results when using larger slice thicknesses (3.0 mm). In general, the use of a smaller slice thickness further reduces the difference between CT-data and EBT-data. The best approximation to the physical amount of calcium was found using a small slice thickness, where 64-slice MDCT and DSCT show similar results. The best approximation of Ca-scoring on EBT is observed for DSCT with a slice thickness of 0.6 mm.

calcium score
64-slice mdct
dual source ct
heart rate
electron beam ct


A database for medicinal plants used in the treatment of diabetes and its secondary complications

Effective treatment of diabetes is increasingly dependent on active constituents of medicinal plants capable of controlling hyperglycemia as well as its secondary complications. Sensing the importance of documenting such medicinal plants, here we describe a web database containing information (name, literature citation, active compounds and few related full text articles) of the diabetes medicinal plants exhibiting hypoglycemic, antioxidant and antimicrobial effects.
Background
Diabetes mellitus is possibly the world's largest growing metabolic disorder and around 30 million people around the world suffer from this disorder.[1] Diabetes can be defined
as a group of syndromes characterized by hyperglycemia, altered metabolism of lipids, carbohydrates and proteins.[2] The complications of diabetes include vascular diseases, eye
disorders, renal disorders and a host of secondary infections. [3] The treatment of diabetes with synthetic drugs is generally not preferred because of its high cost and the range
of side effects caused. Hence development of traditional or alternative medicine is needed. Herbal drugs constitute an important part of traditional medicine and literature shows
that there are more than 400 plant species showing antidiabetic activity. [4,5] Our interest is to study these anti diabetic medicinal plants. Research findings suggest that many
of these plants control diabetes by exhibiting hypoglycemic and antioxidant effects. The secondary infections associated with diabetes are also restricted to antimicrobial effect by
plant products. Sensing the opportunities provided and in an effort to translate research into technology, this database ‘DIAB’ was created to document the research literature
available on these medicinal plants and their active compounds.
Methodology
Step 1: Data collection
Data of antidiabetic plants were collected from various literature sources such as PubMed [6], ScienceDirect [7], Biomed Central [8]
Springerlink [9],
Scirus [10], Wiley journals [11] and also from folklore medicinal usage. The current dataset contains information for about
258 genus and 287 species of plants exhibiting antidiabetic effect. Of these 287 antidiabetic plants, 129 species show proven antioxidant effect and 53 species exhibit
antimicrobial effect besides their hypoglycemic activity.
Step 2: Database Design
The database is developed using MS Access as back-end and ASP.NET as front-end on a Windows platform and updated regularly.
Step 3: Features of DIAB
Three separate links (antidiabetic, antioxidant, and antimicrobial) are provided to access the literature citations of antidiabetic plants showing hypoglycemic, antioxidant and
antimicrobial properties respectively. The record entry consists of the following literature: plant name, part investigated, authors and abstract. The ‘Active principles’ link provides
information (plant name, part investigated, active compounds and properties) on the 46 compounds isolated from the antidiabetic medicinal plants. The ‘Articles’ link provides few free
text articles for further reading. A screen shot of the database is given in figure 1.
Utility
In the current scenario there are several other databases which give information on medicinal plant name, distribution, drugs formulated and usage in treatment of diabetes. We believe
that the freely available database called DIAB will give supplementary information on the antidiabetic plants capable of controlling some secondary complications. This database finds
utility to the scientific community for a quick review on plants for diabetes medicinal plant research and provides enormous scope for development of drugs.
Future development
We plan to refine and keep updating this database and hopefully a link solely for indigenous plants will be made available in the nearest future. The database will also be modified
to develop provisions to search the database to identify plants of interest using keywords.

database
medicinal plants
diabetes
literature
antioxidant
antimicrobial properties
active principles


Interferon and lamivudine vs. interferon for hepatitis B e antigen-positive hepatitis B treatment: meta-analysis of randomized controlled trials

Aims To compare interferon monotherapy with its combination with lamivudine for hepatitis B e antigen (HBeAg)-positive hepatitis B treatment.
Chronic hepatitis B is a common medical condition, affecting more than 400 million individuals worldwide, leading to hepatic inflammation and injury (1–4). This viral-triggered, immune-mediated condition predisposes those affected to cirrhosis and hepatocellular carcinoma, thus necessitating treatment (1–3). The treatment consists of individualized, single-agent therapy with interferon-α or nucleoside analogues. Unfortunately, this treatment fails to yield long-lasting outcomes in majority of the treated population, prompting the notion of their use in combination to enhance the therapeutic efficacy (4–8).
The notion of combination therapy for chronic hepatitis B treatment has been previously examined, yielding inconclusive results (9–16). In our study, we aim to elucidate this topic comparing interferon monotherapy to its combination with the best-studied antiviral agent for that purpose, lamivudine. Furthermore, the focus of our analysis is hepatitis B e antigen (HBeAg)-positive patients, a subset of the patient population in which disease activity, risk of complications and the subsequent need of efficacious therapy are more pronounced.
Methods
Literature search and study design
Two independent researchers conducted the literature search, study selection and data extraction, with any disagreements resolved by consensus among them.
The researchers conducted a systemic literature search using the electronic databases MEDLINE (1966 to January 2006), EMBASE (1980 to June 2006), OVID (1966 to January, week 3, 2006) and the Cochrane library clinical trials registry (issue 1, 2007). The following keywords were used: ‘Hepatitis B’, ‘Interferon’, ‘Lamivudine’ and ‘combination therapy’. In addition, a manual search using citations in previous publications was preformed. The following inclusion criteria were used: (i) study design: randomized controlled trials; (ii) study population: HBeAg-positive patients; (iii) intervention: interferon vs. interferon and lamivudine therapy. Our search was not restricted by language. The following exclusion criteria were used: (i) examining the nonadult population; (ii) not reporting any of the primary efficacy measures as defined by the authors. When several publications pertaining to a single study were identified, the most recent and complete publication was used.
The included studies were divided into two groups according to their use of conventional (CON) or pegylated (PEG) interferon-α, with patients within each group given interferon monotherapy, or interferon and lamivudine combination therapy. Data were extracted for study methodology and for the defined efficacy measures. Only data pertaining to the regimens in question were extracted, while data concerning other regimens were reviewed, and if found to be of significance to our study, were noted and discussed. Separate meta-analyses examining the defined efficacy measures were preformed. In addition, we compared the rates of sustained responses across groups aiming at the identification of a preferable regimen. Intention to treat analysis was used throughout this study, excluding histological response analysis, because of its low outcomes reporting rates.
Efficacy measures and definitions
End-of-follow-up (sustained) virological and biochemical response rates, and sustained HBeAg clearance and seroconversion rates were used as primary efficacy measures. Histological response, emergence of YMDD mutations, liver-related and all-cause mortality, and treatment safety were used as secondary efficacy measures. Virological response was defined as attainment of undetectable (or below 400 copies/mL) levels of hepatitis B virus DNA, as determined by polymerase chain reaction, which was previously found to be the most accurate measure of virological response monitoring (2). Biochemical response was defined as normalization of alanine aminotransferase levels, HBeAg clearance as HBeAg disappearance and seroconversion as HBeAg antibodies appearance. Histological response was defined as a two-point reduction or increase in the histologic activity index score, signifying histological improvement and worsening respectively. Treatment safety was assessed using the occurrence rate of adverse effects necessitating treatment discontinuation.
Study quality and homogeneity
The included studies methodological quality was assessed using the Jadad quality scale (15), an established composite score evaluating randomization, concealment and reporting of patient withdrawal and dropout rates, with scores ≥3 signifying high-quality studies. Heterogeneity was assessed for each analysis.
Statistical analysis
Quantitative meta-analyses were performed to assess differences between monotherapy and combination groups. Statistical analysis was performed and the Forest plots were generated using the comprehensive meta analysis® software application, Version 2.0 (Biostat, Englewood, NJ, USA). The odds ratios (OR) were calculated along with their, respective, 95% confidence intervals (CI) and presented for each individual study as well as interferon type and across all studies. Subgroup analyses were presented using OR and their corresponding 95% CI. Heterogeneity was assessed for each of the meta-analyses by means of Q-statistics and their corresponding P-values.
Results
Study selection and characteristics
The literature search yielded 13 studies. Five studies were excluded on account of the following: (i) examining the nonadult population (n=1); and (ii) not examining or reporting the sustained response rates (n=4). The eight remaining trials, involving 1321 patients, were included. Six trials used conventional interferon-α (n=503) (16–21) and two trials used pegylated interferon-α (n=808) (22, 23). Two of the CON group trials exclusively studied treatment-naïve patients (16, 19), whereas the others studied a mixture of treatment-naïve and previously treated patients (17, 18, 20–23). The majority of patients were treated for a period of 1 year (n=6) (16–23), with some treated for 6 months (n=2) (17, 18). Sustained response rates were obtained at 6 months following treatment completion in most studies (n=6) (17, 19–23), and at 40 or 54 weeks following treatment completion in some (n=2) (16, 18). The studies were of heterogeneous methodological quality (Jadad scores of 2–5). All studies were published as full publications, with six studies published in English (16–19, 22, 23) and two in Chinese (20, 21). One study used sequential therapy (18), six used concurrent therapy (16, 17, 19,21–23) and one used both (20) (see Tables 1 and 2).
Table 2
Patient selection criteria of studies included in the meta-analysis
Study
Inclusion criteria
Exclusion criteria
Ayaz (2006)
1. HBsAg positive for >6 m and anti-HBeAg and HBsAg negative 2. Presence of HBV DNA 3. Evidence of inflammation on biopsy within 6 m of enrolment and ALT>1.5 NL
1. Previous treatment with INF, antiviral or immunosuppressive agents 2. HIV, hepatitis C or D 3. Other aetiologies of liver disease, alcohol intake >40 g/day, decompensated liver disease or cancer 4. No informed consent 5. Pregnancy 6. Any contraindications for INF use 7. Leucocytes, neutrophil or platelet count of <2500, <1000 and <100 000/mL, respectively, or haemoglobin <10 g/dL
Song (2004)
1. 19–65 years old 2. HBsAg positive for >6 m and HBeAg positive 3. HBV DNA>500 000 copies/mL 4. Evidence of inflammation by 2 NL <ALT <500
Not reported
Deng (2003)
1. 15–60 years old 2. HBeAg and HBV DNA positive for >6 m 3. HBV DNA>103 000 copies/mL 4. Evidence of inflammation by ALT>2 NL
1. Immunosuppressive or antiviral therapy within 6 m 2. Hepatitis of other aetiologies 3. Decompensated liver disease 4. Pregnancy or breast feeding
Yalcin (2002)
1. 16–80 years old 2. HBeAg and HBsAg positive 3. HBV DNA positive 4. Evidence of inflammation by histology and 1.5<ALT<10 NL, on three occasions within 6 m
1. Previous INF therapy, antiviral or immunosuppressive therapy, or contraindication for INF therapy 2. HIV, hepatitis C or D 3. Decompensated liver disease or carcinoma 4. Alcohol consumption >40 g/day or other liver disease causes 5. Pregnancy 6. Leucocytes <2500/mm3, neutrophils <1000/mm3, platelets <100 000/mm3, or haemoglobin <10g/dL 7. Unable to obtain consent
Cindoruk (2002)
1. Adults 2. HBeAg positive 3. HBV DNA positive 4. Evidence of inflammation by histology and by abnormal ALT levels for >6 m
1. Previous INF therapy 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Diabetes, autoimmune, or other psychiatric or serious medical illness 5. High alcohol intake or current drug abuse 6. Pregnancy
Schalm (2000)
1. 16–70 years old 2. HBsAg and HBeAg positive at screening and at >6 and >3 m prior respectively 3. HBV DNA>500 000 copies/mL 4. Evidence of inflammation by histology or persistently elevated ALT for >3 m
1. Contraindication to or previous INF therapy, or antiviral therapy within 6 m 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Liver disease of other aetiology
Lau (2005)
1. Adults 2. HBsAg positive for >6 m and HBeAg positive 3. HBV DNA>500 000 copies/mL 4. Evidence of inflammation on biopsy and 1<ALT<10 NL
1. Treatment within 6 m 2. HIV, hepatitis C or D 3. Decompensated liver disease 4. Serious medical or psychiatric illness 5. Alcohol or drug use within 1 y 4. Neutrophils <1500 g/dL, platelets <90 000/mm3, or creatinine >1.5 NL
Janssen (2005)
1. >16 years old 2. HBsAg positive for >6 m and HBeAg positive on two occasions within 8 w of randomization 3. Evidence of inflammation by two measurements of ALT>2 NL within 8 w of randomization
1. Antiviral or immunosuppressive therapy within 6 m 2. HIV, hepatitis C or D 3. Advanced liver disease or carcinoma 4. Serious medical or psychiatric illness, or uncontrolled thyroid disease 5. Substance abuse within 2 y 6. Pregnancy or inadequate contraception 7. Leucocytes <3000/mm3, neutrophils <1800/mm3, or platelets <100 000/mm3
ALT, alanine aminotransferase; HBV, hepatitis B virus; HBsAg, hepatitis B surface antigen; INF, interferon; m, months; NEG, HBeAg negative; NL, upper limit of normal; POS, HBeAg positive; w, weeks; y, years.
Table 1
Characteristics of studies included in the meta-analysis
Study
n
Study design
Jadad score
Therapy period
Follow-up period
Therapy regimen
Conventional interferon-α
 Ayaz (2006)
 68
RCT
2
12 m
 6 m
INF-α-2a 9 MU × 3/w with or without LMV 100 mg/day
 Song (2004)
 90
RCT
2
12 m
 6 m
INF-α 3 MU × 3/w with or without LMV 100 mg/day
 Deng (2003)
 62
RCT
2
48 w
24 w
INF-α-1b 5 MU × 3/w with or without LMV 100 mg/day
 Yalcin (2002)
 49
RCT
2
52 w
52 w
INF-α-2b 10 MU × 3/w with or without LMV 100 mg/day
 Cindoruk (2002)
100
RCT
2
6 m
 6 m
INF-α 9 MU × 3/w with or without LMV 100 mg/day
 Schalm (2000)
144
RCT, DB
4
24 w
40 w
INF-α 10 MU × 3/w with or without LMV 100 mg/day
Pegylated interferon-α
 Lau (2005)
542
RCT, DB
5
48 w
24 w
PegINF-α-2a 180 μg × 1/w with or without LMV 100 mg/day
 Janssen (2005)
266
RCT, DB
4
52 w
26 w
PegINF-α-2b 100 μg × 1/w with or without LMV 100 mg/day
DB, double blind; INF, conventional interferon; LMV, lamivudine; m, months; PegINF, pegylated interferon; RCT, randomized controlled; w, weeks.
Sustained virological response
Greater sustained virological response rates were observed for patients given combination as compared with monotherapy in the CON group [61.1 vs. 35.4%, OR=11.7, 95% CI (7.8–17.6), P<0.0001], and overall [28.9 vs. 18.5%, OR=2.1, 95% CI (1.3–3.3), P=0.002], although not in the PEG group [12.2 vs. 11.8%, OR=1.1, 95% CI (0.5–2.3), P=0.8]. Heterogeneity was assessed and not found to be a concern (Q=3.5, P=0.06) (see Fig. 1).
Fig. 1
Sustained virological response. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration.
Sustained biochemical response
Greater sustained biochemical response rates were observed for patients given combination as compared with monotherapy in the CON group [46.2 vs. 34.0%, OR=1.8, 95% CI (1.2–2.7), P=0.007], although not in the PEG group [37.9 vs. 38.1%, OR=1.0, 95% CI (0.7–1.3), P=0.94], or overall [36.1 vs. 36.7%, OR=1.2, 95% CI (0.9–1.5), P=0.15]. Heterogeneity was assessed and not found to be a concern (Q=3.3, P=0.07) (see Fig. 2).
Fig. 2
Sustained biochemical response. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine.
Sustained hepatitis B e antigen clearance
No significant differences in sustained HBeAg clearance rates were observed between patients given combination and monotherapy in the CON group [33.5 vs. 24.0%, OR=1.6, 95% CI (0.9–2.7), P=0.09], PEG group [30.6 vs. 34.4%, OR=0.8, 95% CI (0.6–1.1), P=0.26] and overall [31.5 vs. 31.9%, OR=1.0, 95% CI (0.7–1.3), P=0.88]. Heterogeneity was assessed and not found to be a concern (Q=2.6, P=0.11) (see Fig. 3).
Fig. 3
Sustained hepatitis B e antigen (HBeAg) clearance. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration.
Sustained seroconversion
Greater sustained seroconversion rates were observed for patients given combination as compared with monotherapy in the CON group [30.0 vs. 18.9%, OR=1.8, 95% CI (1.1–2.8), P=0.01], although not in the PEG group [27.9 vs. 31.0%, OR=0.9, 95% CI (0.6–1.2), P=0.34], or overall [28.7 vs. 27.1%, OR=1.1, 95% CI (0.8–1.4), P=0.59]. Heterogeneity was assessed and not found to be a concern (Q=3.5, P=0.06) (see Fig. 4).
Fig. 4
Sustained seroconversion. CON, conventional interferon monotherapy vs. its combination with lamivudine; PEG, pegylated interferon monotherapy vs. its combination with lamivudine. *Concurrent and sequential administration.
Histological response
Greater histological improvement rates were observed for patients given combination as compared with monotherapy in the CON group [83.8 vs. 26.6%, respectively, n=1, OR=14.3, 95% CI (3.3–61.3), P<0.001], although not in the PEG group [48.1 vs. 53.4%, respectively, n=1, OR=0.8, 95% CI (0.4–1.7), P=0.70], or overall [61.4 vs. 47.9%, respectively, n=2, OR=1.7, 95% CI (0.9–3.3), P=0.11]. No significant differences in histological worsening rates were observed between patients given combination and monotherapy in the CON group [0 vs. 6.7%, respectively, n=1, OR=0.23, 95% CI (0.02–3.3), P=0.16], PEG group [9.6 vs. 10.3%, respectively, n=1, OR=0.9, 95% CI (0.3–3.1), P=0.9] and overall [6.0 vs. 9.6%, respectively, n=2, OR=0.6, 95% CI (0.2–1.9), P=0.55].
All-cause and liver-related mortality
There were no reported deaths of any aetiology for either group (0% for both).
Safety
No significant differences in safety rates were observed between patients given combination and monotherapy in the CON group [1.6 vs. 0.9%, OR=1.7, 95% CI (0.3–9.0), P=0.55, n=6], PEG group [6.0 vs. 4.2%, OR=1.7, 95% CI (0.9–3.2), P=0.12, n=2] and overall [4.1 vs. 2.7.%, OR=1.7, 95% CI (0.9–3.1), P=0.10]. Heterogeneity was assessed and not found to be a concern (Q=0.001, P=0.98).
YMDD mutation emergence
Greater YMDD mutation emergence rates were observed for patients given combination as compared with monotherapy in the PEG group [4.0 vs. 0%, OR=18.1, 95% CI (2.4–136.6), P=0.005, n=2] and overall [4.5 vs. 0%, OR=14.8, 95% CI (2.8–77.6), P=0.001], although not in the CON group [5.9 vs. 0%, OR=9.9, 95% CI (0.5–177.1), P=0.12, n=1]. Heterogeneity was assessed and not found to be a concern (Q=0.11, P=0.74).
Conventional interferon combination therapy vs. pegylated interferon monotherapy
Excluding virological response [61.1 vs. 11.8%, OR=11.7, 95% CI (7.8–17.6), P<0.0001], no significant differences in rates of biochemical response [46.2 vs. 38.1%, OR=1.4, 95% CI (1.0–1.9), P=0.052], HBeAg clearance [33.5 vs. 34.9%, OR=0.9, 95% CI (0.6–1.4), P=0.77] or seroconversion [30.0 vs. 31.4%, OR=0.9, 95% CI (0.7–1.3), P=0.73] were observed between patients given combination therapy in the CON group and those given monotherapy in the PEG group. Significantly greater virological response rates were observed with monotherapy in the CON as compared with the PEG group [35.4 vs. 11.8%, OR=4.1, 95% CI (2.6–6.4), P<0.0001].
Discussion
The suboptimal outcomes of current hepatitis B therapies have prompted the notion of their use in combination to achieve a synergistic effect and decreased mutagenicity (2). Furthermore, it has been suggested that the enhanced efficacy of the combination will allow for the dose reduction of its components, thus decreasing the risk of potential adverse effects (2). In our study, we explored this notion in the subset of HBeAg-positive patients. Our study is the first to examine the combination of interferon and lamivudine for chronic hepatitis B treatment, pooling data from all pertinent randomized-controlled trials into meta-analysis. This analysis will aid in achieving evidence-based conclusions on the matter, resolving the controversy in its regard and directing future investigational efforts.
In our analysis, we found pegylated interferon monotherapy to be of comparable efficacy to its combination with lamivudine, providing similar rates of sustained virological and biochemical responses, and HBeAg clearance and seroconversion (P=0.66, 0.94, 0.26 and 0.34 respectively). Furthermore, while the pegylated interferon trials predominantly involved treatment-naïve patients, analysis of previously treated patients within one of those studies yielded similar outcomes (24). In contrast, the addition of lamivudine to conventional interferon resulted in superior sustained virological, biochemical and seroconversion rates (P<0.001, P=0.007, 0.01 and 0.09 respectively), similarly observed with sequential and concurrent administration (20). A similar trend was observed with HBeAg clearance rates, although the sample size was insufficient to detect this effect (P=0.09). As with pegylated interferon, treatment-naïve patients comprised the majority of the studied population and to a greater extent. Nonetheless, a controlled, nonrandomized trial of previously treated patients reported similar outcomes (25). These outcomes are corroborated by those of our histological analysis (P<0.001 and P=0.70 for histological improvement in the CON and PEG groups respectively) and by those of others (26). Importantly, our analysis provides an explanation to the discordance between the combinations' effectiveness with conventional and not with pegylated interferon, with lamivudine-induced mutagenicity suppressed with the former, while not with the latter (P=0.12, and 0.05 respectively).
Accordingly, two possible regimens emerged from our analysis: pegylated interferon monotherapy, and conventional interferon and lamivudine combination therapy. A comparison between the two found them to be of comparable efficacy (P>0.05), with the exception of virological response (P<0.001). That said, it is the authors opinion that this combinations' favourable virological response should not prompt its use as the regimen of choice, as a greater portion of treatment-naïve and thus easier to treat patients, comprised the CON as compared with the PEG group, with three CON group studies exclusively examining this patient population (16, 17, 19). Our hypothesis is further supported by the superior virological outcomes of conventional as compared with pegylated interferon monotherapy (P<0.0001), which is in conflict with current knowledge (27), and is easily explained by this hypothesis. Accordingly, we suggest that in comparable populations, pegylated interferon monotherapy is likely to be similarly or more efficacious than lamivudine and conventional interferon combination therapy. More so, the thrice-weekly injection therapy required with conventional interferon poses a risk of low-patient compliance rates (1–3, 28), with the risk further exacerbated by the addition of a second agent. The weekly administration of pegylated interferon monotherapy is likely to alleviate this concern, while carrying similar economic costs (29). Consequently, we conclude that pegylated interferon monotherapy is likely to be the treatment of choice for HBeAg-positive chronic hepatitis B, with this conclusion being supported by others (27). That said, when conventional interferon therapy is considered, particularly in highly compliant patients, its combination with lamivudine should be entertained.
Similarly, studies examining the HBeAg-negative hepatitis B population did not find the addition of lamivudine to pegylated (30), or conventional (31), interferon to be advantageous. In addition, while the superiority of the combination over lamivudine monotherapy was suggested in previous studies, this effect is likely to represent interferon's greater inherent efficacy as compared with lamivudine, rather than the enhanced properties of the combination, as demonstrated in those very studies (22, 30).
Our study contains several limitations. Firstly, our use of intention to treat analysis, the methodological heterogeneity of the included studies, and the heterogeneity of their treatment and follow-up protocols, may have introduced some inaccuracies in our analysis. Notably, while the PEG group comprised large, carefully planned, well-executed studies, the CON group involved smaller, lower quality ones, thus weakening our conclusions in its regard. Secondly, the absence of adequate controls precluded the authors from studying the subsets of treatment-naïve and previously treated populations. Those concerns, however, were alleviated by the low patient lost for follow-up rates, the lack of statistically significant heterogeneity across studies, the beneficial effects of the combination with conventional interferon across the measured indicators and the agreement between our conclusions and those of other studies.
While the focus of our study was lamivudine and interferon combination therapy, a plethora of other combinations have been explored as well. Among those studied were combinations of interferon and various antiviral agents (32, 33), interleukin-12 (34) and prednisone (35). All yielded disappointing results. Additionally, studies investigating various antiviral combinations resulted in conflicting outcomes (36–40). Those results indicate the need for further study, as the goal of a safe and efficacious therapy is yet to be attained.
Conclusion
Pegylated interferon-α monotherapy is the treatment of choice for HBeAg-positive chronic hepatitis B, with no added benefit with lamivudine addition. However, when conventional interferon therapy is considered, its combination with lamivudine should be entertained.

interferon
lamivudine
hepatitis b
meta-analysis
therapy
combination