Double-antibody sandwich ELISA using biotinylated antibodies for the detection of Echinococcus granulosus coproantigens in dogs

Here we present the diagnostic evaluation of an improved double-antibody sandwich ELISA for detecting Echinococcus granulosus antigens in dog faecal samples (coproantigens). A purified rabbit IgG fraction against protoscolex excretory-secretory products was used as primary antibody, and the same fraction conjugated with biotin as secondary antibody. In order to validate the sandwich ELISA, intra- and inter-assay precision, linearity, and recovery percentages were calculated. The diagnostic evaluation of the method was carried out by investigating faecal samples from 37 dogs naturally infected with E. granulosus, 15 Echinococcus-free dogs infected with Taenia spp., 82 dogs with non-taeniid helminths and 66 dogs free of helminth infections. An overall sensitivity of 78.4% and specificity of 93.3% were determined. Positive and negative predictive values were 72 and 95%, respectively, and the diagnostic efficiency was 90.5%. In addition, the sandwich ELISA detection limit was estimated in 5.12 ng ml(-1). These results are highly satisfactory, allowing the use of this methodology in surveillance and control programs for intestinal echinococcosis in dogs.     

An update on immunodiagnosis of cystic echinococcosis

Immunological parameters are increasingly investigated as possible markers for the development of cystic echinococcosis. Among the newer immunologic tests for assessing the host-parasite relationship, assay of immunoglobulin isotypes with the use of distinct parasite antigens and detection of Th1/Th2 cytokine expression are an interesting new approach. The findings upon which we have constructed our immunological hypothesis of the host-parasite relationship are: (1) immunoglobulin isotype profiles differ in patients with distinct clinical outcomes of the disease; in particular, antigen B is the antigen of choice to detect specific IgG4, which is the immunoglobulin isotype most clearly associated with the progression of the disease; (2) the isolation and characterisation of recombinant parasite proteins that behave as molecular markers of allergic reactions associated with cystic echinococcosis; (3) Th1/Th2 cell activation is involved in the clinical outcome of Echinococcus granulosus infection and, in particular Th2 response, is associated with susceptibility to the disease, whereas a Th1 response is associated with protective immunity.     

Augmented bioavailability and cysticidal activity of albendazole reformulated in soybean emulsion in mice infected with Echinococcus granulosus or Echinococcus multilocularis

The anthelminthic drug, albendazole (Abz), was reformulated in a soybean oil emulsion and evaluated as a therapeutic agent for the treatment of Echinococcus granulosus and Echinococcus multilocularis in mice. Abz emulsified with 30% soybean oil (AbzE-30) resulted in higher circulating plasma concentrations of the major bioactive Abz metabolite, Abz sulfoxide (AbzSOX), after oral administration, compared with an Abz suspension. The soybean oil-emulsified Abz compound was also noted to penetrate into the hydatid cyst wall and produced higher hydatid cyst concentrations of AbzSOX. The emulsion was superior to Abz suspension in reducing the size of hydatid cysts caused by E. granulosus protoscolices collected from naturally infected sheep in Urumchi, Xinjiang Uygar Autonomous Region. In contrast, the reformulated compound's ability to reduce E. multilocularis cyst masses was only marginally superior to Abz suspension. AbzE-30 exhibited increased bioavailability and bioactivity in the treatment of murine Echinococcus hydatid cyst infections. The compound has the potential for improving therapeutic outcomes for human echinococcosis.     

Morphological and genetic characterization of Echinococcus granulosus in the Slovak Republic

The present study was undertaken to characterize more thoroughly aetiological agent of cystic echinococcosis that still represents a health problem in the Slovak Republic. Sequencing of the mitochondrial ND1 gene, Random Amplified Polymorphic DNA (RAPD) analysis and isoenzyme analyses were used to characterize genetically protoscolices of 37 isolates from pigs and cattle and two isolates from humans collected in different regions of the Slovak Republic. Solely the G7 genotype (pig strain) was identified by ND1 sequences in all 14 pigs isolate examined by this method. This genotype was also found in two human patients and in two cattle. The exclusive finding of the G7 variant has implications for the implementation of control strategies given its unique developmental and biological properties. Despite striking morphological variability, a low degree of the intra-strain variability was found in the Slovak Echinococcus granulosus isolates with the genetic methods used. This was particularly associated with the shape and size of hooks, number of testes and shape of ovaries and uterus. GPI and MDH enzymes were found suitable for the discrimination of G7 and G1 (=cosmopolitan sheep strain) variant.     

Past to present: echinococcosis in Turkey

Echinococcosis is a zoonotic infection caused by Echinococcus spp. and is one of the most important helminthic diseases worldwide. Two forms of echinococcosis occur in Turkey, i.e. Echinococcus granulosus and E. multilocularis. The life cycle of E. granulosus is predominantly in dogs and sheep, and most sheep farmers in Turkey keep a dog or two. Stray dogs are numerous and prevention or treatment of infection in these dogs is very difficult. Cystic echinococcosis (CE) occurs throughout Turkey whereas alveolar echinococcosis (AE) predominantly occurs in the eastern Anatolian region of the country. Both CE and AE are known to be endemic but few surveys have been performed. Most data on human CE and AE have been collected from hospital records. The first reference of echinococcosis, "Kyste hydatique multiloculaire", in Turkey dates as far back to 1872 by an Ottoman Physician, C.R. Katibian. The results of the first Turkish study on E. granulosus were published in 1928. According to Ministry of Health records, 21303 patients had operations to treat or confirm CE in the period 1987-1994 which corresponds to approximately 2663 patients per year. The estimated surgical case rate of CE is 0.87-6.6 per 100000 in Turkey. The prevalence of E. granulosus infection in dogs in Turkey is between 0.32 and 40% and varies widely with geographical location. The reported prevalence of CE in domestic animals in Turkey has ranged from 11.2 to 50.7% and has varied widely with geographical location. Although no detailed information has been published on AE in domestic and wild animals in Turkey, the main definitive and intermediate hosts of E. multilocularis are assumed to be rodents and red foxes, respectively. However, there has been only a single published report of E. multilocularis in a wild animal (fox) in the northwest in 1965 by Merdivenci. The first human case of AE in Turkey was reported by Mutlu in 1939, and total cumulative reported case number is 202 between 1980 and 1998.     

Genetic identification and host range of the Spanish strains of Echinococcus granulosus

Three strains of Echinococcus granulosus have been previously identified in Spain (namely 'sheep', 'horse' and 'pig'), but these Spanish strains have not been properly genotyped yet. The aim of the present research was to identify the genotype to which they correspond to. Cyst isolates were obtained from different host species, and the strain to which each belonged was established by analysis of its random amplified polymorphic DNA (RAPD) banding patterns. These results were compared to those obtained with the analysis of two mitochondrial fragment sequences (cytochrome oxidase 1 (CO1) and NADH dehydrogenase 1 (ND1)) from each isolate. The Spanish 'sheep' strain corresponded with the genotype 1 (G1) of the parasite, infecting Spanish sheep, cattle, goat, pig, wild boar and human; the Spanish 'horse' strain corresponded with the genotype 4 (G4), only infecting Spanish horses; and the Spanish 'pig' strain corresponded with the genotype 7 (G7), infecting Spanish goat, pig and wild boar. Goat, pig and wild boar can be infected by two genotypes, G1 and G7. This circumstance, and especially the possibility of sylvatic intermediate hosts serving as reservoirs of the G1 genotype of the parasite, must be taken into consideration by authorities in order to develop and evaluate effective anti-hydatidosis programmes.     

A hundred years of controversy about the taxonomic status of Echinococcus species

The parasitic diseases which we know today as cystic and alveolar echinococcosis are zoonoses known since antique times, and 1855, respectively. Whether the two clinically and morphologically distinct diseases were caused, according to a “unicistic” and a “dualistic” theory, by only one or two different cestode species was the subject of a fierce, 100 years long debate involving scientists from many countries. The natural life cycle of Echinococcus granulosus was fully clarified in 1855 after successful animal experiments. In contrast, the natural final and intermediate hosts of Echinococcus multilocularis remained unknown, and the advocates of either theory had to draw on a number of surrogate arguments to defend their positions. The seesaw of reasoning and mutual defeats of the two theories, and the final recognition of E. multilocularis as an independent species in the 1950s are described in this article.     

Worldwide epidemiology of liver hydatidosis including the Mediterranean area

The worldwide incidence and prevalence of cystic echinococcosis have fallen dramatically over the past several decades. Nonetheless, infection with Echinococcus granulosus (E. granulosus) remains a major public health issue in several countries and regions, even in places where it was previously at low levels, as a result of a reduction of control programmes due to economic problems and lack of resources. Geographic distribution differs by country and region depending on the presence in that country of large numbers of nomadic or semi-nomadic sheep and goat flocks that represent the intermediate host of the parasite, and their close contact with the final host, the dog, which mostly provides the transmission of infection to humans. The greatest prevalence of cystic echinococcosis in human and animal hosts is found in countries of the temperate zones, including several parts of Eurasia (the Mediterranean regions, southern and central parts of Russia, central Asia, China), Australia, some parts of America (especially South America) and north and east Africa. Echinococcosis is currently considered an endemic zoonotic disease in the Mediterranean region. The most frequent strain associated with human cystic echinococcosis appears to be the common sheep strain (G1). This strain appears to be widely distributed in all continents. The purpose of this review is to examine the distribution of E. granulosus and the epidemiology of a re-emerging disease such as cystic echinococcosis.     

International classification of ultrasound images in cystic echinococcosis for application in clinical and field epidemiological settings

The increased knowledge of the natural history of cystic echinococcosis (CE) by the recent expansion in the use of ultrasound (US) in field and clinical studies has necessitated the development of a new WHO standardised classification of US images. Use of such a classification will enable clinicians to examine recommended clinical procedures for the different cyst types. It will also allow scientists to compare data on the occurrence of cyst types in different parts of the world providing evidence to examine strain differences in the causative organism Echinococcus granulosus. The classification proposed follows that of the first classification developed by Gharbi et al. [Radiology 139 (1981) 459] which has been widely used, but in modified forms, since its publication. The classification presented here is intended for use in field epidemiological studies as well as for clinical investigators. The classification is intended to follow the natural history of CE and starts with undifferentiated simple cysts, as presumably hydatid cysts evolve from these structures. These simple cysts, however, may be due to a number of different aetiologies (parasitic lesions, congenital disorders, biliary cysts or neoplasms) and, therefore, require further diagnostic tests to reveal their identity. As their origin is uncertain they are not given the designation of a CE type lesion, and, in the proposed classification, should be recorded as cystic lesions (CL). The first clinical group starts with cyst types CE 1 and 2 and such cysts are active, usually fertile cysts containing viable protoscoleces. CE Type 3 are cysts entering a transitional stage where the integrity of the cyst has been compromised either by the host or by chemotherapy and this transitional stage is assigned to the second clinical group. The third clinical group comprises CE Types 4 and 5 which are inactive cysts which have normally lost their fertility and are degenerative. The use of the standardised US classification will facilitate the application of uniform standards and principles of treatment currently recommended for each cyst type.     

Could thymol have effectiveness on scolices and germinal layer of hydatid cysts?

Scolicidal solutions remain indispensable in the treatment of hydatid cyst disease. Properties of an ideal solution would be inexpensiveness and the promotion of a rapid and complete scolicidal effect with an absence of local and systemic side effects. From this point of view, no ideal solution and agents have been described yet. The aim of the present work was to determine the in vitro effect of high concentrations of thymol against protoscoleces, microcyst and cyst of Echinococcus granulosus and to evaluate its possible role as a scolicidal agent during surgery or PAIR. After short exposure times, a rapid effect was observed depending on the parasitic material. After 2 min of exposure to thymol, viability of protoscoleces was approximately 1.3% at a concentration of 250 μg/ml. The protoscolicidal effect is dose and time dependent. The results of the in vitro treatment with thymol were similar in both microcysts and secondary murine cysts. The employment of SEM and TEM allowed us to examine, at an ultrastructural level, the effects induced by thymol on E. granulosus protoscoleces, microcysts and murine cysts. In conclusion, the data obtained clearly demonstrated that thymol caused severe damages to the parasite even after short incubation times. This fact and the lack of toxicity at the evaluated concentrations, allow us to propose it as a possible scolicidal agent during hydatid cysts surgery and/or PAIR.     

A discrepancy between cystic echinococcosis confirmed by ultrasound and seropositivity in Turkish children

In three randomly selected villages of Manisa, Turkey, 630 primary school children were examined for cystic echinococcosis (CE) by a portable ultrasound scanner (US) and chest microfilm. Sera samples of 483 (76.7%) children were tested for anti-E. granulosus antibodies by ELISA and indirect hemagglutination (IHA) tests. Hepatic CE were detected in two cases (0.3%) by US, while 43 (8.9%) and 49 (10.1%) cases were found to be positive for CE by ELISA and IHA, respectively. The high seropositivity levels could have been attributed to extra-abdominal or abortive Echinococcus infections, but probably most of them were false-positives. Due to the discrepancy in results, US should be selected as the primary test in field studies and serologic tests should be performed in all cases with suspected lesions. We could not find any reported community based study on CE in Turkey, in which US was applied; but our results suggest that extensive epidemiological studies are required.     

Development and sexual maturation of Echinococcus granulosus adult worms in the alternative definitive host, Mongolian gerbil (Meriones unguiculatus)

In the present study for the first time an alternative definitive host model has been established for the complete development of Echinococcus granulosus in Meriones unguiculatus (Mongolian gerbils) up to shelled eggs production, as happened in the natural host. Animals treated with methylprednisolone acetate were orally inoculated with approximately 3000 protoscoleces and the development of parasites in the small intestine observed up to 46 days post-infection (p.i.). The cestodes survive, strobilate, mature sexually and complete their development in the intestine up to production of shelled embryonated eggs containing fully formed six-hooked oncospheres, similarly to the natural canid host (stage 7 by Smyth). Shelled eggs are first observed in the rodent/E. granulosus model at 44 days p.i. Somatic growth in gerbils appears to follow a similar trend to dogs, length increasing to over 3mm at 44 days p.i., while, compared to the natural host, organogenesis is observed only to progress at a slower rate in the early developmental stages. Parasite burden was found to vary among non-inbred animals and developmental stages of worms recovered from the same animal differed widely. Worm localisation follows the pattern observed in the natural host, the parasites appearing for the most part distributed in the proximal third of the small intestine. The development of alternative definitive hosts may facilitate research in this field, overcoming legislative impediments that restrict the use of dogs for experimental purposes.     

Molecular genetic characterization of different isolates of Echinococcus granulosus in east and southeast regions of Turkey

We used PCR-RFLP analysis of ribosomal ITS1 fragment using four different restriction enzymes and DNA sequencing of mitochondrial CO1 gene to investigate the genetic characteristics of isolates of Echinococcus granulosus obtained from different hosts (179 sheep, 19 cattle, 7 goat, 1 camel, 1 dog and 1 human) and regions (Elazig, Malatya, Erzurum, Van, Diyarbakir and Sanliurfa) of Turkey. The report represents the most comprehensive genotypic investigation of E. granulosus isolates undertaken in Turkey, with Turkish samples of cattle, goat, camel and dog origin being characterized for the first time. We show that the predominant genotype involved in E. granulosus transmission in Turkey is the common sheep strain (G1 genotype) infecting humans, cattle, sheep, goats, camels as well as the dog definitive host. Nevertheless, we urge that coordinators of local control programs in Turkey should take into consideration the potential occurrence and risk of additional strains of E. granulosus infecting humans and animal hosts, and plan accordingly.     

Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and cross-reactivities in human infections and immunized animals

Helminth antigens were investigated in the search for accessible heterologous antigens capable to discriminate different helminthiases, by the enzyme linked immunosorbent assay (ELISA) and the immunoblot assay (IB). Antigens used were: Taenia solium cysticercus total saline (Tso); Taenia crassiceps cysticercus vesicular fluid (Tcra-VF); T. crassiceps cysticercus glycoproteins (Tcra-GP and Tcra-(18-14)-GP); Toxocara canis larva excretory-secretory (TES); Schistosoma mansoni adult total saline (Sm) and Echinococcus granulosus hydatid fluid (Eg). The assayed sera were from patients with: cysticercosis (n = 18); toxocariasis (n = 40); schistosomiasis (n = 19) and hydatidosis (n = 50) with proven clinical and laboratory diagnosis, and sera from rabbits immunized with Tso, Tcra-VF, TES and Eg. Cross-reactivity occurred mostly between infections caused by Taenia and Echinococcus or in immunized rabbits, by ELISA. Moreover, the cross-reactivity among helminthiases was found with the use of antigens belonging to phylogenetically related parasite species, Eg, Tso and Tcra-VF, by sharing same antigenic components. Lower cross-reactivities were obtained by IB technique, when only peptides were considered as antigens, and the use of T. crassiceps purified glycoproteins demonstrated high sensitivity and specificity in the diagnosis of human cysticercosis, similarly to that using homologous antigen (Tso) by the same technique.     

细粒棘球绦虫抗原B重组蛋白的免疫反应性

目的 利用基因工程方法表达细粒棘球绦虫抗原B重组蛋白（rAgB）,并分析其免疫反应性。 方法 将rAgB基因片段插入原核表达载体pET41a（+）中,转化大肠埃希菌BL21（DE3）菌株,经异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达,获得重组蛋白rAgB-GST。用谷胱甘肽琼脂糖树脂亲和层析柱（GST-sepharose 4B）纯化,经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析重组蛋白的表达情况。用蛋白质印迹（Western blotting）分析其免疫反应性,并用免疫胶体金包虫诊断试剂盒作为对照。 结果 PCR、双酶切及DNA测序结果均显示重组质粒pET41a-rAgB构建成功。SDS-PAGE结果表明,重组蛋白rAgB-GST的相对分子质量（Mr）为40 800,纯化蛋白含量为78.4%。Western blotting分析结果显示,重组蛋白rAgB-GST检测细粒棘球蚴病和多房棘球蚴病患者血清的阳性率分别为79.2%（95/120）和51.1%（23/45）,但与卫氏并殖吸虫病患者、华支睾吸虫病患者血清以及健康人血清反应均为阴性。rAgB-GST的敏感性和特异性分别为79.2％（95/120）和81.0%（98/121）,均略高于免疫胶体金棘球蚴病诊断试剂盒的敏感性(72.8%,75/103）和特异性（76.9%,30/39）。 结论 重组rAgB-GST蛋白可被细粒棘球蚴病和多房棘球蚴病患者血清识别,有较好的免疫反应性。     

囊型包虫病流行区犬只细粒棘球绦虫感染季节性规律研究

目的了解犬只感染细粒棘球绦虫(Eg)的时间规律,探索更加易行的犬驱虫策略。方法采取整群抽样的方法,在马尔康和松潘各抽取了1个乡,初始纳入398只犬,使用吡喹酮进行每3个月1次的驱虫,ELISA检测驱虫前犬粪感染情况。结果在3月和6月连续两次驱虫后,9月两县犬只感染水平已分别降至1.25%和1.18%,下降率分别为94.21%和91.23%。但在12月,犬只感染率大幅却上升。犬只感染主要发生在9月至次年3月。原因可能是初冬(11、12月)是屠宰季节,而初春时寒冷及草料缺乏导致牲畜大量死亡,犬进食较多感染性内脏,从而导致感染机会大幅增加。结论马尔康县初冬、初春为主要感染季节,松潘主要感染季节为冬季;应根据实际感染季节有针对性地优化防治策略。     

In vitro effect of 5-fluorouracil and paclitaxel on Echinococcus granulosus larvae and cells

Human cystic echinococcosis is a zoonosis caused by the metacestode stage of the tapeworm Echinococcus granulosus. Although benzimidazole compounds such as albendazole and mebendazole have been the cornerstone of chemotherapy for the disease, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of 5-fluorouracil (5-FU) and paclitaxel (PTX) against E. granulosus germinal cells, protoscoleces and cysts. 5-FU or PTX inhibited the growth of E. granulosus cells in a time dependent manner. Although both treatments had a protoscolicidal effect, 5-FU had a considerably stronger effect than PTX. 5-FU produced a dose- and time-dependent effect, provoking the complete loss of viability after 24 days of incubation. Moreover, cysts did not develop following the inoculation of treated protoscoleces into mice. The loss of viability was slower in PTX treated protoscoleces, reaching to approximately 60% after 30 days. The results of the in vitro treatment with 5-FU and PTX were similar in secondary murine cysts. The employment of SEM and TEM allowed us to examine, at an ultrastructural level, the effects induced by 5-FU and PTX on E. granulosus germinal cells, protoscoleces and murine cysts. In conclusion, the data obtained clearly demonstrated that 5-FU and PTX at clinically achievable concentrations inhibit the survival of larval cells, protoscoleces and metacestodes. In vivo studies to test the antiparasitic activities of 5-FU and PTX are currently being undertaken on the murine model of cystic echinococcosis.     

Human cystic echinococcosis in South Africa

Cystic echinococcosis is recognised as causing considerable morbidity and even mortality in South Africa, but the epidemiology of the disease is to date unknown. From current evidence there are also concerns that co-infections with HIV and tuberculosis (TB) considerably increase CE-associated morbidity. The aim of this analysis was to characterise clinical features and disease burden of CE in South Africa in preparation for a prospective study.Retrospective case note analysis of patients presenting to two large academic hospitals in Johannesburg, South Africa, for clinical and demographic data was performed. In addition, data of the National Health Laboratory Service were accessed for requested serological and microscopic investigations for CE and the numbers of positive results evaluated.According to a recently published definition, 14 cases of confirmed CE and 9 cases of probable CE were identified at both hospitals. When accessing the national database it became apparent that even with the most conservative estimate at least 137 patients per year present with CE in South Africa. However, numbers are likely to be much higher for a variety of reasons. Further prospective analysis is necessary to shed more light on the epidemiology, clinical presentation and risk factors for CE, which is currently underway.     

The use of mathematical models to simulate control options for echinococcosis

In many parts of the world Echinococcus granulosus is a widespread infection in sheep and dogs with a consequential spill over into the human population. In the past, mathematical models have been derived to define the transmission dynamics of this parasite, principally in the sheep-dog life cycle. These models have characterized the cycles of infection as lacking in density dependent constraints in both the definitive or intermediate hosts. This suggested that there was little, if any, induced host immunity by the parasite in either host in natural infections. However, recent evidence from both Tunisia and Kazakhstan, where young dogs are the most heavily parasitised, suggests the possibility of significant definitive host immunity. This may have an effect on the control effort needed to destabilize the parasite. A preliminary computer simulation model (based on an Excel spreadsheet) to attempt to predict the results of a control programme has been written. This demonstrates that there could be significantly different results if there is indeed protective immunity in the dog than in the absence of immunity. In the former the parasite needs a greater control effort to push the parasite towards extinction than in the latter. The computer simulation is based on a mathematical model of the parasite's life cycle and is flexible so that different values of parameters can be used in different situations where the transmission of the parasite may be at different levels. Because of the flexibility of the computer simulation it is anticipated that this programme can be applied in most situations, although initial parameters for a particular location or strain of the parasite will have to be first predetermined with base line field surveys and possibly experimental infections. The programme also has an additional flexibility to enable simulations if some parameters cannot be accurately estimated through Monte-Carlo techniques. In the latter situation, worst and best case scenarios can be estimated and likely frequency distributions of the unknown parameters can be included in the model.     

Cystic echinococcosis in Argentina: evolution of metacestode and clinical expression in various Echinococcus granulosus strains

Echinococcus granulosus hydatid cysts were examined in 41 patients from Neuquén and Tucumán provinces in Argentina. Sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) revealed in 19 patients common sheep strain (G1), in 6 patients Tasmania sheep strain (G2), in 1 patient cattle strain (G5), and in 15 patients camel strain (G6). In Argentina the only known is the domestic cycle that affects dogs and herbivorous, including ovine, swine, cattle and goats. These strains produced a total of 58.6% of primary liver infections, 29.2% primary in lung, 2.4% primary in spleen and 9.8% were multiorgan abdominal infections. The metacestode was classified using the evolutive stages proposed by WHO-IWGE (from CE1 to CE5). We estimated that CE1 cyst has a duration of about 22 years, CE2 of 14 years, CE3 of 10 years, CE4 of 19 years and CE5 was not determined. The active types CE1 and CE2 reached 75% of all cases from all strains. In 36 patients with cysts from G1, G5 and G6 strain, there were only two asymptomatic cases. The strains of the E. granulosus complex do not present important clinical differences; only G6 seems to have higher growth rate.