Development of Em18-immunoblot and Em18-ELISA for specific diagnosis of alveolar echinococcosis

Extensive experience has documented that Em2(plus)-ELISA, Em10-ELISA and Em18-immunoblot and Em18-ELISA are reliable serologic methods for detection of alveolar echinococcosis (AE) caused by the metacestodes of Echinococcus multilocularis. Among these, tests based on detection of antibodies to the specific Em18 antigen, either immunoblot or ELISA, appears to be the most specific for AE. Between 90 and 97% of AE cases with characteristic hepatic lesions detectable by image analysis have been positive in Em18-serology. In contrast Antigen B (8 kDa)-immunoblot is the most sensitive for all forms of echinococcosis, although it can not differentiate AE from cystic echinococcosis (CE). Primary serologic screening for echinococcosis, especially for CE using hydatid cyst fluid of Echinococcus granulosus appears to be highly sensitive in endemic areas. Glycoproteins (GPs) purified from cyst fluid of Taenia solium are highly specific for diagnosis of T. solium neuorcysticercosis (NCC). Using currently available antigens it is not difficult to differentiate these three larval cestodiases serologically. We recommend that (1) primary screening of CE in endemic areas should be carried out using hydatid cyst fluid of E. granulosus prepared from cysts in either sheep, human or mouse for immunoblot and from sheep or mouse for ELISA, (2) both primary screening and confirmation of AE in endemic areas should be carried out using Em18-ELISA, Em18-immunoblot or Em2(plus)-ELISA. Serodiagnosis in areas where both AE and CE are endemic, such as in China, should be carried out as a combination of (1) and (2), and (3) serology of NCC should be carried out using GP-ELISA or GP-immunoblot. All samples showing antibody to Em18 are exclusively from echinococcosis cases. There have been no false positive test reactions with sera from other diseases. Strongest Em18 responders are all from patients with AE but some weaker responses may be found in sera of persons with advanced complex lesions of CE. These highly reliable serodiagnostic methods using native, recombinant and synthetic antigens are briefly summarized and experiences with these methods in Japan is reviewed. We believe that use of these specific antigens in screening and confirmation programs for AE in Japan will improve specificity and reduce the confusion, anxiety and expense in persons whose sera give false positive reactions with crude echinococcal antigens.     

Proposal of an ultrasonographic classification for hepatic alveolar echinococcosis: Echinococcosis multilocularis Ulm classification-ultrasound

AIM: To establish an ultrasonographic classification based on a large sample of patients with confirmed hepatic alveolar echinococcosis(AE).METHODS: Clinical data and ultrasonography(US) findings of 185 patients(100 males; 85 females; mean age at diagnosis: 51.4 ± 17.6 years; mean age at time of US examination: 58.7 ± 18.2 years) were retrospectively reviewed with respect to the US morphology of hepatic AE lesions. The sonomorphological findings were grouped according to a five-part classification scheme.RESULTS: Application of the new classification resulted in the following distribution of sonomorphological patterns among the patients examined: hailstorm(54.1%); pseudocystic(13.5%); ossification(13.0%); hemangioma-like(8.1%); and metastasis-like(6.5%). Only 4.9% of lesions could not be assigned to a sonomorphological pattern.CONCLUSION: The sonomorphological classification proposed in the present study facilitates the diagnosis,interpretation and comparison of hepatic alveolar echinococcosis in routine practice and in the context of scientific studies.     

白黎芦醇体外对多房棘球蚴原头节和微囊的作用研究

目的 研究白藜芦醇(Resveratrol,RES)体外对多房棘球蚴原头节(Protoscoleces,PSCs)和微囊的作用效果,为多房棘球蚴病的临床用药提供新的依据.方法 PSCs体外经不同浓度(5、10、20、40、80、100、200和400 μmol/L)的RES作用7 d,采用台盼蓝染色观察其活力与形态变化...     

Review: Molecular tools for the diagnosis of cystic and alveolar echinococcosis

In the past years, the diagnostic tools applied to identify alveolar (AE) and cystic echinococcosis (CE) in human patients have not only increased in number but also substantially improved in quality. The identification and characterization of species-specific parasite proteins/antigens allowed to generate subsequently recombinant or synthetic polypeptide antigens, as well as corresponding monoclonal antibodies. Some of these new tools have already demonstrated operating characteristics superior to conventional tests used for the immunodiagnosis of CE and AE, and thus may be suggested for routine laboratory application. Powerful molecular techniques, such as the polymerase chain reaction (PCR), have been developed and adapted to advance laboratory diagnosis of AE and CE. Detecting minute amounts of parasite DNA and mRNA, not only to identify but also to characterize the biological status of parasite material, thus becomes a complementary method to synergize immunodiagnostic techniques. This review focuses on recent developments of molecular tools, discussing their potential use as a primary or a supporting diagnostic element. We also outline some future developments to be undertaken in the field of molecular diagnosis, linked to clinical and laboratory problems.     

泡球蚴组织的体外培养及在药物筛选中的应用

目的 建立泡球蚴组织的体外培养模型，对4种药物的体外抗泡球蚴疗效进行初步筛选。方法 泡球蚴组织体外培养9周后，分为7组：阿苯达唑组、汉防己甲素组、己酮可可碱组、苦豆碱组、氧氟沙星组、未用药组和二甲基亚砜组。在加药24h和18d后，分别取样固定并在电子显微镜下观察超微结构。实验期间对囊泡计数。实验结束后，将培养物接种BALB／c小鼠进行活力测试。结果 汉防己甲素和己酮可可碱在体外能抑制泡球蚴的生长（P〈0．005），氧氟沙星对泡球蚴没有作用（P〉0．005），苦豆碱能促进泡球蚴生长。接种各药物组培养物的BALB／c小鼠饲养80d后处死并解剖，发现所有小鼠的腹腔中均有泡球蚴生长。结论 汉防己甲素和己酮可可碱在体外对泡球蚴有一定的抑制作用，氧氟沙星和苦豆碱对寄生虫无治疗作用。活力测试提示所选药物只能抑制寄生虫生长而无杀灭作用。     

In pursuit of the abnormal serum alkaline phosphatase

The serum alkaline phosphatase (ALP) is often included among the tests used for case-finding among ambulatory patients. To determine the positive predictive value of the ALP, test results for all adults screened by a health maintenance organization between March and December 1969 were obtained by computer. The authors reviewed the charts of all 661 patients with abnormal tests whose primary source of medical care was at this facility. Complete two-year follow-up data were available for 91% of these patients. There were 56 patients (9%) with a diagnosis that could have explained an abnormal ALP. Of those cases in which ALP would have been clinically useful all but one could have been diagnosed by a simple, noninvasive work-up, and in that one case, no management change would have occurred. The authors conclude that in the absence of a small number of specific indications, extensive testing need not be performed to evaluate an isolated abnormal ALP obtained from a screening examination. Received from the Department of Medicine and the School of Public Health, University of California, Los Angeles, Los Angeles, California. Supported in part by the Robert Wood Johnson Clinical Scholars Program. The views expressed herein do not necessarily represent those of the Robert Wood Johnson Foundation.     

多房棘球蚴病新型诊断标志物研究进展

影像学与血清学技术在多房棘球蚴病诊断、治疗与评价中扮演着重要角色,但其在临床应用中的一些不足促使研究者对潜在诊断标志物进行探索。基于血清学、基因组学及蛋白质组学的新型诊断标志物单独或联合应用有望提高多房棘球蚴病早期诊断的灵敏度与特异度,在后续病程监测与预后评估中发挥重要作用。本文主要就多房棘球蚴病新型诊断标志物研究进展作一综述。     

Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans

The earlier recommendations of the WHO-Informal Working Group on Echinococcosis (WHO-IWGE) for the treatment of human echinococcosis have had considerable impact in different settings worldwide, but the last major revision was published more than 10 years ago. Advances in classification and treatment of echinococcosis prompted experts from different continents to review the current literature, discuss recent achievements and provide a consensus on diagnosis, treatment and follow-up. Among the recognized species, two are of medical importance - Echinococcus granulosus and Echinococcus multilocularis - causing cystic echinococcosis (CE) and alveolar echinococcosis (AE), respectively.For CE, consensus has been obtained on an image-based, stage-specific approach, which is helpful for choosing one of the following options: (1) percutaneous treatment, (2) surgery, (3) anti-infective drug treatment or (4) watch and wait. Clinical decision-making depends also on setting-specific aspects. The usage of an imaging-based classification system is highly recommended.For AE, early diagnosis and radical (tumour-like) surgery followed by anti-infective prophylaxis with albendazole remains one of the key elements. However, most patients with AE are diagnosed at a later stage, when radical surgery (distance of larval to liver tissue of >2 cm) cannot be achieved. The backbone of AE treatment remains the continuous medical treatment with albendazole, and if necessary, individualized interventional measures. With this approach, the prognosis can be improved for the majority of patients with AE.The consensus of experts under the aegis of the WHO-IWGE will help promote studies that provide missing evidence to be included in the next update.     

Flow cytometry of leucocyte alkaline phosphatase in normal and pathologic leucocytes

Leucocyte alkaline phosphatase (LAP) is an enzyme expressed on the external aspect of the neutrophilic granulocyte plasma membrane, and represents a specific marker for the fully differentiated granulocyte. In this report we characterize 1B12.1, a monoclonal antibody raised against human bone alkaline phosphatase, by its ability to recognize the LAP protein. As assessed by Western blot analysis, following electrophoresis under non-reducing conditions, the antibody specifically reacts with LAP upon forced expression of the protein in simian COS-7 fibroblasts. In addition, the 1B12.1 antibody recognizes partially purified LAP isolated from peripheral blood granulocytes. With this antibody we developed a quantitative flow-cytometry-based method for the determination of LAP. Double fluorescence flow cytometry demonstrated that the LAP protein was present in relatively high amounts in neutrophilic granulocytes, but not in monocytes, natural killer cells, or B and T lymphocytes of normal individuals. The protein was completely absent in granulocytes obtained from chronic myeloid leukaemia and paroxysmal nocturnal haemoglobinuria patients. Higher than normal levels of LAP protein were evident in neutrophilic granulocytes of patients suffering from polycythaemia vera, essential thrombocythaemia and severe aplastic anaemia. However, the highest amounts of LAP protein were present in the granulocytes of normal individuals treated with G-CSF for the isolation of peripheral blood stem cells.     

小鼠肝细粒棘球蚴和多房棘球蚴病不同时期病灶周围组织纤维化对比

目的 本实验通过研究对比不同时间两种肝棘球蚴病灶周围组织纤维化情况,进一步了解肝棘球蚴病的病理生理发展过程,为肝棘球蚴病的诊治提供参考。方法 建立动物模型,使用HE,Masson染色以及COL1,COL3、α-SMA、TGF-β1免疫组化染色对比观察两种肝棘球蚴病在不同时间纤维化情况的不同。结果 随着时间的变化肝细粒棘球蚴病灶周围纤维化由弥漫到聚集,可形成连续致密的纤维外膜;肝多房棘球蚴病灶周围组织纤维化始终为弥漫性,无法形成连续质密的纤维外膜。细粒棘球蚴组病灶周围COL1(r=-0.768,P<0.05)、COL3(r=-0.781,P<0.05)、α-SMA(r=-0.867,P<0.05)、TGF-β1(r=-0.854,P<0.05)的表达强度与时间呈负相关,多房棘球蚴组病灶周围COL1(r=-0.349,P>0.05)、COL3(r=-0.037,P>0.05)、α-SMA(r=-0.107,P>0.05)、TGF-β1(r=-0.148,P>0.05)的表达强度与时间无相关性。 无相关性同时观察到两种包虫周围细胞外基质胶原含量不同,细粒棘球蚴组I、III型胶原比高于多房棘球蚴组(Z=-3.23,P<0.05)。结论 相较于多房棘球蚴,细粒棘球蚴病灶周围可产生连续致密的纤维外囊。细粒棘球蚴在外囊形成后纤维化进程减弱或停止,多房棘球蚴在整个病程中均有活跃的纤维化反应。细粒棘球蚴相较于多房棘球蚴外囊的I/III型胶原比值较高。     

骨质疏松症大鼠血液中碱性磷酸酶来源方式探讨

目的探讨骨质疏松症大鼠的血液碱性磷酸酶（ALP）的来源方式。方法 40只4个月龄雌性SD大鼠,分为两组,模型组和假手术组（对照组）,模型组进行双侧卵巢摘除,对照组只打开腹腔。实验动物颈椎脱臼处死后,取股骨,去尽肌肉与结缔组织,截取相应的骨组织浸入4%多聚甲醛中固定3 d,然后浸入4%EDTA溶液中,室温脱钙4周,每周换液1次。采用原位杂交和免疫组化法分别检测骨组织中ALP表达情况。结果对照组骨组织中的ALP蛋白及ALP mRNA表达含量均明显高于模型组,P均〈0.01。结论骨质疏松症大鼠血液中的ALP主要来源于成骨细胞破坏;骨组织ALP水平可作为其他细胞向成骨细胞分化的一个鉴定指标。     

包虫病影像诊断技术和手术治疗进展

包虫病是严重危害中国西部人群的人兽共患寄生虫病,手术仍为包虫病临床最主要的治疗手段.该文通过对囊型包虫病和泡型包虫病等两型肝包虫病的影像学诊断和各种术式回顾性分析总结后,综述如下:1)超声检查主要用在两型包虫病的诊断与鉴别诊断,尤其是在农牧区进行流行病学调查和普查时,超声以其便携低耗易行的特点成为首选.近来用超声对肝两型包虫病病灶在二维超声、彩色多普勒血流成像的基础上进行灰阶超声造影检查,超声造影可以更好地显示其病灶的确切范围,周边增值带微循环血供状态,为肝包虫病外科手术提供更多的诊断治疗信息.2)囊型包虫病CT/MRI均表现为境界清楚的囊肿,病程发展中出现子囊、囊膜剥离、囊壁及内容物钙化等特征;泡型包虫病CT/MRI均表现为非均质实性肿块,内部有小圈状和颗粒状钙化,中心常出现液化坏死,形成"地图状"外观.其类似肿瘤的浸润生长可压迫和侵蚀肝血管和胆管并伴远处血行转移.MRI水成像技术对不典型包虫病有独特的诊断效果;CT/MRI血管成像和胆系成像有利于诊断包虫并发症;CT/MRI灌注技术、MRI扩散成像和波谱成像有望实现活体无创的分子影像诊断.3)肝囊型包虫病:(1)肝包虫外囊完整剥除术是理想的治疗方法,值得推广的首选术式;(2)外囊次全切除术可有效消减残腔,避免胆瘘等,能减少手术时间及外囊完整剥除术所致手术风险;(3)肝内囊摘除术仍是主要方法,术中应经胆道或胆囊管美蓝造影以确认是否胆漏.4)肝泡型包虫病:(1)根治性肝切除是治疗首选方法;(2)姑息性病灶切除手术,因继发难治性肝切面胆瘘而宜慎重采用;(3)肝移植是治疗两型包虫病终末期最后手段,术后仍存在复发的可能,需酌情服用抗包虫药,如阿苯达唑片剂或其他剂型(乳剂、脂质体口服液等).     

Utility of lavage alkaline phosphatase in detection of isolated small intestinal injury

The ability of lavage alkaline phosphatase (LAP) to detect small intestinal injury was studied in 81 patients who underwent diagnostic peritoneal lavage following blunt and penetrating abdominal trauma. Patients with a grossly positive lavage were excluded. The LAP of five patients with small intestinal injury (79.0 +/- 41.7 IU/L) was significantly greater than in 76 cases without (1.2 +/- 0.4 IU/L; P less than .05). LAP greater than or equal to 3 was seen in six patients. Four had isolated small intestinal pathology, and one had combined small and large bowel injury. In three of these five, other lavage values were normal. One possible false positive occurred. The sensitivity of LAP greater than or equal to 3 (100%) exceeded that of usual lavage parameters in the detection of small intestinal injury. LAP may be a sensitive marker for small intestinal pathology in the immediate postinjury period.     

Associations of serum alkaline phosphatase with metabolic syndrome and mortality

Background

Recent data suggest that elevated serum alkaline phosphatase levels are associated with increased mortality, but the mechanisms for this association are unknown. As metabolic syndrome is associated with higher serum alkaline phosphatase levels, we examined the joint association of mortality with metabolic syndrome and serum alkaline phosphatase levels in the US general population.

Methods

Retrospective observational study of 15,234 adult participants in the National Health and Nutrition Examination Survey III. Multivariable Cox regression analyses were used to jointly relate mortality risk to serum alkaline phosphatase and indicators of metabolic syndrome.

Results

Prevalence of metabolic syndrome was 14% to 41% among patients in lowest and higher quartiles, respectively, for baseline serum alkaline phosphatase. The mortality hazard ratio for each doubling of serum alkaline phosphatase was 1.52 (95% confidence interval [CI], 1.35–1.72) adjusting only for demographic factors, and 1.37 (95% CI, 1.21–1.56) adjusting for both demographic and metabolic syndrome factors in the full cohort, and was 1.83 (95% CI, 1.36–2.46) adjusting for demographic factors in the subgroup without any of the component conditions of metabolic syndrome.

Conclusions

In the US general population, higher levels of serum alkaline phosphatase is a predictor of mortality independent of the baseline prevalence of metabolic syndrome. Further studies are warranted to unravel the mechanisms of this association.     

快速诊断多房棘球蚴病胶体金免疫层析试条方法的建立与评价

目的建立快速、简便诊断多房棘球蚴病的胶体金免疫层析试条方法。方法提取多房棘球蚴原头节总RNA,通过RT-PCR获得编码Em18基因片段并克隆入pGEX-3X表达载体,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达得到重组蛋白;采用柠檬酸三钠还原法制备胶体金,标记抗人IgG单克隆抗体;将重组Em18抗原包被于硝酸纤维素膜适当位置,制成检测特异抗体的胶体金免疫层析试条。用该试条检测多房棘球蚴病(56份)、细粒棘球蚴病(87份)、囊尾蚴病(30份)、日本血吸虫病(10份)和弓形虫病(10份)患者血清,以及健康人(50份)血清,以评价其检测的敏感性和特异性,同时用ELISA法进行平行检测,以评价该试条的诊断性能。结果以重组蛋白Em18为抗原胶体金免疫层析试条检测多房棘球蚴病患者血清,敏感性为92.9%(52/56)。与细粒棘球蚴病和囊尾蚴病患者血清分别存在9.2%(8/87)和3.3%(1/30)的交叉反应,与健康人血清存在8.0%(4/50)的假阳性率,与日本血吸虫病和弓形虫病患者血清则无交叉反应,特异性为93.0%(174/187)。ELISA法检测的敏感性和特异性分别为94.6%(54/56)和92%(172/187),与试条法比较两者差异均无统计学意义(P>0.05)。kappa分析结果显示,试条法与ELISA法检测多房棘球蚴病患者血清的结果高度一致(κ=0.98)。结论以重组Em18抗原建立的快速诊断胶体金免疫层析试条,检测多房棘球蚴病的敏感性和特异性均较高。     

Neutrophil alkaline phosphatase levels after induction of hypersegmented neutrophil release into blood of the rat

Summary Hypersegmentation of nuclei was induced in rat circulating neutrophils after cyclophosphamide (CY) administration and neutrophil alkaline phosphatase (NAP) activity was measured. It was shown that changes in NAP levels and segmentation of neutrophil nuclei were parallel. Relationship between age of neutrophils and NAP levels is discussed.     

Analysis of specific IgE and IgG subclass antibodies for diagnosis of Echinococcus granulosus

The potential roles of specific antibodies of different immunoglobulin G (IgG) subclasses and IgE in serological diagnosis of cystic echinococcosis (CE) were investigated by an enzyme linked immunosorbent assay (ELISA) based on Antigen 5 (Ag5). Presence of IgG1 was demonstrated in all sera from 58 patients with CE. The most discriminatory and specific antibodies found in this study belonged to IgG4 and IgE. Only one false-positive reaction was observed with IgG4 and no IgE cross-reactivity occurred with 40 sera from healthy controls. In 36 sera from patients infected with parasites other than CE two false-positive reactions with IgG4 were observed but none occurred with IgE. In immunoblotting, it was shown that IgG1 subclass was responsible for cross-reactivity of human antibodies that reacted with a 38 kDa subunit of Ag5. IgG4 and IgE antibodies could not recognize the 38 kDa subunit and under non-reducing conditions reacted with the 57 kDa subunit without any cross-reactivity to other parasites. The results demonstrated that IgG4 and IgE are the most important antibodies for serological diagnosis of hydatid cyst in an Ag5 based immunoassay system.     

实验性牙周炎大鼠牙龈组织中碱性磷酸酶水平变化及意义

目的观察碱性磷酸酶（ALP）水平变化在牙周炎发病中的作用。方法选用40只健康SD大鼠随机分为对照组和观察组各20只,观察组建立牙周炎模型,对照组常规饲养;分别于造模术后4周和8周各处死20只,采用酶标仪测定牙龈组织中ALP水平,观察牙槽骨吸收情况及牙周组织学改变。结果造模术后4周和8周时观察组牙龈组织中ALP水平均明显高于对照组（P〈0．01）,且术后8周高于4周（P〈0．01）;观察组有明显牙槽骨吸收及牙周组织炎症细胞浸润,对照组则无。结论ALP水平变化可能在牙周炎发病过程中起重要作用。