多聚酶链反应鉴别致病性与非致病性的溶组织内阿米巴虫株

多聚酶链反应是体外DNA增殖的新技术。在多聚酶、dNTP和引物的参与下在数小时内使特异性的DNA顺序大量增殖。用B、C、G、T4株溶组织内阿米巴的DNA增殖30周期后,作凝胶电泳分析,发现致病性虫株的引物不能使B、C、G、T虫株的DNA增殖,凝胶电泳不出现条带;而非致病性虫株的引物能使B、C、G、T虫株的DNA增殖,凝胶电泳出现特异性条带。用辣根过氧化物酶标记致病性虫株DNA探针与B、C、G、T的DNA进行斑点杂交呈阴性反应;而用非致病性虫株DNA探针与B、C、G、T的DNA斑点杂交呈阳性反应。实验结果显示B、C、G、T为非致病性虫株,与同工酶分析一致。多聚酶链反应是鉴别致病性和非致病性溶组织内阿米巴虫株的高特异性和敏感性方法,为从分子生物学研究溶组织内阿米巴的致病机理提供一新技术。     

A survey of Entamoeba histolytica and Entamoeba dispar (Brumpt) infections on Mahé, the Seychelles.

E. histolytica, pathogenic, and E. dispar, non-pathogenic, being morphologically identical are, when recovered as cysts in feces, diagnosed as E. histolytica. A survey on The Seychelles demonstrated that when culture and zymodeme characterization was used to compare against microscopy alone, the risk of overdiagnosis of E. histolytica infections was drastically reduced. From a total of 313 subjects tested 21 cultures grew amebas. By zymodeme analysis eight were E. histolytica, 40 were E. dispar and the remainder were various species of non-pathogenic intestinal amebas. Two further small comparative surveys confirmed these findings.     

A DNA probe specific to pathogenic Entamoeba histolytica.

A DNA sequence, IE-gen1 (3.1 kb), was isolated from the pathogenic strain of E. histolytica NIH-200. IE-gen1 was identified by the subtractive hybridization of a genomic library to a cDNA probe prepared from NIH-200 trophozoites. The IE-gen1 probe specifically detected pathogenic E. histolytica in slot blots of genomic DNA and Northern blots, but not other Entamoeba species and additional human parasites. This genomic probe could detect with complete specificity DNA from about 10(3) organisms. The IE-gen1 probe could be related to highly specialized loci in pathogenic E. histolytica, and is likely to be a valuable DNA reagent for clinical diagnosis and epidemiological investigations.     

Collagenase activity in clinical isolates of Entamoeba histolytica maintained in xenic cultures.

Several isolates of pathogenic and non-pathogenic E. histolytica from xenic cultures were tested for their capacity to digest native type I collagen gels. The results demonstrate that the pathogenic isolate HM48:IMSS has a high collagenolytic activity. The non-pathogenic isolates, HM43:IMSS, HM44:IMSS and HM46:IMSS showed significantly lower collagenolytic activity. Six groups showing different degrees of collagenase activity or non-activity were separated from the non-pathogenic isolates by a Percoll gradient. The groups obtained from the pathogenic isolate HM48:IMSS always displayed enzymatic activity.     

Diagnosis of amebiasis in Bangladesh

Diagnosis of amebiasis by microscopic identification of the parasite in stool and liver abscess pus is insensitive and unable to distinguish the invasive parasite E. histolytica from the commensal parasites such as E. dispar and E. moshkovskii. New approaches to the detection of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests for diagnosis of amebiasis have been developed and used to diagnose E. histolytica in Bangladesh. We have compared the TechLab E. histolytica-specific antigen detection test with PCR assays and with isoenzyme analysis of cultured amebas. The PCR assays are based on amplification of the multi-copy small subunit ribosomal RNA gene of E. histolytica and E. dispar. PCR assays and antigen detection test had comparable sensitivities when performed directly on fresh stool specimens. The correlation of antigen detection with PCR assays for identification of E. histolytica was excellent. TechLab's E. histolytica- specific antigen detection test was both rapid and simple to perform, making it appropriate for use in the developing world, where amebiasis is most prevalent.     

The effect of axenic versus xenic culture conditions on the total and secreted proteolytic activity of Entamoeba histolytica strains.

Proteolytic activity was measured in lysates of four axenic and five xenic cultures of different strains of pathogenic E. histolytica, and in five non-pathogenic strains ("E. dispar") growing in xenic culture. There was no significant difference in total proteolytic activity, using either gelatin or albumin as substrate, between the two sets of xenic cultures; the axenic pathogens however had significantly higher levels of activity than the xenic pathogens, the levels appearing to correlate with virulence. An axenic strain (NIH 200) reassociated with mixed bacterial flora reverted to close to xenic levels of activity. There was no evidence of secretion of an elevated proportion of the total enzymic activity by pathogenic strains, and expression levels may owe more to culture conditions than to genotype.     

Organization of repeated sequences in the region downstream to rRNA genes in the rDNA episome of Entamoeba histolytica.

The E. histolytica rDNA episome consists of a 3.7 kb HindIII fragment located downstream of the rDNA inverted repeats. We have determined the complete nucleotide sequence of this fragment and have shown that it is comprised of two families of short tandem repeats, the 170 bp DraI repeat and the 144 bp ScaI repeat. Each DraI repeat unit consists of 12-mer sequences with near complete homology to yeast consensus autonomously replicating sequence. In addition, a 21-mer subrepeat structure is also present in each unit. The sequence of the ScaI repeat is about 90% homologous with the sequence of another family of 145 bp tandem repeats, the PvuI repeats reported to be present upstream of the rRNA genes (3). Compared with most other parts of the rDNA episome, the downstream region showed frequent restriction fragment length polymorphism. This was due to changes in the number of tandem DraI repeat units. The loss of these repeats might explain how rDNA length heterogeneity observed in the clones of HM1:IMSS could have arisen. On the other hand, the number of ScaI repeat units in these clones remained unchanged. The repeat units found in the 3.7 kb HindIII fragment show superficial resemblance to equivalent regions of the intergenic spacers of higher eukaryotes. Moreover, these repeated sequences seem to be specific for the pathogenic strain of E. histolytica.     

Amebiasis. An increasing problem among homosexuals in New York City.

During a five-year period at The New York Hospital, Entamoeba histolytica was identified in the stools of 20 men who had not traveled outside the New York area. All of the patients were found subsequently to homosexual. During this same period amebiasis was diagnosed in 30 men who had traveled; only two were homosexual. Of ten patients with E histolytica infection seen during the first year of this study, none were homosexual whereas eight of 11 patients in the fifth year were homosexual, suggesting a gradual increase during this period of this disease in the homosexual community.     

Effect of hamster liver passage on the isoenzyme patterns of Entamoeba histolytica.

The effect of hamster liver passage on the isoenzyme patterns of isolates of Entamoeba histolytica was investigated. Three isolates, F, G and T were originally obtained from patients with acute amebic dysentery and another strain, C, was obtained from an asymptomatic carrier. All these strains were maintained for over two years in axenic culture. The isoenzyme pattern (zymodeme) of hexokinase (HK), phosphoglucomutase (PGM) and glucose phosphoisomerase (GPI) of these strains was found to belong to non-pathogenic group X, but the isoenzyme pattern of GPI resembled less pathogenic zymodeme XX and might be an intermediate type. Following inoculation of trophozoites into hamster livers and recovery after abscess formation, their isoenzyme pattern changed and revealed that they belonged to pathogenic type XIV. Liver passage caused an enhancement in amebic virulence as evidenced by their increased ability to destroy leukocytes. The results indicate that isoenzyme pattern is not a stable property of E. histolytica.

     

Characterization of two long vesicle-associated membrane proteins or longins genes from Entamoeba histolytica

BACKGROUND: This study characterizes two long vesicle-associated membrane protein (VAMP) genes (SYBL-1 and SYBL-2) obtained from DNA of Entamoeba histolytica by PCR amplification. (Nucleotide sequences of the Entamoeba histolytica SYBL-1 and SYBL-2 genes appear in the GeneBank under accession numbers AY256852 and AY309014.) METHODS: The two cDNA products include one of 663 bp with sequence of 220 deduced amino acids, and a second of 693 bp with 230 deduced amino acid residues. Both products possess corresponding sequences for longin domain at N-terminus, a soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor (SNARE) coiled-coil region, a transmembrane domain (TM), and an intravesicular tail C-terminal, characteristics of all long VAMPs or longins. The current study identified presence of deduced peptide bonds in SYBL-1 and -2, which indicates that these two long VAMPs from E. histolytica could be appropriate substrates for zinc endopeptidases from tetanus and botulinum neurotoxins with specific activity toward neuronal synaptobrevins. RESULTS: Alignment by Basic Local Alignment Search Tool (BLAST) of deduced amino-acid sequence of long VAMPs genes SYBL-1 and -2 showed identity of between 20 and 40% with Caenorhabditis elegans, Dictyostelium discoideum, Drosophila melanogaster, Arabidopsis thaliana, Mus musculus, Homo sapiens, and Plasmodium falciparum. CONCLUSIONS: It is possible that these two putative transport proteins are involved in endocytosis/exocytosis during a biological membrane fusion process, which may make them suitable candidates as targets for new drugs. According to published data, this is the first time that two such genes have been isolated from E. histolytica.     

Value of stool examination in patients with diarrhoea

Findings of stool examinations in 1593 patients with diarrhoea due to a single enteric pathogen--enterotoxigenic Escherichia coli rotavirus, Shigella, Campylobacter jejuni, Vibrio cholerae 0:1, Entamoeba histolytica, or Giardia lamblia--were reviewed to determine how well they predicted the agent associated with the diarrhoea. Specimens were examined visually for blood and mucus, tested for pH, and examined under a microscope for the presence of red and white blood cells, parasites, and stool fat. Although visible blood was more common in specimens from patients infected with Shigella (51%) and Ent histolytica (39%) than in those from patients infected with other agents (6%; p less than 0.01), patients infected with Shigella were most likely to have numerous faecal leucocytes (greater than 50/high power field: 39% v 8% of all patients and 7% of patients infected with Ent histolytica, p less than 0.01 in both cases). Patients infected with enterotoxigenic E coli, rotavirus, V cholerae 0:1, or C jejuni had loose stools with fewer red or white cells. Patients infected with rotavirus and C jejuni were more likely to have acid stools with 3 to 4+ fat, but these findings were related to young age and breast feeding. Stool examination is most useful in establishing a diagnosis of dysentery and in helping to distinguish between patients infected with Shigella and Ent histolytica; it is of limited usefulness in discriminating between pathogens causing watery diarrhoea.     

Cloning and partial characterization of an antigen detected on membrane surfaces of non-pathogenic strains of Entamoeba histolytica.

A novel and unique mAb (318-28) which specifically interacts with a 60 kDa antigen that is found only on the surfaces of non-pathogenic (NP) strains of E. histolytica has been recently characterized. The antigen appears to be present also on (NP) cyst forms of amebas but was not detected on any of the various (P)-strains tested. It was also not found on other Entamoeba species such as Moshkovskii, Laredo, Huff, Coli, Gingivalis, or Invadens. Clinical trails for the differentiation of (P) and (NP) amebas directly from stools using this mAb are in progress. Cloning of the gene encoding for the (NP)-specific antigen was achieved after screening with mAb 318-28 a lambda gt11 expression library of NP strain SAW 1734R. No sequence homology to any known protein was found in data bases.     

A unique cysteine proteinase gene of pathogenic Entamoeba histolytica correlates with virulence.

Extracellular neutral cysteine proteinases are an important virulence factor of E. histolytica. Experimental evidence supporting its role in invasion includes the ability to degrade components of the extracellular matrix and activate complement by specifically cleaving C3. We had previously reported the isolation of fragments encoding cysteine proteinase genes from HM-1 (ACP1) and a nonpathogenic strain (REF291, ACP2) by PCR using consensus sequences based on conserved structural motifs of eukaryotic cysteine proteinases. Using similar techniques, we have now identified a third gene encoding a cysteine proteinase which is present in both pathogenic and nonpathogenic strains and have correlated cysteine proteinase specific-mRNA levels with enhanced proteolytic activity and cytopathic effect on a fibroblast cell monolayer, a quantitative assay of virulence.     

应用聚合酶链反应-膜芯片技术快速鉴定分支杆菌菌种

目的建立一种简便、快速、灵敏、特异的分支杆菌菌种鉴定方法。方法通过 1 6SrDNA聚合酶链反应 单链构象多态性 (polymerasechainreaction singlestrandedconformationpolymorphism ,PCR SSCP)分析鉴定 1 4 0株分支杆菌临床分离株 ;设计与合成用于鉴定分支杆菌菌种的 1 6SrDNA寡核苷酸探针 ,制作膜芯片 ,与待测菌株生物素标记的 1 6SrDNA基因PCR产物进行反向斑点杂交。结果 1 4 0株分支杆菌临床分离株中 ,经 1 6SrDNAPCR SSCP初步鉴定 ,1 33株为结核分支杆菌复合群 ,7株为非结核分支杆菌。经膜芯片杂交分析 ,1 33株结核分支杆菌分离株鉴定为结核分支杆菌复合群 ;7株非结核分支杆菌中 ,1株鉴定为戈登分支杆菌 ,1株为土分支杆菌 ,1株为偶发分支杆菌 ,1株为胞内分支杆菌 ,另 3株与分析探针杂交阴性。分析 2 8种分支杆菌标准菌株和 9种非分支杆菌菌株 ,结果显示寡核苷酸探针是特异的。结论 1 6SrDNAPCR 膜芯片技术灵敏度高、特异性强、简便、快速 ,可用于鉴定分支杆菌菌种     

Intestinal amoebiasis for 36 years.

A case is reported of amoebic dysentery in a former soldier who had symptoms of Entamoeba histolytica infection for 36 years. It emphasizes the need for careful search for parasites in the stools of any patient with bowel symptoms because the consequences of wrong diagnosis are potentially catastrophic.     

Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes

目的　检测蚊体内间日疟原虫DNA。方法　以疟原虫SSurRNA为模板 ,采用套式PCR扩增间日疟原虫特异性 12 1bp片段。结果　在实验感染蚊中 ,套式PCR可检测到低至 3个间日疟原虫子孢子的DNA或 10 0只蚊中的一只阳性蚊 ,而其它 3种人疟原虫或正常蚊DNA未扩增出条带。结论　套式PCR的敏感性及特异性表明 ,该方法在检测蚊体内少量疟原虫感染时 ,比DNA探针或直接PCR具有更大优越性。     

PCR诊断细菌性痢疾的临床价值

目的建立一种快速、特异、敏感的诊断方法,对痢疾杆菌的致病基因进行基因检测研究。方法分别用PCR和多重聚合酶链反应(multiplex PCR)诊断方法,扩增2种痢疾杆菌的致病基因(iali、paH基因),扩增产物经电泳,出现与已知扩增片段大小一致的条带,并经测序与GENE库中标准菌株比较序列相同,即判断为该标本志贺菌的iali、paH基因阳性。结果对1组常规培养生化检测后的40份腹泻患者标本进行iali、paH基因特异片段扩增,ipaH基因阳性率为75%,ial基因阳性率为35%,iali、paH基因双基因阳性率为35%,较常规培养生化检测志贺菌阳性率(7.5%)高4倍多,iali、paH基因双基因阴性率为25%。结论PCR检测痢疾杆菌的致病基因具有简便、快速、特异、敏感和不需培养等特点,适于临床应用。     

PCR法检测粪便样本溶组织内阿米巴的初步研究

  目的  建立直接检测粪便样本中溶组织内阿米巴的PCR方法。  方法  根据溶组织内阿米巴标准株基因组中小亚基核糖体RNA（small subunit ribosome RNA,SSU rRNA）序列合成4对特异性引物（2对自主设计引物和2对参考引物）,建立PCR检测方法,用该方法对221例临床腹泻患者粪便标本进行检测,同时采用病原学碘染涂片镜检法和酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测抗原,对3种方法的阳性率进行比较,并采用Kappa检验进行一致性分析,分析3种检测方法结果的准确性。  结果  4对引物均扩增出溶组织内阿米巴特异的目的片段,建立了粪便样本溶组织内阿米巴检测的PCR法。选择其中2对引物（1对自主设计引物和1对参考引物）对221例粪便样本进行PCR扩增,同时用碘染涂片镜检法查病原体,用ELISA法进行抗原检测,3种方法溶组织内阿米巴阳性检出率分别为2.26%、0.90%和9.50%,差异有统计学意义（χ2=23.34, P<0.01）。PCR法与碘染涂片镜检法比较,Kappa值为0.216,一致性微弱;PCR法与ELISA法比较,一致性差,Kappa值为–0.134。PCR法的结果与临床诊断的一致性最好。  结论  本研究通过自行设计引物建立的粪便样本溶组织内阿米巴PCR检测法在准确性上优于镜检法和ELISA抗原检测法,为该方法用于临床诊断提供了实验基础。     

APPLICATION OF POLYMERASE CHAIN REACTION FOR DIAGNOSING AMEBIC LIVER ABSCESS

Polymerase chain reaction (PCR) has been applied in diagnosing amebic liver infection by detecting pathogenic Entamoeba histolytica DNA in liver aspirates. Oligonucleotide primers found to he specific for the gene encoding the 30 kDa molecule of this pathogenic ameba were used in the test. Liver aspirates obtained from 23 patients with amebic liver abscess substantiated by typical clinical rnanifastation or with very high titres of anti-E histtolytica antibodies by ELISA were found to he positive by PCR. Fourteen controlsamples (3 cases of bacterial liver abscess, I of liver cancer and 10 of other abscess) were all found to be negative to this reaction. The results suggested PCR to he a specific and sensitive tool for diagnosing amebic liver abscess infections.