Preliminary observations using a multi-layer ELISA method for the detection of Entamoeba histolytica trophozoite antigens in stool samples

A method for detecting Entamoeba histolytica trophozoite antigens in aqueous solution is described. This involves coating plastic microtitre plates with a 'catching antibody', specific rabbit anti-E. histolytica antibody (SRAE). After adding the test material the presence of antigen is determined using two additional heterologous antibody layers, one of 'developing antibody', in this case human anti-E. histolytica immunoglobulin (HAE), which is followed by a final layer of peroxidase conjugated sheep anti-human immunoglobulin antibody (SH-HRP). The specificity and sensitivity of the assay was investigated both in the model system and using stool samples from infected patients. In the model system, the test had a sensitivity equivalent to detection of approximately one amoeba per microscope coverslip (18 mm X 18 mm). Little cross reaction was observed with other intestinal parasites common to the area of Bangladesh from which the stool samples were taken. The possible use of this method in large scale screening of stool samples and in the detection of circulating antigens is discussed.     

Molecular detection of kobuvirus sequences in stool samples collected from healthy pigs in Japan

This study reports the detection and genetic characterization of porcine kobuvirus, a new member of the genus Kobuvirus in the family Picornaviridae. Among 293 fecal specimens collected from healthy pigs in 2009, in Japan, 133 (45.4%) were positive for kobuvirus using RT-PCR screening method. Of the positive specimens, 124 were obtained from pigs ≤6 months old, while 9 samples were from pigs >6 months old. Fifty-two representative strains of kobuviruses detected in this study were randomly selected and analyzed for their phylogenetic relationships with those other kobuvirus reference strains. The phylogenetic tree confirmed that 51 strains belonged to porcine kobuvirus and formed the exclusive branch with other porcine kobuvirus reference strains. In addition, the nucleotide sequence of H023/2009/JP shared very low levels of sequence identity with those of other porcine kobuvirus strains, but showed the highest level of sequence identity with bovine kobuvirus U-1 prototype strain. Our study demonstrated clearly that, porcine kobuvirus infection was common in healthy pigs and high prevalence of this virus was found in younger age of <6 months old of porcine populations in Tokyo and Hokkaido, Japan.     

辛酯酶荧光试验快速检测粪便标本中沙门菌的研究

辛酯酶荧光试验是一种检测样品中沙门菌属的新方法,具有简便、快速、灵敏的特点。该文从培养基的选择、细菌培养时间、底物用量等方面对辛酯酶荧光试验的条件进行了优化,并将此试验引入临床医学专业本科生的病原生物学实验教学,取得了较为满意的效果。     

Entamoeba moshkovskii and Entamoeba dispar-associated infections in pondicherry, India

The prevalence of Laredo strain--Entamoeba moshkovskii--and non-pathogenic E. dispar in patients attending the Jawaharlal Institute of Postgraduate Medical Education and Research hospital, Pondicherry, India, is reported here. E. moshkovskii is reported for the first time in India. The species are morphologically indistinguishable from pathogenic E. histolytica. Of 746 stool samples screened, 68 showing cyst or trophozoite stage of E. histolytica, E. dispar, or E. moshkovskii were subjected to small subunit (SSU) rRNA gene-based polymerase chain reaction, which revealed a higher prevalence of E. dispar (8.8%) and E. moshkovskii (2.2%) compared to E. histolytica (1.7%) in patients. Only 19% of the 68 stool samples, resembling E. histolytica by microscopy, were actually E. histolytica, implying that 81% of suspected infections were misdiagnosed and would have been treated unnecessarily with anti-amoebic drugs.     

A dot-blot hybridization procedure for the detection of astrovirus in stool samples.

We have developed a nucleic acid dot-blot hybridization test for the detection of astroviruses in stool samples. The test was not as sensitive as electron microscopy for the detection of low numbers of well preserved astrovirus particles, but was able to identify astroviruses in stools containing particles of indistinct morphology. In total, this procedure identified astroviruses in more samples than did electron microscopy, and the data indicate that the incidence of astroviruses may be substantially underestimated.     

Rapid detection of toxigenic Clostridium difficile from stool samples by a nested PCR of toxin B gene

Toxigenic Clostridium difficile is the aetiologic agent of most cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present standard method for C. difficile diagnosis is a cytotoxicity assay, performed on human fibroblast cultures. It is time consuming and requires special facilities. A nested-PCR assay detecting toxin B gene within a few hours was designed. One hundred and two stool samples were collected during four months. All samples were processed for toxin B-PCR, cultured for C. difficile and tested for cytotoxicity. This approach achieved 99% concordance with the cytotoxic assay. The sensitivity and specificity for the new PCR assay were 96.3% and 100% respectively. The procedure described is easy to perform, does not require special equipment and has produced excellent results. It deserves serious consideration for routine clinical microbiology laboratory use.     

Genotyping of Cryptosporidium spp. isolated from human stool samples in Switzerland

In a study to estimate the frequency of Cryptosporidium infections in Switzerland, stool samples from patients found to be positive for Cryptosporidium spp. by modified Ziehl-Neelson staining and fluorescence microscopy were used for genotyping experiments. With 9 of 12 samples, DNA extraction and subsequent genotyping was successful. All Cryptosporidium-isolates belonged to the bovine genotype. In one stool sample, two strains of Cryptosporidium were demonstrated, suggesting a mixed infection. In comparison with reference strains from calves, one of the isolates showed a full sequence identity and the other a similarity of 97.5%. The fact that only bovine genotypes were detected suggests, that cryptosporidiosis must primarily be considered as a zoonotic disease in Switzerland. This is in contrast to other countries, where the human genotype of C. parvum was shown to dominate the epidemiological situation. The results of our study are supported by the previous finding, that two of the analysed strains originated from patients who used to consume raw milk or raw cream, a known risk factor for cryptosporidiosis.     

大便标本的采集条件对EV71病毒核酸检测结果的影响因素分析

目的比较不同标本采集条件对EV71病毒核酸检测结果的影响,加强手足口病标本采集工作。方法对菏泽市11家手足口病治疗定点医院采集的标本做RT-PCR检测;依据不同条件对EV71阳性率做统计学分析。结果 EV71阳性率在不同病例年龄关系、病例所在地、是否重症3种情况下,差异无统计学意义;而在不同病例标本采集时间、采集地、病例性别差异三种条件下,EV71阳性率出现了明显的统计学差异。结论 EV71阳性率与病例标本的采集时间、采集地以及病例性别关系三者之间有明显的关系。     

Contribution of second stool specimen to increased sensitivity of poliovirus detection in India, 1998-2000

Acute flaccid paralysis (AFP) surveillance data from India were analysed to examine sensitivity of poliovirus isolation from stool specimens and the added sensitivity obtained from collection of a second stool specimen. Analysis was restricted to Indian AFP cases, 1998-2000, with two adequate stool specimens. The proportion of cases confirmed with wild poliovirus isolation by the second specimen only was calculated, regardless of specimen quality. Overall specimen sensitivity (1998-2000) was 81% using the first specimen, 78% using the second, and 96% using both. Sensitivity increased from 1998 to 2000, with slightly higher sensitivity each year for the first specimen. The second specimen increased sensitivity by 15% overall and contributed more when the first specimen was collected late or was in poor condition. As wild poliovirus disappears, increased sensitivity provided by a second stool specimen may reduce the risk of missing circulating virus.     

Selenium in selected species of mushrooms from Poland

The selenium was quantified in the caps, stalks or a whole fruiting bodies of king bolete (Boletus edulis), brown birch scaber stalk (Leccinum scabrum), parasol mushroom (Macrolepiota procera), fly agaric (Amanita muscaria) and poison pax (Paxillus involutus) collected at the various regions of Poland in 1998-2001. King bolete, parasol mushroom and fly agaric were a much more abundant in selenium than brown birch scaber stalk or poison pax. Some differences were observed between the selenium content of the particular species collected at different sites as well as depending on anatomical part of the fruiting body.     

核桃低聚肽对６０Ｃｏγ射线照射小鼠氧化损伤 保护作用的实验研究

摘要：目的　探讨核桃低聚肽（ＷＯＰｓ）对６０Ｃｏγ射线辐照小鼠氧化损伤的保护作用。方法　将９６ 只ＳＰＦ 级雌性ＢＡＬＢ／ｃ小鼠随机分为６ 组：空白对照组、模型对照组、乳清蛋白组（０．４４ｇ／ｋｇ）、ＷＯＰｓ组低、 中、高组（０．２２ｇ／ｋｇ、０．４４ｇ／ｋｇ、０．８８ｇ／ｋｇ）,连续饮水途径给予不同溶液第１４ 天,除空白对照组外, 所有小鼠全身照射３．５ Ｇｙ剂量６０Ｃｏγ 射线,分别在辐照后第３ 天和第１４ 天检测小鼠血清及肝脏ＳＯＤ、 ＧＳＨ Ｐｘ活性及ＭＤＡ 含量。结果　辐照降低了小鼠肝脏及血清ＳＯＤ、ＧＳＨ Ｐｘ含量,增高了ＭＤＡ 含量。 辐照后第３ 天,ＷＯＰｓ中组小鼠肝脏及血清ＳＯＤ 活性均高于模型对照组和乳清蛋白组（犘＜０．０５）;辐照后 第１４ 天,ＷＯＰｓ低组小鼠肝脏及血清ＳＯＤ 活性均高于模型对照组和乳清蛋白组（犘＜０．０５）。辐照后第 ３ 天,ＷＯＰｓ低组小鼠肝脏及血清ＧＨＳ Ｐｘ 活性均高于模型对照组和乳清蛋白组（犘＜０．０５）; 辐照后第 １４ 天,ＷＯＰｓ低组小鼠肝脏及血清ＧＳＨ Ｐｘ活性均高于模型对照组和乳清蛋白组（犘＜０．０５）。辐照后第 ３ 天,ＷＯＰｓ低、中组小鼠肝脏和血清ＭＤＡ 含量均低于模型对照组和乳清蛋白组（犘＜０．０５）; 辐照后 第１４ 天,ＷＯＰｓ低、中、高组小鼠肝脏及血清ＭＤＡ 含量均低于模型对照组和乳清蛋白组（犘＜０．０５）。结 论　ＷＯＰｓ对辐照所致氧化损伤具有一定的保护作用。 关键词：核桃低聚肽;辐照;氧化损伤;超氧化物歧化酶;谷胱甘肽过氧化物歧化酶;丙二醛 中图分类号：Ｒ１５１　　文献标识码：Ａ　　文章编号：１００９ ６６３９ （２０１９）０３ ０２１２ ０５     

Genetic diversity of human parechoviruses in stool samples,Germany

Human parechoviruses (HPeV) are ubiquitous and mainly occur in early infancy. They are known to cause various clinical manifestations including acute gastroenteritis. To gain insight into the diversity of circulating HPeV genotypes, stool samples from patients (n = 539) with clinical signs of infectious gastroenteritis which showed negative results for other common viral and bacterial enteric pathogens were obtained during three years, 2008 to 2010. Real-time RT-PCR showed HPeV RNA in 34 (6.3%) of the samples. The HPeV detection rate was highest (8.8%) in samples derived from infants and young children under the age of two years. Genotyping was based on VP3/VP1 junction nucleic acid sequences and revealed predominant HPeV-1B (n = 16) and HPeV-3 (n = 12) strains. Those prevailed minor HPeV-6 (n = 3) as well as HPeV-2, −4 and −5 (n = 1, each) strains. To ascertain the assigned HPeV-2 genotype of uncommon strain LPZ04-2008, analysis of complete coding sequences was performed. In complete VP1 analysis strain LPZ04-2008 showed 81.2% nucleic acid identity with HPeV-2 reference strain Williamson. In phylogenetic analysis VP1 of strain LPZ04–2008 clustered with a recent HPeV-2 strain from the UK. Regarding clinical manifestations, severe disease occurred HPeV-1B, −3 and − 6 infections. In conclusion, this paper a high genetic diversity of HPeV in stool samples, including rare strains. The investigation adds data on the whole coding sequences of the rare HPeV-2 strain. Genotyping results confirm previously reported association of more severe illness with HPeV-3 and HPeV-1B strains.     

Use of the enzyme-linked immunosorbent assay (ELISA) for detection of Entamoeba histolytica antigen in faecal samples

A promising new ELISA method developed by Root et al. to diagnose amoebiasis by the detection of Entamoeba histolytica coproantigen was tested in Mexico City and was reported to be more than twice as sensitive as microscopy. In this study the same procedure was compared to microscopy for paired specimens from 107 patients in San Francisco. Microscopy was more than twice as sensitive as ELISA: of 12 specimens positive by microscopy only 5 were also positive by ELISA. The ELISA did not detect a single specimen not detected by microscopy. Over-all, there was a 93% agreement between the two tests: 5% were positive and 89% negative by both tests. Possible reasons for the differences in the San Francisco and Mexican City findings are discussed.     

2008年-2009年鞍山地区手足口病患儿粪便样本中肠道病毒的检测结果分析

目的:通过实时荧光RT-PCR法检测2008年-2009年手足口病患儿粪便样本,了解鞍山地区手足口病病原特征,为手足口病防治工作提供科学依据。方法:分别于2008年6月-9月收集657份手足口病患儿粪便样本及2009年5月-9月收集274份手足口病患儿粪便样本进行检测。结果:2008年EV71阳性31份,CA16阳性58份2,009年EV71阳性75份,CA16阳性55份。结论:鞍山地区手足口病病原体以EV71和CA16为主,2008年以CA16为主要病原,2009年以EV71为主要病原。重症手足口病患儿病原均为EV71。     

Multiple regression analysis of twin data obtained from selected samples

The multiple regression analysis of twin data in which a cotwin's score is predicted from that of a proband (the member of a twin pair selected because of a deviant score) and the coefficient of relationship provides a powerful test of genetic etiology (DeFries and Fulker: Behav Genet 15:467-473, 1985). Moreover, when an augmented model containing an interaction term is fitted to the same data set, direct estimates of heritability (h2) and the proportion of variance owing to shared environmental influences (c2) are also obtained. In the present paper, the expected partial regression coefficients estimated from these models are derived, and the flexibility of the general approach is illustrated. An extended model is formulated for the analysis of data from combined samples of affected and control twin pairs that yields tests for differential h2 and c2 in the two groups as well as pooled estimates of these parameters. The application of these models is illustrated by an analysis of data from reading-disabled and control twin pairs. Because of the ease, flexibility, and utility of the multiple regression analysis of twin data, it is an appealing alternative to more traditional model-fitting approaches.