Entamoeba histolytica and Entamoeba dispar: comparison of two PCR assays for diagnosis in a non-endemic setting

Detection of Entamoeba histolytica, the causative agent of amoebiasis, is an important goal of the clinical parasitology laboratory. The identification of Entamoeba dispar as a morphologically identical but non-pathogenic species has highlighted the need for non-microscopic detection methods able to differentiate between the two organisms. In this study we evaluated the utility of conventional PCR and real-time PCR as methods for identification and differentiation of E. histolytica and E. dispar. The second aim of this study was to determine the relative proportions of infections caused by E. histolytica and the non-pathogenic E. dispar, allowing a picture of the epidemiological situation in a non-endemic setting to be obtained. One hundred and sixty-six clinical samples (faecal and liver abscess samples and one intestinal biopsy) belonging to 108 patients were analysed. More patients with E. dispar infection (8.3%) than patients with E. histolytica infection (5.6%) were found by both PCR assays. It is concluded that routine diagnosis of invasive amoebiasis performed by a combination of microscopy, culture and serology should be complemented with a PCR assay such as real-time PCR that offers a practical and clinically acceptable alternative for rapid and accurate diagnosis of amoebic infection in patients presenting with symptoms indicative of this disease.     

Risk factors for infection by the Entamoeba histolytica/E. dispar complex: an epidemiological study conducted in outpatient clinics in the city of Manaus, Amazon Region, Brazil

An epidemiological study was conducted on a population attending outpatient clinics in Manaus, Amazon, Brazil to determine the prevalences of infection by the Entamoeba histolytica/E. dispar complex and by E. histolytica alone, as well as to identify the risk factors involved in transmission. The study was conducted in two phases: survey and case-control. Face-to-face interviews were carried out and faecal samples collected from 1578 individuals. Faeces were examined by optical microscopy and tested for the pathogenic E. histolytica specific antigen. Positivity to E. histolytica/E. dispar was 21.5% (340 cases). Cases were compared with 340 control samples, negative for the E. histolytica/E. dispar complex based on examination by optical microscopy. The analysis was conducted by logistic regression. The risk factors identified were: place of residence, age, ingestion of raw vegetables, quality of water consumed, number of rooms and bedrooms per house, and having other protozoan infections. Specific antigen detection tests identified 22 participants infected by E. histolytica (6.8%) among those positive for E. histolytica/E. dispar. There was a higher proportion of males among participants infected by E. histolytica than among those with E. dispar infections. The study population was asymptomatic or presented non-specific symptoms that could be attributed to amoebiasis.     

Entamoeba histolytica encephalitis diagnosed by PCR of cerebrospinal fluid

Extra-intestinal amebiasis is secondary to invasive intestinal infections and usually results in an amebic liver abscess. Other organs, including lungs, brain, skin, spleen and kidneys, may be involved. Diagnosis of the cerebral infection is of the utmost importance and is most often made by detection of the organism at the time of brain biopsy or at autopsy. We report the first case of Entamoeba histolytica encephalitis diagnosed by PCR of the cerebrospinal fluid. The patient was treated successfully with metronidazole. PCR is an increasingly useful tool for the diagnosis of central nervous system infection and can provide rapid diagnosis.     

Overdiagnosis of amoebiasis in the absence of Entamoeba histolytica among patients presenting with diarrhoea in Wonji and Akaki, Ethiopia

To confirm the high reported incidence of intestinal amoebiasis among study participants at 2 cohort sites in Ethiopia where an HIV/AIDS study is taking place, stool samples of 232 patients with complaints of diarrhoea were examined for the presence of Entamoeba histolytica and E. dispar DNA between April and December 2001. By microscopy, 91 (39%) of the study participants were reported to harbour Entamoeba trophozoites and/or four-nucleated cysts. Using specific E. histolytica and E. dispar DNA amplification and detection, none of the study participants were found to be infected with E. histolytica and only 21 (9%) with E. dispar. The consequences of the overdiagnosis of E. histolytica are briefly discussed.     

Sequence Diversity in tRNA Gene Locus A-L among Iranian Isolates of Entamoeba dispar

Background

A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates.

Methods

A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced. The sequences obtained were edited manually and aligned using Gene Runner software.

Results

With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified.

Conclusion

The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran.     

Multiplex Real-Time Polymerase Chain Reaction-Based Method for the Rapid Detection of gyrA and parC Mutations in Quinolone-Resistant Escherichia coli and Shigella spp.

Two real-time polymerase chain reaction assays were developed to detect mutations in codons 83 and 87 in gyrA and in codons 80 and 91 in parC, the main sites that causes quinolone resistance in pathogenic Escherichia coli and Shigella spp. isolates. These assays can be employed as a useful method for controlling infections caused by quinolone-resistant E coli and Shigella isolates.     

Validation of PCR Assay for Identification of Sarcoptes scabiei var. hominis

Background

Infestation of the skin by the “itch mite” Sarcoptes scabiei var. hominis results in a contagious skin infection in humans called “scabies”. By resolving morphology issues, the present study was designed to be acquainted with itch mite by molecular markers.

Methods

The mite samples were collected from scabies patients by visiting government hospitals of twin City, Pakistan. For successful molecular detection approach, preparation of Sarcoptes mite DNA by commercial DNA extraction kit method. Furthermore, two primers i.e. Sarms 15 F/R and 16S D1/D2 were used to amplify target sequence by using PCR. The amplified products were then separated by agarose gel, electrophoresis and analyzed after staining and visualizing in UV transilluminator.

Results

Analysis of PCR product showed one specific band of 178 bp with primer Sarms 15 F/R, while, with primer 16S D1/D2 bands of 460 bp and 600 bp were observed on 2% agarose gel. The appearance of different band of 600 bp revealed that it might be due to heteroplasmy state present in the Pakistani Sarcoptes mites population.

Conclusion

Current study adds validity to the claim that PCR is more accurate, specific and sensitive in the detection of the ectoparasites even in smallest amount.     

Efficacy of a Gal-lectin subunit vaccine against experimental Entamoeba histolytica infection and colitis in baboons (Papio sp.)

To determine the efficacy of a Gal-lectin based intranasal synthetic peptide vaccine, we developed a new experimental primate model of Entamoeba histolytica intestinal infection. Release of xenic E. histolytica trophozoites (5×10(6)) into the small bowel of baboons (Papio sp.) resulted in a rapid intestinal anti-amebic antibody response and a brief infection; however, release of trophozoites directly into the cecum (5 baboons) elicited a sustained E. histolytica infection, as determined by quantitative fecal PCR, and an ulcerative, inflammatory colitis observed on colonoscopy and histopathology. In three controlled experiments, baboons received four immunizations at seven day intervals of 1600 μg of the vaccine/nostril, with Cholera toxin, 20 μg/nostril as adjuvant; vaccinated (n=6) and control baboons (n=6) baboons were then challenged via colonoscopy with xenic trophozoites (5×10(6)). During 90 days of follow up, 250 of 415 (60.24%) fecal samples in control baboons had a (+) PCR for E. histolytica, compared to only 36 of 423 (8.51%) samples from vaccinated baboons (P<0.001). All 6 vaccinated baboons were free of infection by the 51st day after challenge, 5 of 6 controls positive had (+) fecal PCRs for up to 126 days post-challenge (P=0.019). Inflammatory colitis developed in 4 of 6 control baboons post-challenge, with invasive E. histolytica trophozoites present in 2 of the 4 on histopathology. There was no evidence of inflammatory colitis or parasite invasion in any of the vaccinated baboons; there was a strong inverse correlation between positive ELISA OD value indicating the presence of intestinal anti-peptide IgA antibodies and baboons having a positive fecal PCR CT value, P<0.001. In conclusion, we developed a novel primate model of E. histolytica intestinal infection and demonstrated that a Gal-lectin-based intranasal synthetic peptide vaccine was highly efficacious in preventing experimental E. histolytica infection and colitis in baboons.     

呼吸道病原体多重实时荧光定量PCR的检测

目的 建立多重实时荧光定量PCR检测方法,为实现对呼吸道病原体的快速、灵敏特异性检测.方法 采用经报道的引物和探针对合胞病毒、腺病毒等8种呼吸道病原体进行多重实时荧光定量PCR检测.结果 多重荧光定量PCR阳性检测率高达57.8%,无假阳性及假阴性,特异性强.结论 多重实时荧光定量PCR具有经济、快速、特异性强等优点,在呼吸道病原体检测方面有巨大应用价值.     

Evaluation of newer diagnostic methods for the detection and differentiation of Entamoeba histolytica in an endemic area

We evaluated the colorimetric polymerase chain reaction (PCR)-based method for the detection and differentiation of Entamoeba spp. and compared the efficacy of E. histolytica-specific antigen detection in faeces with the detection of specific antibodies to E. histolytica-specific antigen in faeces, by enzyme-linked immunosorbent assay. Faecal samples were obtained from patients attending hospital in Chandigarh, India, from March 2001 to February 2002. The PCR-based colorimetric method was found to be the most sensitive (100%) and it could differentiate between pathogenic and non-pathogenic Entamoeba spp. The present study also emphasized that the antigen detection system may prove to be a better diagnostic tool than the antibody detection system in endemic areas.     

多重PCR检测水中肠道总病毒及甲型肝炎病毒

PCR技术用于病毒的检测已有过较多的研究报道，国内主要将PCR技术用于脊髓灰质炎病毒（简称脊灰病毒）的检测与分型，以及野毒株的鉴定[1。2]；国外已将常规PCR技术用于脊灰病毒及甲型肝炎病毒（简称甲肝病毒）的检测[3-5]。常规PCR技术一次只能检测一种病毒的核酸，如果反应体系中含有两种以上的病毒核酸，则需进行多次PCR，不但操作复杂，费用相对也较高。近年来，人们发展了一种新的PCR技术—多重PCR技术，即在一个反应体系中含两对以上引物，通过PCR反应可同时检测多种目的核酸[6。7]。我们利用肠道病毒具有保守的5非编码区核…     

Detection of Infection with Larval Stages of Ornithobilharzia turkestanicum using PCR in Field-Collected Snails of Lymnaea gedrosiana from Northwestern Iran

Background

Infection with Ornithobilharzia turkestanicum has been reported in a wide range of animals worldwide. This study was undertaken to assess the utility of polymerase chain reaction (PCR), for detecting the infection with O. turkestanicum larvae stages in Lymnaea gedrosiana.

Methods

A total of 6,759 Lymnaeidae snails were collected from six aquatic habitats in West Azarbaijan, northwest Iran. Of these, the snails of L. gedrosiana were identified. To detect infected L. gedrosiana with the larval stages of O. turkestanicum, they were subjected for cercarial shedding and molecular examinations. The genomic DNA was extracted and PCR was performed to specifically amplify a fragment of the nuclear 28SrRNA gene of O. turkestanicum.

Results

Of all collected snails, 5.4% (365/6,759) were the snails of L. gedrosiana. The cercarial shedding method revealed that 23.56% (86/365) of the snails were infected. The PCR patterns confirmed that 28.77% (105/365) snails of L. gedrosiana were infected with larval stages of O. turkestanicum. The infected snails were observed in five studied sites. The highest infection rate (66.66%, 20/30) was recorded in the snails of Ghargologh in the northern part. Only 35.24% (37/105) of the infected snails were from the plain areas, whereas the remaining existed in high altitudes.

Conclusion

It was concluded PCR method could be an efficient and fast method for uncovering the actual rate of infection with larval stages of O. turkestanicum in the snails of L. gedrosiana. This method can be also useful for the domestic animals and public health management programs in the country.     

Detection of Asymptomatic Carriers of Plasmodium vivax among Treated Patients by Nested PCR Method in Minab,Rudan and Bashagard,Iran

Background

Plasmodium vivax is the most widespread species of Plasmodium in humans and causing about 80 million clinical cases annually. This study was undertaken to detect P. vivax in asymptomatic treated vivax malaria patients to trace latent/sub-patent malaria infection.

Method

The venous blood of all detected cases with P. vivax in Bashagard, Minab and Roodan Districts in Hormozgan Province from 2009 to 2010 was examined by microscopic and nested PCR methods for presence of the parasite.

Results

In microscopic examination of peripheral blood smears, all samples were negative for the presence of the parasites. But, we detected two P. vivax related bands in the electrophoresis of the nested PCR products (120 bp).

Conclusion

Following up the malaria cases after treatment by a combination of methods, or new diagnostics such as RDTs can be included in the priorities of malaria elimination program in Iran.     

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples

Background

Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples.

Methods

A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar''s χ² test, with consideration of a P-value of <0.05 as indication of significant difference.

Results

In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture.

Conclusion

Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.     

转基因食品多重定性PCR及实时荧光定量PCR检测方法的研究

多重PCR方法是建立在传统PCR检测技术基础上的快速简便的检测手段。本研究以国际市场上转基因食品的主流产品转抗除草剂大豆 (RR大豆 )、Bt176玉米及我国自行研发的抗菌肽辣椒为材料 ,以多重PCR方法为基础 ,通过基因片段筛选及引物比例调整 ,进行了转基因食品的调控元件、外源结构基因及物种内源特性基因三种片段的单管同时扩增。结果与常规PCR方法检测结果完全相符。同时 ,针对RR大豆 ,对几类大豆产品运用实时定量PCR方法进行定量分析 ,得到相应的转基因成分的含量。操作简便快速 ,检测结果与标准相符 ,检测灵敏度较常规PCR更高。     

Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

Background:

Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.

Methods:

A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.

Results:

Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.

Conclusion:

The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.     

Noninvasive Test for Tuberculosis Detection among Primates

Traditional testing methods have limited epidemiologic studies of tuberculosis among free-living primates. PCR amplification of insertion element IS6110 of Mycobacterium tuberculosis from fecal samples was evaluated as a noninvasive screening test for tuberculosis in primates. Active tuberculosis was detected among inoculated macaques and naturally exposed chimpanzees, demonstrating the utility of this test.     

新型冠状病毒双重荧光RT-PCR检测方法

目的为快速检测新型冠状病毒,防控疫情的输入,建立一种快速的双重实时荧光RT-PCR检测方法。方法对WHO公布的单重实时荧光RT-PCR检测引物和探针进行重新设计和优化,建立双重荧光RT-PCR反应体系,分别采用羧基荧光素(FAM)和绿色荧光蛋白(VIC)荧光基团标记探针,实现双基因的同时检测。结果经优化的双重实时荧光RT-PCR方法有较好的灵敏度和特异性,对阳性对照质粒检测灵敏度为31拷贝/μl,检测健康和普通发热人员咽拭子以及流感病毒无交叉反应。结论建立了双重实时荧光RT-PCR快速检测方法,提高检测效率和准确度的同时降低了成本,可用于新型冠状病毒的快速应急检测。     

多重聚合酶链反应检测儿童常见下呼吸道感染细菌

目的 探讨多重聚合酶链反应(PCR)检测儿童急性下呼吸道感染细菌的应用价值.方法 对383例急性下呼吸道感染患儿的深部呼吸道吸引物进行细菌培养及多重PCR检测,检测肺炎链球菌、流感嗜血菌、金黄色葡萄球菌、肺炎克雷伯菌、大肠埃希菌、嗜肺军团菌、铜绿假单胞菌等9种病原菌.结果 细菌培养的总检出率为42.04％,多重PCR检出率45.95％,两者差异无统计学意义;以细菌培养为标准,多重PCR检测的总敏感性为77.02％,特异性为76.58％,其中大肠埃希菌和表皮葡萄球菌的检出率明显高于培养法.结论 多重PCR检测可作为下呼吸道致病菌快速检测的有用工具.