Development of multiplex real-time polymerase chain reaction for detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in clinical specimens

Multiplex real-time polymerase chain reaction (PCR) was developed for differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii. Specific primers were designed for all three species, and then differentiation of E. histolytica and E. dispar was achieved simultaneously using a hybridization probe and melting curve analysis, whereas E. moshkovskii was detected with a separate probe under the same condition. This assay detected as little as 0.2 pg of E. histolytica DNA and 2 pg each for E. dispar and E. moshkovskii DNA. Thirty-five clinical samples suspected to be E. histolytica infection by microscopy were tested. The results showed 32 positive samples; four samples were E. histolytica and 28 samples were E. dispar. Interestingly, one E. dispar positive sample showed a mixed infection with E. moshkovskii. This is the first report of E. moshkovskii infection from Thailand and this assay is currently the most rapid and sensitive method to differentiate these human amoebas.     

An improved PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in formalin-fixed stools

Detection of infectious agents in faecal samples by polymerase chain reaction (PCR) can be limited by the presence of substances which inhibit DNA amplification. Here, an improved protocol is reported for directly isolating DNA from fresh and aged formalin-fixed stools, after concentration by formalin-ethyl acetate (FEA). The protocol was successfully applied to detect DNA of Entamoeba histolytica/Entamoeba dispar complex in stools by nested PCR, showing high specificity and low detection limit. Extended time of specimen storage in formalin had no influence on PCR yields. This PCR-based method offers technical advantages for routine detection and discrimination of invasive E. histolytica and non-invasive E. dispar.     

PCR differentiation of Entamoeba histolytica and Entamoeba dispar from patients with amoeba infection initially diagnosed by microscopy

Amoebiasis is a notifiable disease in Sweden and 400-500 cases are reported annually to the Swedish Institute for Infectious Disease Control (SMI). The true number of patients with Entamoeba histolytica infection is unknown as diagnosis mainly relies on cyst detection by microscopy. The main purpose of this study was to estimate the proportions between E. histolytica and E. dispar in patients with amoebic infection, using established PCR technologies. Secondly, we aimed to evaluate the usefulness of ethanol as a transport medium for samples forwarded for Entamoeba-PCR. Faecal samples from 207 patients with initial diagnosis of E. histolytica/E. dispar were referred to SMI for species differentiation. The PCR analysis showed that 165 patients were positive for E. dispar, whereas only 10 patients were positive for E. histolytica. No mixed infections were observed. The remaining 32 patients were negative both by microscopy and by PCR. Ethanol fixation was evaluated on 168 paired samples (transported unfixed or fixed in ethanol). Ethanol was found to be a useful transport medium as in 8 cases only the fixed sample was PCR-positive. This study shows that few patients in Sweden are infected with E. histolytica. The ability to differentiate E. dispar from E. histolytica should reduce the number of unnecessarily treated patients.     

Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp

Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. are not only three of the most important and common diarrhea-causing parasitic protozoa, but they often have similar clinical presentations. Microscopic diagnosis of these parasites is neither sensitive nor specific. Recently, more specific and sensitive alternative molecular methods (polymerase chain reaction [PCR] and antigen detection tests) have been introduced for all three of these parasitic infections. The use of these molecular diagnostic tests in routine diagnostic laboratories is still limited. In this study, we developed a multiplex real-time PCR assay for the simultaneous detection of E. histolytica, G. intestinalis, and Cryptosporidium spp. in one reaction using species-specific probes. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. The reagents used in this multiplex PCR assay can also be used for detection of these parasites individually. The use of this real-time PCR multiplex assay in developing countries at present will have limited scope for routine diagnosis because the cost will be high for a single test, although in the developed world, the test could see immediate application.     

Usefulness of multiplex-PCR for identification of Entamoeba histolytica and Entamoeba dispar

We evaluated the usefulness of a multiplex-PCR method for differentiation of Entamoeba histolytica and Entamoeba dispar, which are morphologically indistinguishable species. Cultured trophozoites of E. histolytica HM-1: IMSS and E. dispar SAW were used as the positive control. Seven human fecal samples, from which E. histolytica-like cysts were detected by microscopic examination, and three intestinal protozoan parasites, Cryptosporidium parvum HNJ-1, Giardia intestinalis Portland-1, and Blastocytis hominis Nand II, were used for the evaluation of sensitivity and specificity of the PCR method. The other PCR method, which has been used for the diagnosis of amebic infections in Japan, was also performed by using the same samples for the evaluation. In comparison with the conventional PCR method, the multiplex-PCR showed 1) higher sensitivity, 2) the size of diagnostic fragments of PCR products was clearly different in both Entamoeba species, 3) it was possible to perform PCR using a single tube per sample, and then to save the amount of DNA polymerase, 4) no diagnostic amplification products were found in other intestinal protozoan parasites, and 5) E. histolytica specific fragment was amplified in all clinical samples examined. In conclusion, it is considered that the multiplex-PCR method is a useful tool for detection of both Entamoeba species DNA from fecal samples and for the distinction between E. histolytica and E. dispar.     

Validation and utilization of PCR for differential diagnosis and prevalence determination of Entamoeba histolytica/Entamoeba dispar in Salvador City,Brazil

Amoebiasis is an infection caused by Entamoeba histolytica and is a potential health risk in countries in which health barriers are inappropriate. Since the discovery of Entamoeba dispar, the prevalence of amoebiasis has been modified.ObjectiveThis study has standardized the PCR technique applied for the diagnosis of different species of the E. histolytica/E. dispar complex and has evaluated the prevalence of infection among patients attending private and public clinical laboratories in Salvador City, Bahia State, Brazil.ResultsAnalysis of 52,704 stool samples by microscopic examination demonstrated that 1,788 (3.4%) were positive for the E. histolytica/E. dispar complex and infection occurred more often in samples originated from public clinical laboratories (5.0%) than those that came from private laboratories (3.2%). PCR performed in approximately 15% (262) E. histolytica/E. dispar complex positive samples, randomly chosen, amplified 227 samples (86.6%), all of them positive for E. dispar. The non-amplified 35 samples (13.4%) were also negative for E. histolytica-specific galactose adhesin. Moreover, to exclude a probable infection caused by E. hartmanni, morphometric analysis demonstrated that non-amplified samples had cyst sizes comparable to E. histolytica/ E. dispar ( > 10 μm).ConclusionThe absence of amplification of these samples indicates the presence of PCR inhibitors in the stool samples or the presence of DNA from Entamoeba species other than E. dispar, E. histolytica or E. hartmanni.     

Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples.

Due to the clinical importance of differentiating the two species of the Entamoeba histolytica/Entamoeba dispar complex, we developed a multiplex polymerase chain reaction (PCR) method that overcomes time-consuming and laborious procedures. We report here a DNA extraction protocol using non-fixed stool samples that avoid long lysis-incubation periods through the combined use of zirconium beads and a lysis-supporting buffer. We characterized 49 of 52 stool specimens from Cuban patients with amoebiosis. Among them, 36 (75.5%) were infected only with E. dispar (the nonpathogenic species), while 13 (24.5%) displayed a mixed infection with both E. dispar and E. histolytica. The multiplex PCR protocol showed a specificity of 1.00 and a sensitivity of 0.94. Furthermore, the entire procedure can be performed in one day. This approach is therefore reliable and applicable in the field for epidemiologic studies.     

Sensitivity of Entamoeba histolytica and Entamoeba dispar patient isolates to human complement

Twenty-one Entamoeba histolytica and 56 Entamoeba dispar patient isolates were investigated for their sensitivity to the classical and alternative pathway of human complement. E. histolytica and E. dispar patient isolates were differentiated by polymerase chain reaction and hexokinase isoenzyme typing. It was found that 90.3% (±12.0%) of the trophozoites of E. histolytica were lysed after 30 min by the alternative pathway of complement in the presence of 50% human serum (19 isolates showed lysis rates higher than 80%), whereas E. dispar cells were less susceptible to the alternative pathway as 68.8% (±28.2%) of lysis occurred. However, 23 of the E. dispar isolates were lysed between 100 and 80% (90.9%±9.1%), demonstrating that about half of the tested E. dispar isolates were highly sensitive to complement lysis. Only 11 of the E. dispar isolates were proven to be 'resistant' to the alternative pathway of complement and were lysed less than 40%. These results are in conflict to earlier publications, describing resistance of E. dispar to complement lysis (Hamelmann et al. 1992, 1993).     

A multiplex PCR for the simultaneous detection of species of filarioids infesting dogs

The present study reports the applicability of a multiplex PCR for the simultaneous detection and differentiation of common filarioids infecting dogs, i.e., Dirofilaria immitis, Dirofilaria repens, Acanthocheilonema reconditum and Cercopithifilaria sp. Amplicons of different sizes (i.e., 170 bp, 480 bp, 590 bp and 300 bp, respectively) of regions within the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene were amplified on a single-step multiplex PCR using a mix of species-specific forward primers coupled with a single reverse primer. Experiments were carried out by amplifying genomic DNA extracted from blood or skin samples test-positive for microfilariae (mff). The number of mff present in each blood sample was quantified (from 800 to 25,000 mff/ml for A. reconditum and D. repens, respectively) and mixed blood samples were tested for the simultaneous detection of DNA from these mff. Specific amplicons for blood-circulating mff of A. reconditum, D. immitis and D. repens and for those whose adults are localized in skin (i.e., A. reconditum and Cercopithifilaria sp.) were simultaneously detected on agarose gel up to a dilution of 250 mff/ml for D. repens. The specific identity of the amplicons was confirmed by sequencing. The multiplex PCR assay reported herein represents a new tool for the molecular detection and differentiation of canine filarioids in blood and skin samples.     

Specific detection of Entamoeba histolytica DNA by hemolysin gene targeted PCR

Diagnostic differentiation of pathogenic Entamoeba histolytica from non-pathogenic Entamoeba dispar is of great clinical importance. We have developed and evaluated a new polymerase chain reaction (PCR) assay (haemo-PCR) based on the novel E. histolytica hemolysin gene HLY6. The specificity of this assay was confirmed by analyzing different Entamoeba species, faeces samples, human and bacterial DNA, and digestion of amplification products with appropriate restriction enzymes. The sensitivity was confirmed by serial dilutions of E. histolytica HM-1:IMSS DNA in the excess of human DNA. Totally, 45 clinical samples were analyzed by the haemo-PCR assay including amoebic liver abscess (ALA) fluids from 23 patients suspected for amoebiasis, four faeces samples containing E. histolytica and E. dispar, and positive and negative controls. The results were compared with those obtained with PCRs for cystein-rich surface protein (P30) and small subunit ribosomal RNA (ssu rRNA) genes. The haemo-PCR gave a positive result in 18 (89%) ALA fluids compared with 14 (77%) and five (28%) by PCR for p30, and ssu rRNA, respectively. PCR products were obtained only from specimens containing E. histolytica DNA. The haemo-PCR assay was therefore found to be a valuable diagnostic tool for identification of E. histolytica infections both in faeces and ALA samples.     

Entamoeba histolytica/Entamoeba dispar infections in human immunodeficiency virus-infected patients in the United States.

We describe the incidence of and laboratory and clinical characteristics associated with Entamoeba histolytica/Entamoeba dispar infection diagnosed in human immunodeficiency virus (HIV)-infected persons enrolled in the Adult and Adolescent Spectrum of HIV Disease Project. From 1 January 1990 to 1 January 1998 (82, 518 person-years of follow-up), 111 patients (98% men) were diagnosed with E. histolytica/E. dispar infection. Among HIV-infected patients in the United States, the incidence of diagnosed E. histolytica disease is low (13.5 cases per 10,000 person-years [95% confidence interval, 7.7-22.2], with diagnosis most common in those patients exposed to HIV through male-male sex.     

一种检测胆螺杆菌套式PCR方法的建立

目的 建立一种敏感性高、特异性强的检测胆螺杆菌的套式PCR方法。方法 基于17种胆螺杆菌亚种的16S rRNA 基因设计筛选出1套套式引物(2条内引物、2条外引物),优化反应条件后,通过模拟粪便标本、动物模型标本和临床标本的检测对方法的敏感性和特异性进行评估。结果 模拟粪便标本中,该方法 检测胆螺杆菌的敏感性达到10 CFU/100 μL。3例感染胆螺杆菌的SPF级BALB/c小鼠模型中,3例小鼠粪便和盲肠中均可检测到胆螺杆菌,1例肝脏标本中检测到了胆螺杆菌。10例胆石病患者中,有2例患者的胆汁、胆囊粘膜和粪便标本中检测到了胆螺杆菌。结论 本研究建立的套式PCR方法 ,敏感性高、特异性强,可用于检测胆螺杆菌的感染。     

Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity.

Recent studies suggest that stool antigen assays are more sensitive and specific than microscopy for the diagnosis of Entamoeba histolytica infection. One hundred twelve patients presenting at 3 centers with symptoms or risk factors of E. histolytica infection were prospectively enrolled in this study to evaluate new diagnostic tests for infections with E. histolytica and Entamoeba dispar. Four ELISA-based stool antigen kits for detecting E. histolytica or E. dispar were blindly compared with stool microscopy. Amebic serology was assessed by indirect hemagglutination. When antigen assays were used as the reference standard, microscopy performed at referral centers was more specific (68.4% vs. 9.5%) but less sensitive (70.4% vs. 92.1%) than microscopy performed in community laboratories. Diagnosis with the E. histolytica test and Merlin Optimun S ELISA indicated that only 3 (4.2%) of 72 coproantigen-positive stools were positive for E. histolytica. Indirect hemagglutination was a good predictor of E. histolytica infection when titers of antibody to ameba were >/=1:512.     

肝螺杆菌多重PCR检测方法的建立及应用

目的建立肝螺杆菌的多重PCR检测方法,对中国动物肝螺杆菌进行检测。方法以毒力基因flaB基因、ureA基因和cdtB基因和cdtC基因作为靶基因,建立检测肝螺杆菌的多重PCR方法。对肝螺杆菌、空肠弯曲菌、幽门螺杆菌、沙门氏菌、志贺氏菌、小肠结肠炎耶尔森菌、大肠埃希氏菌和铜绿假单胞杆菌抽提的DNA进行多重PCR扩增。应用本研究建立的多重PCR检测方法对482只动物(其中:海南30只猴、江苏34只小鼠和北京32只猴、30头猪、66只犬、13只兔、29只豚鼠、69只大鼠、213只小鼠)进行检测,对扩增出的阳性结果进行测序。同时,对所有样本采用选择性培养基进行肝螺杆菌培养以作对照。结果肝螺杆菌能扩增出各自的特异性条带,而其他参考菌株均未扩增出条带,这表明该方法具有较强的特异性。482份样本中检出43份肝螺杆菌阳性样本,阳性率8.92%(43/482),其中:2只犬(3.03%,2/66)、1只兔(7.69%,1/13)、5只大鼠(7.25%,5/69)和35只小鼠(16.43%,35/247)均扩增出肝螺杆菌毒力基因片段。结果显示,肝螺杆菌flaB基因、ureA基因和cdtB基因和cdtC基因序列与GenBank中的肝螺杆菌ATCC51449的相应基因序列核苷酸同源性高达99%。用选择性培养基能培养出肝螺杆菌。结论中国动物中的犬、兔、大鼠和小鼠均能检出肝螺杆菌flaB基因、ureA基因、cdtB基因和cdtC基因。建立的多重PCR检测方法可作为肝螺杆菌大规模检测的新技术,这为在中国开展肝螺杆菌流行病学调查提供了有效科学工具。     

Isoenzyme and molecular characterization of Entamoeba histolytica and Entamoeba dispar isolates from symptomatic and asymptomatic subjects.

AIM: To correlate the clinical features of amebic infections with the characteristics of Entamoeba culture isolates of stools. METHODS: Isolates from seven irritable bowel syndrome (IBS) patients, four asymptomatic cyst passers (ACP) and five patients with invasive amebic disease were subjected to hexokinase polyacrylamide electrophoresis (HK-PAGE) and their DNA subjected to restriction fragment (RF) analysis of amplified polymerase chain reaction (PCR) products. These findings were correlated with anti-amebic serology. Two axenic pathogenic strains (HM1:IMSS, NIH:200) and one xenic nonpathogenic strain (SAW1734) were used as standards. RESULTS: All isolates from IBS patients as well as ACP had slow-moving (nonpathogenic) band pattern, whereas those from patients with invasive disease had fast-moving (pathogenic) band pattern on HK-PAGE. Serological data using EIA and RF patterns of PCR-amplified genome corroborated these results. CONCLUSIONS: Our results support the view that there are two species of Entamoeba infecting humans--E. histolytica(pathogenic) and E. dispar (nonpathogenic), and HK-PAGE of culture isolates can differentiate between them.     

The use of real-time PCR to identify Entamoeba histolytica and E. dispar infections in prisoners and primary-school children in Ethiopia

In Ethiopia, it is generally unknown what proportion of the amoebic infections commonly found, by microscopy, in humans are caused by non-invasive Entamoeba dispar rather than the potentially invasive E. histolytica. Faecal samples were therefore collected from 363 primary-school students and 409 prisoners from various regions of Ethiopia. Each of these samples was checked for Entamoeba infection by the microscopical examination of formol-ether concentrates. DNA was then extracted from the 213 samples (27.6%) found Entamoeba-positive, and run in a real-time PCR with primers, based on the SSU-rRNA gene sequences of E. histolytica and E. dispar, that allow DNA from the two species to be distinguished. Although E. dispar DNA was identified in 195 (91.5%) of the 213 samples checked by PCR, no E. histolytica DNA was detected. This finding is consistent with the conclusion of a previous, smaller investigation: that many amoebic infections in Ethiopia are incorrectly attributed to E. histolytica and then treated, unnecessarily, with amoebicidal drugs.     

牛源贾第虫套式PCR检测方法的建立

目的建立牛源贾第虫套式PCR检测方法。方法根据牛源贾第虫TPI基因序列,设计1对保守引物和1对特异性引物,建立套式PCR检测方法;通过阳性参照摸索出该检测方法的最佳反应条件,进行特异性和敏感性试验;并用临床样品进行验证。结果该套式PCR能特异性地扩增出牛源贾第虫基因组DNA中相应片段,与艾美耳球虫、隐孢子虫、环孢子虫、弓形虫、毛首线虫和纤毛虫等10种相关原虫基因组DNA无交叉反应;最低能检测到0.395fg的阳性参照DNA。结论建立的套式PCR方法具有高度的特异性和敏感性,可以用于临床样品的检测。