PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia

This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.     

Agglutinating monoclonal antibodies that specifically recognize lipooligosaccharide A of Bordetella pertussis.

Monoclonal antibodies that specifically agglutinate strains of Bordetella pertussis having serotype 1 agglutinogen were uniquely reactive with the electrophoretically slow-migrating A form of lipooligosaccharide. These monoclonal antibodies should be useful for the structural analysis of B. pertussis lipooligosaccharide and for the establishment of a better-defined serogroup for Bordetella species.     

Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits.

Humans are infected by two morphologically identical species of Entamoeba: Entamoeba histolytica causes amebic colitis and liver abscess, and Entamoeba dispar is noninvasive. Several weeks of culture and isoenzyme (zymodeme) analysis are required to differentiate E. histolytica from E. dispar. Here we report a field trial of commercial antigen detection kits designed to rapidly detect and differentiate E. histolytica from E. dispar in stool specimens. Stool specimens from 202 patients with diarrhea were examined for E. histolytica and E. dispar by microscopy, culture, and antigen detection. Compared with culture, microscopic identification of the E. histolytica-E. dispar complex was 60% sensitive and 79% specific, while the screening antigen detection test for the E. histolytica-E. dispar complex was 80% sensitive and 99% specific. Differentiation of E. dispar from E. histolytica by the E. histolytica-specific test was 95% sensitive and 93% specific compared with zymodeme analysis. We conclude that the antigen detection test for the E. histolytica-E. dispar complex is more sensitive and specific than microscopy and that the E. histolytica-specific antigen detection test is as reliable and much more rapid than zymodeme analysis for the differentiation of E. histolytica from E. dispar.     

Development of monoclonal antibodies that recognize Treponema pallidum.

We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.     

Cellular antigens in nephroblastoma: identification with monoclonal antibodies which recognize hemopoietic cells

The correlation between expression of differentiation antigens and morphogenesis was examined in 11 human nephroblastomas using monoclonal antibodies which recognize both hemopoietic and renal cells. Analysis of frozen tissue sections by indirect immunofluorescence revealed that blastema cells and some tubules were recognized by BA-1 and OKB2, which identify unstimulated B cells and granulocytes. Stroma, some tubules, and focal blastema were recognized by BA-2, which identifies a 24-kDa antigen on leukemic cells and platelets. Mature epithelium in glomerular bodies was identified by C3bR and J5, which recognize CR1 and the common acute lymphoblastic leukemia antigen, respectively. Tissue sections from a clear cell sarcoma and a mesoblastic nephroma were notably reactive only with BA-2. These observations demonstrate that the relationship between antigen expression and morphology in nephroblastomas is similar to that observed in fetal kidney and suggest that expression of these cell surface antigens may have a role in morphogenesis.     

Phorbol esters specifically enhance the cytolytic activity of Entamoeba histolytica.

Entamoeba histolytica causes invasive amebiasis by lysis of host tissue and inflammatory cells. The in vitro cytolysis of target Chinese hamster ovary (CHO) cells by axenic E. histolytica trophozoites (strain HM1:IMSS) is a calcium- and phospholipase A-dependent event initiated by the binding to the target cell of the galactose-inhibitable surface lectin of the parasite. We utilized phorbol esters as a probe to determine whether an amebic protein kinase C has a role in the cytolytic event. The addition of phorbol 12-myristate 13-acetate (PMA) at 10(-6) or 10(-7) M resulted in a greater than twofold enhancement of amebic killing of target CHO cells over 30 min (P less than 0.01). Prior exposure of only the amebae, but not the CHO cells, to PMA produced a similar effect (P less than 0.01). The inactive analog 4-alpha-phorbol had no effect on amebic killing of CHO cells. The PMA-mediated enhancement of amebic cytolysis persisted for up to 60 min after a 5-min exposure; however, after a 30-min exposure to PMA (10(-6) M) there was no augmentation of amebic killing of CHO cells. PMA (10(-6) M) did not promote adherence of parasites to CHO cells but did enhance amebic cytolysis of previously adherent target cells (P less than 0.01). Sphingosine, a specific inhibitor of protein kinase C, abolished both the PMA-stimulated and the basal cytolytic activity of E. histolytica. PMA enhanced CHO cell cytolysis by the less virulent wild-type strain H-303:NIH (P less than or equal to 0.02) but did not augment the activity of the less virulent strain H-200:NIH or two avirulent clones of HM1 (L6 and C919). In summary, these experiments with the phorbol esters and sphingosine as probes to modulate the activity of protein kinase C indicate participation of a parasite protein kinase C in the cytolytic activity of virulent, axenic E. histolytica trophozoites and thus in the pathogenesis of amebiasis.     

Production of species-specific murine monoclonal antibodies against Cryptococcus neoformans which recognize a noncapsular exoantigen

Three monoclonal antibodies (MAbs), designated 7C5, 7C9, and 5G8, against a cytoplasmic antigen of Cryptococcus neoformans were produced. MAbs 7C5 and 7C9 recognize culture filtrate antigen (exoantigen) of both encapsulated and nonencapsulated isolates of this pathogen, which suggests that they do not recognize capsular polysaccharide material. This is supported by immunofluorescence data which show reactivity of all 3 MAbs to cytoplasm and cell membranes only. MAb 7C9 also recognized C. neoformans var. gattii antigens but no other fungal pathogens tested in an enzyme-linked immunosorbent assay, while 7C5 and 5G8 recognized antigens of the cross-reactive pathogen Trichosporon beigelii but did not recognize either C. neoformans var. gattii isolates or any other fungal antigens. By Western blot (immunoblot), 7C9 detected antigen at 110 to 120, 65 to 70, 45 to 50, and 36 to 38 kDa; in addition to the latter band, the other two MAbs recognized a band at approximately 30 kDa. All three MAbs were of the immunoglobulin G1 subclass. The two MAbs which are capable of reacting with noncapsular culture supernatant antigen have possible uses in serodiagnosis, particularly in AIDS patients infected with C. neoformans, since in this group the present latex agglutination test has some limitations.     

Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.     

Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction

 An attempt to identify cysts of Entamoeba histolytica and E. dispar in human stool was conducted by polymerase chain reaction (PCR) using two sets of primers (p11 plus p12 and p13 plus p14) specific for either species of ameba. The cysts in stool specimens obtained from 12 infected individuals were concentrated, freeze-thawed, and treated with Triton X-100 before their examination by PCR. The results of PCR on the cysts were generally consistent with data obtained by PCR on ameba trophozoites hatched from the cysts, by zymodeme analysis, and by enzyme-linked immunosorbent assay (ELISA) and with clinical findings. This PCR was negative for the stool containing large numbers of cysts of either E. coli, E. hartmanni, or Giardia lamblia as well as for the stool specimens obtained from uninfected individuals. The ameba cyst in stool processed using the present method was effective for the PCR analysis even after 1 month of storage at 4°C. The present PCR was sensitive enough to detect ten cysts of either of the amebae. Received: 20 February 1996 / Accepted: 16 July 1996     

Differentiation of Entamoeba histolytica from E. dispar facilitated by monoclonal antibodies against a 150-kDa surface antigen

Murine monoclonal antibodies (mAbs) were produced against an n-octyl-β-D-glucopyranoside- extracted fraction of trophozoites of Entamoeba histolytica HM-1:IMSS. Four of the mAbs were reactive with a 150-kDa surface antigen characterized by Western-immunoblot analysis under nonreducing conditions. When the reactivity of the four mAbs with nine reference strains of E. histolytica was examined by an indirect fluorescence antibody test, two of the mAbs (EH3015 and EH3023) were found to react with all nine strains and the other two mAbs (EH3056 and EH3126) reacted with seven strains. The four mAbs did not react with any E. dispar reference strain or with other enteric protozoan parasites. The reactivity of EH3015 and EH3023 with numerous isolates of E. histolytica and E. dispar collected in our laboratories was also examined. The 2 mAbs reacted with all of the 37 E. histolytica isolates tested but did not react with any of the 33 isolates of E. dispar. These results indicate that common antigenic epitopes of E. histolytica are on the 150-kDa surface molecule and that mAbs can distinguish between E. histolytica and E. dispar. Received: 12 October 1996 / Accepted 13 December 1996     

Murine monoclonal antibodies which recognize adenosine deaminase from calf, mouse, rat and man

Three monoclonal antibodies against calf adenosine deaminase were obtained from mice. All three strongly cross-react with the rat and human forms of adenosine deaminase, and two of them with the mouse enzyme. We show that these reagents can be useful for the preparation of adenosine deaminase-free cell culture media and consider their potential interest for the early immunofluorescence detection of T-cell acute lymphoblastic leukemias.     

Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR.

Amoebiasis is caused by two distinct species, a pathogenic form (Entamoeba histolytica) and a nonpathogenic form (Entamoeba dispar), which are morphologically identical. Although the distinction between these two species is of great clinical importance, the methods developed for this purpose either are very time-consuming or involve laborious procedures for isolation of the DNA. We report here a simple PCR method starting with fresh stool specimen that allows for the sensitive and reliable distinction between E. histolytica and E. dispar. After initial concentration by the sodium acetate-acetic acid-formalin (SAF) method and digestion with proteinase K, a 0.88-kb sequence of the multicopy 16S rRNA gene served as a target for PCR amplification. The method starting with unpreserved specimens proved to be very sensitive and was not influenced by the quick exposure to SAF fixative during the initial concentration step. However, storage in SAF fixative prior to testing resulted in a decreased sensitivity within 2 days. The detection limit of the method was as low as one copy of the 16S rRNA gene. No cross-reactivity was observed with other common intestinal protozoa. Mixed infections involving both E. histolytica and E. dispar could easily be detected at a ratio of 1:10,000 by agarose gel electrophoresis or a DNA hybridization immunoassay.     

Evaluation of an antigencapture enzyme immunoassay for detection ofEntamoeba histolytica in stool samples

In order to identify the prevalence ofEntamoeba histolytica in tourists with diarrhoea returning from countries of the developing world, sensitivity and specificity of a commercially available enzyme immunoassay (EIA) kit for the detection ofEntamoeba histolytica coproantigen in stool were evaluated. Five hundred seventy-seven specimens from 469 patients were examined by microscopy and EIA. Sixty-two specimens from 49 patients were considered positive forEntamoeba histolytica. Compared with microscopic examination of stool samples, the EIA was found to be slightly more sensitive (90.3% vs. 87.1%) and was 97.7% specific forEntamoeba histolytica.     

Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin.

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme profiles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.     

Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase

The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.     

Production of monoclonal antibodies to rotavirus suitable for detection of rotavirus in stool samples by one-step EIA.

Two stable hybridoma cell clones producing high amounts of monoclonal antibodies (MAb) to rotavirus have been selected. The MAbs were shown to recognize different epitopes of the major inner capsid rotavirus protein VP 6. The two types of MAb in question cross-reacted with human and animal group A rotaviruses. Due to their high affinity and good binding capacity to microtiter plates a simultaneous EIA was developed for detection of rotavirus in stool samples. The sensitivity and specificity of MAb one-step EIA was compared with an approved polyclonal sandwich EIA by testing 1309 stool samples.     

Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.     

Purine-metabolising enzymes in Entamoeba histolytica

The enzymes that catalyse the salvage of purines in Entamoeba histolytica trophozoites have been surveyed. Adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), adenine phosphoribosyltransferase (PRTase) (EC 2.4.2.7), xanthine PRTase (EC 2.4.2.22) and hypoxanthine PRTase (EC 2.4.2.8) were all detected in cell homogenates but only at low activities, whereas AMP deaminase (EC 3.5.4.6) and guanine PRTase (EC 2.4.2.8) were not found. Phosphorylases (EC 2.4.2.1) active in both anabolic and catabolic directions were present and all nucleosides tested were phosphorylated by kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73). 3'-Nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were found, the former being mainly particulate. Nucleotide interconversion enzymes (adenylosuccinate lyase, EC 4.3.2.2; adenylosuccinate synthetase, EC 6.3.4.4; IMP dehydrogenase, EC 1.2.1.14; GMP synthetase, EC 6.3.5.2 and GMP reductase, EC 1.6.6.8) were not detected. The results suggest that in E. histolytica the main route of nucleotide synthesis is from the individual bases through the actions of phosphorylases and kinases.     

Chromatin organization in Entamoeba histolytica

The chromatin structure of Entamoeba histolytica was investigated. It was found that this protozoan organizes its chromatin in nucleosome-like particles 10 nm in diameter, but digestion of the chromatin with micrococcal nuclease did not render a regularly spaced DNA ladder in agarose gels. Southern blot analysis of the products of Entamoeba chromatin digestion using total amebic DNA and a non-transcribed repetitive sequence produced a banding pattern characteristic of eukaryotic chromatin with a repetitive size of approximately 130 bp. Conversely, hybridization with two active gene probes, actin and ribosomal RNA, showed that these sequences are not part of the chromatin organized in nucleosomes. It was also found that the basic nuclear proteins differ from histones of higher eukaryotes in electrophoretic mobility. Screening of an E. histolytica HM1-IMSS genomic library with Saccharomyces cerevisiae H3 and H4 genes and attempts to amplify E. histolytica sequences, homologous to these yeast histone genes, gave negative results suggesting that the Entamoeba proteins involved in chromatin organization are not typical histones.