Regina Helena Saramago Peralta

Use of Taenia crassiceps Cysticercus Antigen Preparations for Detection of Antibodies in Cerebrospinal Fluid Samples from Patients with Neurocysticercosis (Taenia solium)

ABSTRACT Antigen extracts obtained from the vesicular fluid of Taenia crassiceps cysticerci and from fractions purified by affinity chromatography with the lectin concanavalin A and the glycoprotein antigen separated by electrophoresis were used for the detection of Taenia solium anticysticercus antibodies. The sensitivity and specificity obtained for all antigens were 100% in enzyme-linked immunosorbent assay with good reproducibility. Using immunoblotting of the three antigens, low-molecular-mass peptides (18 and 14 kDa) were characterized only in cerebrospinal fluid samples from patients with neurocysticercosis. The results confirm that antigen fractions purified from T. crassiceps cisticerci are important sources of specific peptides and proved to be efficient in detecting anti-T. solium antibodies.

Comparison of multiplex-PCR and antigen detection for differential diagnosis of Entamoeba histolytica

Amebiasis is an infection caused by Entamoeba histolytica. However, differentiation between E. histolytica and Entamoeba dispar, which are morphologically identical species, is essential for treatment decision, precaution of the invasive disease and public health. The purpose of the present study was to evaluate a Multiplex -PCR for detection and differentiation of E. histolytica from E. dispar from fresh stool samples in comparison with the coproantigen commercial ELISA. Microscopic examination of stools using the Coprotest method, detection of stool antigen by enzyme-linked immunosorbent assay kit and a home made Multiplex-PCR, were used for the diagnosis of amoebiasis infection. Analysis of the 127 stools samples by microscopy examination demonstrated that only 27 (21%) samples were positive for E. histolytica/E. dispar complex. Among these stool samples, 11 were positive by Multiplex-PCR, with nine presenting the diagnostic fragment characteristic of E. dispar (96 bp) and two presenting diagnostic fragment of E. histolytica (132 bp). Among negative samples detected by microscopic examination, three positive samples for E. dispar and one positive for E. histolytica by Multiplex-PCR was observed. This denotes a low sensibility of microscopic examination when a single stool sample is analyzed. Assay for detection of E. histolytica antigen was concordant with multiplex-PCR in relation to E. histolytica. Statistical analysis comparing the sensibility tests was not done because of the low number of E. histolytica cases. The results demonstrate the importance of the specific techniques use for the differentiation between E. histolytica and E. dispar.

Evaluation of an antigen from Taeniacrassiceps cysticercus for the serodiagnosis of neurocysticercosis

We report here the evaluation of an antigen from Taenia crassiceps cysticercus as a potential reagent in an enzyme-immunoelectrotransfer blotting assay (EITB) and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of neurocysticercosis (NC) using clinical specimens obtained from patients in different phases of the disease. Serum and cerebrospinal fluid (CSF) samples from 64 patients suspected of having NC according to clinical manifestation and brain computed tomography were tested by ELISA with Taenia solium total saline antigen (ELISA-Tso) and by immunoblotting with T. crassiceps glycoproteins antigen (EITB-gpTcra). Forty-five serum samples were also tested immunoblotting with T. solium glycoproteins antigen (EITB-gpTso) and 30 were tested by ELISA with T. crassiceps 14 kDa glycoprotein (ELISA-gp14Tcra). Serum samples from apparently healthy individuals without any parasitic disease and from patients with other parasitic diseases were included as controls. The results of ELISA-Tso analysis with CSF obtained from 64 patients with NC showed that 53 (83%) were reactive. EITB-gpTcra analysis with serum from the same group of patients showed a sensitivity of 91%. Results of EITB-gpTso and EITB-gpTcra analysis with serum samples demonstrated an agreement of 100% between both tests. ELISA-gp14Tcra was positive in 23 (77%) sera, 22 with paired CSF positive. When ELISA-gp14Tcra results were compared to EITB-Tso results, a relative sensitivity of 95% was observed. All serum samples from the control group were negative in ELISA-gp14Tcra and only one serum from an individual with Taenia saginata was reactive in this assay, showing a specificity of 99% for ELISA-gp14Tcra. This fraction was purified in only one step with a good yield for use in immunoassays. We suggest that the gp14Tcra antigen can be used for detecting anti-cysticercus antibodies in serum samples for epidemiological investigation purposes and also for diagnostic screening of NC patients.

Differential identification of Entamoeba spp. based on the analysis of 18S rRNA

Entamoeba histolytica is known to cause intestinal and extra-intestinal disease while the other Entamoeba species are not considered to be pathogenic. However, all Entamoeba spp. should be reported when identified in clinical samples. Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features overlap. E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii are morphologically identical but can be differentiated using molecular tools. We developed a polymerase chain reaction (PCR) procedure followed by DNA sequencing of specific regions of 18S rRNA gene to differentiate the Entamoeba spp. commonly found in human stools. This approach was used to analyze 45 samples from cases evaluated for the presence of Entamoeba spp. by microscopy and a real-time PCR method capable of differential detection of E. histolytica and E. dispar. Our results demonstrated an agreement of approximately 98% (45/44) between the real-time PCR for E. histolytica and E. dispar and the 18S rRNA analysis described here. Five previously negative samples by microscopy revealed the presence of E. dispar, E. hartmanii, or E. coli DNA. In addition, we were able to detect E. hartmanii in a stool sample that had been previously reported as negative for Entamoeba spp. by microscopy. Further microscopic evaluation of this sample revealed the presence of E. hartmanii cysts, which went undetected during the first microscopic evaluation. This PCR followed by DNA sequencing will be useful to refine the diagnostic detection of Entamoeba spp. in stool and other clinical specimens.

Molecular and serological techniques to detect co-circulation of DENV, ZIKV and CHIKV in suspected dengue-like syndrome patients

BACKGROUND Arboviruses are important emerging viruses worldwide. The signs and symptoms of Zika virus (ZIKV) infection are similar to those presented by infections with dengue virus (DENV) and chikungunya virus (CHIKV). Furthermore, diagnosis of ZIKV infection is particularly challenging in dengue endemic regions and with co-circulation of DENV, CHIKV, and ZIKV, making diagnosis based solely on clinical and epidemiological data unreliable. As these three viral infections share similar clinical manifestations, differential diagnosis is crucial. OBJECTIVES In this study, diagnoses of ZIKV, CHIKV and DENV infections were investigated in 30 patients with suspected dengue fever residing in the area of co-circulation of these three arboviruses. STUDY DESIGN The study included whole blood and/or serum samples obtained from 30 patients with suspected dengue fever. All patients were tested for DENV infection as well as for CHIKV and ZIKV infections. Assays for detecting anti-DENV IgM and DENV RNA by semi-nested RT-PCR and ZIKV and CHIKV RNA by real-time RT-PCR were performed. RESULTS DENV RNA was not detectable in any of the clinical samples, whereas ZIKV RNA was detectable in 17 samples (56.7%). Co-infection by ZIKV and CHIKV was documented in one case. Of the 17 ZIKV-positive individuals, 8 showed reactivity for anti-DENV IgM, which suggested recent DENV infection, cross-reactivity or co-infection. CONCLUSION Our findings confirm that accurate laboratory testing is of paramount importance for differential diagnosis in areas of simultaneous transmission of different arboviruses with similar clinical presentations.

Serodiagnosis of neurocysticercosis in patients with epileptic seizure using ELISA and immunoblot assay

Sera from 88 patients from Santa Catarina and São Paulo states of Brazil, with epileptic seizures who underwent cerebral computed tomography (CT) were analyzed for the detection of antibodies to T. solium cysticercus by ELISA and Immunoblot (IB) with the following antigens: Taenia solium cysticercus total saline (Tso), Taenia crassiceps cysticercus vesicular fluid (Tcra-vf) and T. crassiceps cysticercus glycoproteins (Tcra-gp). ELISA carried out with Tso, Tcra-vf and Tcra-gp antigens showed 95%, 90% and 80% sensitivities, respectively, and 68%, 85% and 93% specificities, respectively. In the epileptic patients group, ELISA positivity was 30%, 51% and 35% with Tso, Tcra-vf and Tcra-gp antigens respectively. Considering the IB as the confirmatory test, the positivity was 16% (14/88) in the epileptic patients total group and 22% (12/54) in the epileptic patients with positive CT and signals of cysticercosis. We found a significant statistical correlation among ELISA or IB results and the phase of the disease when any antigens were used (p < 0.05). We emphasize the need to introduce in the laboratory routine the search for neurocysticercosis (NC) in patients presenting with epileptic seizures because of the high risk of acquiring NC in our region and its potential cause of epilepsy.

LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools

BackgroundSix species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species.MethodsPCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools.ResultsUsing this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli.ConclusionThese results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.

Taenia crassiceps cysticerci: characterization of the 14-kDa glycoprotein with homologies to antigens from Taenia solium cysticerci.

Glycoproteins from the total vesicular fluid of Taenia crassiceps (VF-Tc) were prepared using three different purification methods, consisting of ConA-lectin affinity chromatography (ConA-Tc), preparative electrophoresis (SDS-PAGE) (14 gp-Tc), and monoclonal antibody immunoaffinity chromatography (18/14-Tc). The complex composition represented by the VF-Tc and ConA-Tc antigens revealed peptides ranging from 101- to 14-kDa and from 92- to 12-kDa, respectively. Immunoblotting using lectins confirmed glucose/mannose (glc/man) residues in the 18- and 14-kDa peptides, which are considered specific and immunodominant for the diagnosis of cysticercosis, and indicated that these fractions are glycoproteins. Serum antibodies from a patient with neurocysticercosis that reacted to the 14 gp band from T. crassiceps (Tc) were eluted from immunoblotting membranes and showed reactivity to 14 gp from Taenia solium. In order to determine the similar peptide sequence, the N-terminal amino acid was determined and analyzed with sequences available in public databases. This sequence revealed partial homology between T. crassiceps and T. solium peptides. In addition, mass spectrometry along with theoretical M(r) and pI of the 14 gp-Tc point suggested a close relationship to some peptides of a 150-kDa protein complex of the T. solium previously described. The identification of these common immunogenic sites will contribute to future efforts to develop recombinant antigens and synthetic peptides for immunological assays.

Estudo soroepidemiológico da cisticercose humana em um município do Estado do Piauí, Região Nordeste do Brasil

As part of parasitological studies in the area surrounding the Serra da Capivara National Park, Piaui State, Northeast Brazil, from 1999 to 2001, the current study aimed to evaluate the epidemiological profile of human cysticercosis in the Municipality of Joao Costa. Clinical and epidemiological data were obtained, and blood samples were drawn for immunoenzymatic serological tests (ELISA and Western blot), using Taenia crassiceps as the antigen. The first stage, in 1999, investigated 169 individuals with a confirmed history or suspicion of infection/disease involving the teniasis/cysticercosis complex, along with the family members. Some 13.6% of the individuals were seroreactive for cysticercosis by the ELISA method. The second stage, in 2001, evaluated 92 serum samples of individuals who had been detected as reactive for cysticercosis in the first stage, along with their family members; 24% of the samples were reactive to cysticercosis by ELISA and 29% by Western blot. During this same stage a coprological survey was performed with 701 individuals, including volunteers. Prevalence of intestinal parasites was 51%, with a higher prevalence of protozoans (95%) than helminths (5%). The results indicate the endemicity of cysticercosis in the area, in addition to the high frequency of intestinal protozoan infections.

Evaluation of immunological, parasitological and molecular methods for the diagnosis of Schistosoma mansoni infection before and after chemotherapy treatment with praziquantel in experimentally infected Nectomys squamipes

In low endemicity areas of schistosomiasis, the recommended diagnostic method of coprological examination results in an underestimation of infection cases. Alternative diagnostic methods have been developed, such as immunodiagnostic and molecular techniques. In this study we evaluated three methods used in the diagnosis of Schistosoma mansoni infection: parasitological (Kato-Katz), immunological (ELISA) and molecular (real time PCR), and also investigated the sensitivity of each technique in the cure determination after treatment with praziquantel using the water rat Nectomys squamipes, a natural reservoir for S. mansoni, as an experimental model. Two infection laboratory experiments were carried out. The first experiment aimed to observe the evolution of the immunological response in the first moments after infection and in the first months after treatment. The second experiment aimed to compare the efficacy of the three diagnostic techniques after infection and after treatment over a more extended time period. In the first experiment, 44% of the infected animals showed IgG reactivity after two weeks of infection, and 94% were positive based on serology 30 days after infection. The serological IgG titers increased just after infection but decreased gradually after treatment. In the second experiment, 89% of the animals showed positive IgG titers 22 days after infection. Only 68% of the animals showed positive results on the coproscopic diagnostic analysis and 79% did so by qPCR, 50 days after infection. Treated animals showed negative results on coproscopy one month after treatment but remained positive by serology even 12 months after treatment, although showing a decline in immunologic reaction after treatment. By qPCR analysis, all animals showed negative results three months after treatment, except for one animal. The parasitosis can be detected by coproscopy only six weeks after infection, and by serology 14 days after infection. The qPCR was a better diagnostic method for confirming the infection cure of S. mansoni. In early infection, this method was less efficient than serology but was slightly more efficient than the Kato-Katz method. We suggest that the methods should be used in low endemic areas as follows: serology should be used in the initial diagnosis in a population with potential positive cases; subsequently, coproscopy should be used in IgG positive cases to confirm the current infection; and qPCR should be used to evaluate the infection cure after treatment and is also a very valuable tool when there are cases showing positive IgG and negative coproscopy.