Heloisa Werneck de Macedo

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.

[Intestinal parasite infections in a semiarid area of Northeast Brazil: preliminary findings differ from expected prevalence rates].

We report on intestinal parasite infection prevalence in a population sample from Sao Raimundo Nonato, Southeast Piaui State, Brazil, aimed at comparison with previous studies on Trichuris trichiura and Ascaris lumbricoides infection. A total of 265 stool specimens were collected and examined by spontaneous sedimentation. Approximately 57% of specimens were infected with at least one parasite species. Entamoeba coli (35.8%), Endolimax nana (13.6%), Hymenolepis nana (9.4%), and hookworm (9.4%) were the most frequently observed parasites. Two cases of roundworm infection were detected, probably acquired outside the region. T. trichiura eggs were not found. Interestingly, neither A. lumbricoides nor T. trichiura has been found in local prehistoric human coprolites. Nevertheless, hookworm infection has been present in the region for at least 7,000 years.We report on intestinal parasite infection prevalence in a population sample from S o Raimundo Nonato, Southeast Piau State, Brazil, aimed at comparison with previous studies on Trichuris trichiura and Ascaris lumbricoides infection. A total of 265 stool specimens were collected and examined by spontaneous sedimentation. Approximately 57% of specimens were infected with at least one parasite species. Entamoeba coli (35.8%), Endolimax nana (13.6%), Hymenolepis nana (9.4%), and hookworm (9.4%) were the most frequently observed parasites. Two cases of roundworm infection were detected, probably acquired outside the region. T. trichiura eggs were not found. Interestingly, neither A. lumbricoides nor T. trichiura has been found in local prehistoric human coprolites. Nevertheless, hookworm infection has been present in the region for at least 7,000 years.

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.

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.

Avaliação de testes imunológicos para o diagnóstico da neurocisticercose

Background: The diagnosis of neurocysticercosis (NCC) has been made by association of neuroimaging studies and use of sensitive and specific serological assays. Objectives: Evaluating Elisa and Western blot (Wb) tests using a crude extract of Cysticercus cellulosae (Taenia solium) as antigen and a Wb test using a glycoprotein of Cysticercus longicollis (Taenia crassiceps) as antigen for the diagnosis of NCC. Methods: Serum samples from 43 patients with NCC: 21patients presenting clinical manifestations, cerebral computed tomography findings compatible with cerebral lesions and high levels of anti-cysticercus antibodies in cerebrospinal fluid (CSF); 22 patients with clinical manifestations and cerebral computed tomography findings compatible with cerebral lesions and serum samples from 229 patients with other parasitic infections were tested by Elisa standardized with crude extract of C. cellulosae and Wb standardized with glicoprotein of C. cellulosae and C. longicollis. Results: The Elisa test using crude extract of C. cellulosae showed specificity of 95% and sensibility of 71%. Both tests using glycoproteins of either C. cellulosae or C. longicollis showed specificity of 99% and sensibility of 86%. Conclusions: The use of immunological techniques for antibodies detection in CSF was shown to be an important tool for NCC diagnosis. However, detection of antibodies in serum lacked sensibility when Elisa was evaluated. The Wb assay in serum samples was sensitive and specific and it can be helpful for the diagnosis of the transitional form of NCC frequently not detected by cerebral computed tomography.

Blastocystis sp. in splenic cysts: causative agent or accidental association? A unique case report

BackgroundBlastocystis sp. is one of the most prevalent parasites found in human stool and has been recently considered an opportunistic emerging pathogen in immunocompromised individuals. However, cases of invasive intestinal infections and skin rashes have been attributed to infection by Blastocystis sp in immunocompetent individuals, suggesting that it is an emerging parasite with pathogenic potential.FindingsWe present a case of a 22 year old female patient who complained of pain in the left hypochondrium. Ultrasonography and abdominal computed tomography scans showed two splenic cysts. The cyst fluid analysis demonstrated numerous Blastocystis sp.; PCR and DNA sequencing analyses confirmed the presence of Blastocystis subtype 3.ConclusionsThis is, to our knowledge, the first case report of the presence of Blastocystis subtype 3 in extra-intestinal organs and is strong evidence that Blastocystis sp. is potentially pathogenic and invasive. However, further studies are required to determine the pathogenicity of the parasite.

Experimental amoebic liver abscess in hamsters caused by trophozoites of a Brazilian strain of Entamoeba dispar

It has been claimed that amoebic molecules such as amoebapore, galactose/N-acetyl galactosamine inhibitable lectin, and cysteine proteases are responsible for host tissue destruction and are present in both pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar. Some reports have provided evidence that after infection with E. dispar, pathological changes may occur in some humans. The aim of this study was to evaluate E. dispar pathogenicity by comparing it to the pathogenicity of E. histolytica through liver abscesses induced in hamsters. Syrian golden hamsters were challenged by intrahepatic inoculation with the 03C E. dispar strain or with two strains of E. histolytica (HM1:IMSS and EGG) to compare their virulence grades. As control groups, we used bacterial flora and Pavlovas modified medium. Lesions were verified at 1, 3 and 6 days after inoculation. Multiplex Polymerase Chain Reaction was performed to characterize each strain using EdP1/EdP2 and EhP1/EhP2 primers. The EGG and HM1:IMSS E. histolytica strains and 03C E. dispar were able to cause liver lesions. The EGG strain caused extensive hepatic abscesses, and trophozoites were found in the lesions throughout the three periods of study. The HM1:IMSS strain caused smaller abscesses when compared to EGG lesions; however, trophozoites were observed at 1 and 3 days after inoculation. The 03C E. dispar strain caused intermediate abscesses when compared to the others; trophozoites were observed in all periods analyzed. The EGG strain caused progressive evolution of the injury, which differed from the HM1:IMSS and 03C strains. These results strongly suggest that the 03C E. dispar strain is pathogenic in the experimental hamster model. Additional studies are necessary to identify potential factors that regulate the manifestation of virulence of this strain and others.

Differential diagnosis of Entamoeba spp. in clinical stool samples using SYBR green real-time polymerase chain reaction.

Amoebiasis, a disease caused by Entamoeba histolytica, is usually diagnosed by microscopic examination, which does not differentiate the morphologically identical species of the E. histolytica/E. dispar complex. Furthermore, morphologically similar species such as Entamoeba hartmanni contribute to misidentification. Therefore, there is a need for more sensitive and specific methods. This study standardized a multiplex real-time PCR system for E. histolytica and E. dispar and a single real-time PCR for E. hartmanni. The multiplex protocol detected up to 0.0143 pg of E. histolytica DNA and 0.5156 pg of E. dispar DNA, and the average melting temperature (T m) was 73°C and 70°C, respectively. For E. hartmanni, the T m was 73°C and the amplification was successful down to 0.03 fg of plasmid DNA. Negative controls and other intestinal parasites presented no amplification. Among the 48 samples tested, E. dispar DNA was detected in 37; none exhibited E. histolytica DNA and 11 were negative in the multiplex protocol. In 4 of these 11 samples, however, E. hartmanni DNA was amplified. SYBR Green is demonstrated to be an interesting option and these combined PCR reactions can improve laboratory diagnosis of amoebiasis in developing countries.

Nefropatia induzida por contraste: avaliação da proteção pela n-acetilcisteína e alopurinol em ratos uninefrectomizados

OBJECTIVE: Contrast medium-induced nephropathy is the third most frequent cause of iatrogenic acute renal failure involving inpatients. The present study was aimed at evaluating the protective effect of n-acetylcysteine and allopurinol in both male and female rats receiving diatrizoate. MATERIALS AND METHODS: Thirty-five young adult Wistar rats submitted to hydric restriction were divided into groups as follows: groups 1 and 2 (respectively male and female rats) receiving saline solution; groups 3 and 4 (respectively male and female rats) receiving diatrizoate; group 5 (male rats) receiving diatrizoate and n-acetylcysteine; group 6 (male rats) receiving diatrizoate and allopurinol; and group 7 (male rats) receiving diatrizoate and n-acetylcysteine + allopurinol. The glomerular filtration was evaluated by measurement of creatinine clearance. Students t-test and the test of signal were utilized for statistical analysis. RESULTS: Animal models receiving allopurinol did not present a significant increase in the creatinine levels, while n-acetylcysteine did not prevent the creatinine levels increase. CONCLUSION: Allopurinol has shown to be more effective than n-acetylcysteine in the renal function protection against sodium diatrizoate-induced damages. No difference has been found between male and female groups as regards the intensity of sodium diatrizoate-induced renal damages.