Prevalence of human cryptosporidiosis in the Americas: systematic review and meta-analysis.

Cryptosporidiosis is a disease caused by the Cryptosporidium spp parasite. As some species of Cryptosporidium have a wide host spectrum, the characterization of the pathogen at the species or genotype level is of great importance to define the sources of infection for humans and the potential for public health. This study investigated the diversity of the genus Cryptosporidium spp. in humans from all over the American continent and observed whether the method used to search for the parasite influenced the prevalence found in the Americas. This systematic review was carried out using the Pubmed, Science direct, Lilacs, Scielo, and Scopus databases with publications from January 1, 2010, to December 31, 2020. For data synthesis, the PRISMA flowchart was used and for the meta-analysis we used the MetaXL program. Of the selected publications, 57, 9 and 16 belonged to the region of South, Central and North America, respectively. The prevalence found for South, Central, and North America was 7%, 7%, and 8%, respectively, when analyzing publications that used only the microscopy method. When we analyzed the publications that used immunological and molecular methods, we found prevalences of 10%, 9%, and 21% for South, Central, and North America, respectively. The C. hominis subtype IbA10G2 was the most reported in the American continent, followed by subtype IeA11G3T3 and, for C. parvum, subtype IIaA15G2RI was the most reported. In conclusion, Cryptosporidium spp. is present throughout the American continent and its prevalence is higher when immunological and/or molecular methods are used, in addition to direct microscopic examination.

Molecular characterization of Cryptosporidium spp. obtained from fecal samples of immunosuppressed patients from Brazil.

BACKGROUND: Cryptosporidium spp. are pathogenic protozoans that play an important role in developing diseases in the elderly, children, and immunosuppressed individuals. METHODS: The objective of this study was to detect and genetically characterize Cryptosporidium spp. in kidney transplanted patients (n = 97 samples; group 1) and immunosuppressed individuals from an outpatient clinic suspected of having Cryptosporidium infection (n = 53 samples; group 2). All fecal samples were analyzed by parasitological stool examination, immunochromatographic test, and real-time polymerase chain reaction (real-time PCR). Cryptosporidium-positive samples were tested using nested PCR for the gp60 gene, followed by sequencing for subtype determination. RESULTS: Parasitological examination was negative in all Group 1, and positive in four Group 2 samples. Real-time PCR revealed Cryptosporidium in 13 samples: four in Group 1 (three C. hominis and one C. parvum) and nine in Group 2 (seven C. hominis, one C. parvum, and one mixed C. hominis/C. parvum). The immunochromatographic test was reactive in 11 samples (four in Group 1 and seven in Group 2). All 11 C. hominis isolates were identified as subtype IbA10G2 and one C. parvum as subtype IIbA15G2R1. All C. hominis belonged to subtype IbA10G2, which is recognized as the most prevalent and pathogenic subtype. CONCLUSIONS: This study showed, for the first time, that the presence of Cryptosporidium subtypes is considered more virulent in Brazilian transplanted kidney patients.

Prevalence of human cryptosporidiosis in the Americas: systematic review and meta-analysis

ABSTRACT Cryptosporidiosis is a disease caused by the Cryptosporidium spp parasite. As some species of Cryptosporidium have a wide host spectrum, the characterization of the pathogen at the species or genotype level is of great importance to define the sources of infection for humans and the potential for public health. This study investigated the diversity of the genus Cryptosporidium spp. in humans from all over the American continent and observed whether the method used to search for the parasite influenced the prevalence found in the Americas. This systematic review was carried out using the Pubmed, Science direct, Lilacs, Scielo, and Scopus databases with publications from January 1, 2010, to December 31, 2020. For data synthesis, the PRISMA flowchart was used and for the meta-analysis we used the MetaXL program. Of the selected publications, 57, 9 and 16 belonged to the region of South, Central and North America, respectively. The prevalence found for South, Central, and North America was 7%, 7%, and 8%, respectively, when analyzing publications that used only the microscopy method. When we analyzed the publications that used immunological and molecular methods, we found prevalences of 10%, 9%, and 21% for South, Central, and North America, respectively. The C. hominis subtype IbA10G2 was the most reported in the American continent, followed by subtype IeA11G3T3 and, for C. parvum, subtype IIaA15G2RI was the most reported. In conclusion, Cryptosporidium spp. is present throughout the American continent and its prevalence is higher when immunological and/or molecular methods are used, in addition to direct microscopic examination.

New insights into the detection and molecular characterization of Cryptosporidium with emphasis in Brazilian studies: a review.

Cryptosporidium spp. is a pathogenic protozoan present in the gastrointestinal tract of several hosts. This protozoan was originally classified as within the Coccidia Class and has recently been reclassified to gregarine based on studies that observed the evolutionary phases from the process of excision and sequencing of the 18S rRNA gene. Molecular biology techniques have become diagnostic tools and have also been used to understand the epidemiology of Cryptosporidium spp., since several species of this genus are very similar morphologically and morphometrically. Molecular techniques have been used in the identification of parasites, at the species and subtypes levels and to study disease transmission. The laboratory diagnosis of human cryptosporidiosis can be made by parasite detection methods, such as optical microscopy, antigens or genetic material detection, as well as serum antibodies raised to Cryptosporidium spp. Molecular methods were developed and allowed, not only an extensive revision of the taxonomy, but also an improvement in the laboratory diagnosis. In Brazil, there are few reports of Cryptosporidium spp. outbreaks in humans and all of them took place in nurseries. A few epidemiological studies developed in Brazil have used molecular methods for the detection of Cryptosporidium spp., as well as genotyping studies of their species and subtypes. The use of real-time PCR, together with microscopy and immunochromatography techniques, would result in a more precise diagnosis of cryptosporidiosis. The analysis of genotypes, subtypes and clonality of Cryptosporidium could be useful to understand and define the prognosis and severity of infections.

Performance of an Ultra-Sensitive Assay Targeting the Circulating Anodic Antigen (CAA) for Detection of Schistosoma mansoni Infection in a Low Endemic Area in Brazil.

Techniques with high sensitivity and specificity are required for an accurate diagnosis in low-transmission settings, where the conventional parasitological methods are insensitive. We determined the accuracy of an up-converting phosphor-lateral flow circulating anodic antigen (UCP-LF CAA) assay in urine and serum for Schistosoma mansoni diagnosis in low-prevalence settings in Ceará, Brazil, before and after praziquantel treatment. Clinical samples of a total of 258 individuals were investigated by UCP-LF CAA, point-of-care-circulating cathodic antigen (POC-CCA), soluble worm antigen preparation (SWAP)-ELISA and Kato-Katz (KK); a selection of 128 stools by real-time PCR technique. Three and 6-weeks after treatment, samples were collected and evaluated by detection Schistosoma circulating antigens (CAA and CCA). The UCP-LF CAA assays detected 80 positives (31%) with urine and 82 positives (31.8%) with serum. The urine POC-CCA and serum SWAP-ELISA assays detected 30 (11.6%) and 107 (40.7%) positives, respectively. The Kato-Katz technique revealed only 4 positive stool samples (1.6%). Among the 128 individuals with complete data records, 19 cases were identified by PCR (14.8%); Sensitivities and specificities of the UCP-LF CAA assays, determined versus a combined reference standard based on CCA/KK/PCR positivity, ranged from 60-68% to 68-77%, respectively. In addition only for comparative purposes, sensitivities of the different assays were determined vs. a comparative reference based on CAA/KK/PCR positivity, showing the highest sensitivity for the urine CAA assay (80%), followed by the serum CAA (70.9%), SWAP-ELISA (43.6%), PCR (34.5%), POC-CCA (29.1%), whilst triplicate Kato-Katz thick smears had a very low sensitivity (3.6%). CAA concentrations were higher in serum than in urine and were significantly correlated. There was a significant decrease in urine and serum CAA levels 3 and 6-weeks after treatment. The UCP-LF CAA assays revealed 33 and 28 S. mansoni-infected patients at the 3- and 6-week post-treatment follow-up, respectively. The UCP-LF CAA assays show high sensitivity for the diagnosis of S. mansoni in low-endemicity settings. It detects a considerably higher number of infections than microscopy, POC-CCA or PCR. Also it shows to be very useful for evaluating cure rates after treatment. Hence, the UCP-LF CAA assay is a robust and promising diagnostic approach in low-transmission settings.

A gel-free proteomic analysis of Taenia solium and Taenia crassiceps cysticerci vesicular extracts.

The taeniasis/cysticercosis complex is a zoonosis caused by the presence of the parasite Taenia solium in humans. It is considered a neglected disease that causes serious public health and economic problems in developing countries. In humans, the most common locations for the larval form are the skeletal muscles, ocular system, and the central nervous system, which is the most clinically important. Several glycoproteins of T. solium and Taenia crassiceps cysticerci have been characterized and studied for their use in the immunodiagnosis of neurocysticercosis and/or the development of synthetic or recombinant vaccines against cysticercosis. The aim of this study was to perform a gel-free shotgun proteomic analysis to identify saline vesicular extract (SVE) proteins of T. solium and T. crassiceps cysticerci. After solubilization of the SVE with and without surfactant reagent and in-solution digestion, the proteins were analyzed by LC-MS/MS. Use of a surfactant resulted in a significantly higher number of proteins that were able to be identified by LC-MS/MS. Novel proteins were identified in T. solium and T. crassiceps SVE. The qualitative analysis revealed a total of 79 proteins in the Taenia species: 29 in T. solium alone, 11 in T. crassiceps alone, and 39 in both. These results are an important contribution to support future investigations and for establishing a Taenia proteomic profile to study candidate biomarkers involved in the diagnosis or pathogenesis of neurocysticercosis.

Exploring lipid and apolipoprotein levels in chronic hepatitis C patients according to their response to antiviral treatment.

BACKGROUND: Hepatitis C virus is known to be highly dependent of lipid metabolism to infect new cells and replicate. AIMS: To investigate lipid and apolipoprotein profile in chronic HCV patients according to treatment response. METHODS: Patients recruited from the Hepatitis Treatment Center at Niteroi (Brazil) who received interferon (IFN)-based therapies were separated into two groups, those who achieved sustained virological response (SVR) or not (non-SVR). Another group of patients treated with IFN-free direct-acting antiviral (DAA) therapies was followed from before starting the treatment until one year after therapy. Triglycerides, total cholesterol and fractions were determined by colorimetric and/or electrophoresis techniques. Lecithin cholesterol acyltransferase (LCAT) activity and serum levels of apolipoproteins A1, A2, B, C2, C3 and E were assessed by enzymatic and multiplex assays, respectively. RESULTS: We studied 114 patients, and SVR was reached in 28 (39.4%) patients treated with IFN-therapy and in all (100%) patients who received DAA. Non-SVR patients (n = 43) presented altered liver parameters post-treatment. Levels of total cholesterol, LDL-C, VLDL-C and triglycerides were significant higher in SVR group. In contrast, LCAT activity and HDL-C levels were elevated in non-SVR patients. Only apolipoproteins B, C2 and C3 levels were increased in SVR group. The follow-up of SVR-DAA patients (n = 43) revealed a significant and progressive increase in serum levels of total cholesterol, LDL-C, VLDL-C and triglycerides. CONCLUSIONS: After a successful treatment, chronic hepatitis C patients experienced a reestablishment of lipid metabolism. Our results suggest that the monitoring of serum lipids could be a practical and routine laboratory tool to be applied during the treatment follow-up.

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.

A Luminex-based single DNA fragment amplification assay as a practical tool for detecting and serotyping dengue virus.

Dengue is a mosquito-borne viral infection that can evolve from subclinical to severe forms of disease. Early recognition during initial primary and secondary infections correlates with a reduced case-fatality rate in susceptible groups. The aim of this study was to standardize a DNA hybridization assay based on the Luminex technology for detecting and serotyping dengue virus (DENV). Reference DENVs representing the four different serotypes were used as controls to standardize the test. For validation, 16 DENV isolates obtained from a reference laboratory were analyzed in a double-blind manner to validate the test. Sixty blood samples from patients suspected of having dengue fever were used to evaluate the methodology after the validation step, and the results were compared with the reference semi-nested RT-PCR. Additionally, five human samples of each Zika and Chikungunya confirmed patients were used for specificity analysis. The Luminex-based assay correctly identified all 16 DENV isolates. In the evaluation step, the results of the RT-PCR/Luminex assay showed a concordance of 86.7% with those of the semi-nested RT-PCR. None of other virus infection samples was amplified. This is the first description of a hybridization assay that can discriminate the four DENV serotypes using probes against a single DENV sequence. The results indicated that the RT-PCR/Luminex DENV assay designed and evaluated in this study is a valuable additional tool for the early and rapid detection and serotyping of DENV, which could, in the future, be applied to new targets such as the Zika and Chikungunya viruses.

Validation of western blot for Histoplasma capsulatum antibody detection assay.

BACKGROUND: Histoplasmosis is worldwide systemic mycoses caused by the dimorphic fungus Histoplasma capsulatum. The isolation and identification of H. capsulatum in culture is the reference test for histoplasmosis diagnosis confirmation. However, in the absence of it, serology has been used as a presumptive diagnosis through antibody and antigen detection. The purpose of the present study was to validate an immunoassay method (western blot) for antibodies detection in the diagnosis of histoplasmosis. METHODS: To validate the western blot (WB) a study was conducted using 118 serum samples from patients with histoplasmosis and 118 serum controls collected from January 2000 to December 2013 in residents of the Rio de Janeiro State, Brazil. Diagnostic validation parameters were calculated based on the categorization of results obtained in a 2 × 2 table and subjected to statistical analysis. In addition, the viability of deglycosylated histoplasmin antigen (ptHMIN) onto nitrocellulose membranes previously sensitized was evaluated during the same period. RESULTS: The WB test showed sensitivity of 94.9 %, specificity of 94.1 %, positive predictive value of 94.1 %, negative predictive value of 94.9 %, accuracy of 94.5 %, and almost perfect precision. Besides, the strips have proved to be viable for using at least 5 years after ptHMIN antigen sensitization. CONCLUSION: Western blot test using ptHMIN provides sensitive, specific, and faster results. Therefore, could be considered a useful tool in the diagnosis of histoplasmosis being used by public health system, even in situations where laboratory facilities are relatively limited.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina.

The identification and characterisation of Cryptosporidium genotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity of Cryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays for Cryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.

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.

Frequency of amoebiasis and other intestinal parasitoses in a settlement in Ilheus City, State of Bahia, Brazil

Introduction: This study evaluated the frequency of intestinal parasites, emphasizing the identification and differentiation of Entamoeba spp. Methods: Multiplex polymerase chain reaction (PCR), coproantigen tests and morphometric analysis were performed for Entamoeba spp. differentiation. Results: The overall frequency of intestinal parasites was 65%. Entamoeba histolytica was detected by the coproantigen test, and the PCR showed that Entamoeba dispar predominated in the population. In contrast, morphometric analysis was important for identifying Entamoeba hartmanni. Conclusions: It is possible to identify the causative agent of amoebiasis and to differentiate this agent from other species by combining techniques. .

A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area

The aim of this study was to evaluate the efficacy of a polymerase chain reaction (PCR)-based method to detect Schistosoma mansoni DNA in stool samples from individuals living in a low-endemicity area in Brazil. Of the 125 initial stool samples, 80 were ELISA reactive and eggs were identified in 19 of the samples by parasitological examination. For the PCR evaluations, 56 stool samples were selected and divided into five groups. Groups I-IV were scored negative for S. mansoni eggs by parasitological examination. Groups I and II were ELISA reactive, whereas Groups III and IV were ELISA nonreactive. Groups II and III were positive for other intestinal parasites. PCR testing scored eight samples as positive from these four groups. Group V represented the S. mansoni -positive group and it included ELISA-reactive samples that were scored positive for S. mansoni by one or more parasitological examinations (6/19 were positive by Kato-Katz method, 9/17 by saline gradient and 10/13 by Helmintex®). PCR scored 13 of these 19 samples as positive for S. mansoni . We conclude that while none of these methods yielded 100% sensitivity, a combination of techniques should be effective for improving the detection of S. mansoni infection in low-endemicity areas.

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

BACKGROUND: Six 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. METHODS: PCR 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. RESULTS: Using 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. CONCLUSION: These 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.

The identification and characterization of epitopes in the 30-34 kDa Trypanosoma cruzi proteins recognized by antibodies in the serum samples of chagasic patients.

Trypanosoma cruzi proteins with molecular weight between 30 and 34 kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34 kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools.

A conventional polymerase chain reaction-based method for the diagnosis of human schistosomiasis in stool samples from individuals in a low-endemicity area.

The aim of this study was to evaluate the efficacy of a polymerase chain reaction (PCR)-based method to detect Schistosoma mansoni DNA in stool samples from individuals living in a low-endemicity area in Brazil. Of the 125 initial stool samples, 80 were ELISA reactive and eggs were identified in 19 of the samples by parasitological examination. For the PCR evaluations, 56 stool samples were selected and divided into five groups. Groups I-IV were scored negative for S. mansoni eggs by parasitological examination. Groups I and II were ELISA reactive, whereas Groups III and IV were ELISA nonreactive. Groups II and III were positive for other intestinal parasites. PCR testing scored eight samples as positive from these four groups. Group V represented the S. mansoni -positive group and it included ELISA-reactive samples that were scored positive for S. mansoni by one or more parasitological examinations (6/19 were positive by Kato-Katz method, 9/17 by saline gradient and 10/13 by Helmintex®). PCR scored 13 of these 19 samples as positive for S. mansoni . We conclude that while none of these methods yielded 100% sensitivity, a combination of techniques should be effective for improving the detection of S. mansoni infection in low-endemicity areas.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil.

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.

Detection and differentiation of Cryptosporidium by real-time polymerase chain reaction in stool samples from patients in Rio de Janeiro, Brazil

This study reports the first genetic characterisation of Cryptosporidium isolates in Brazil using real-time polymerase chain reaction (RT-PCR). A total of 1,197 faecal specimens from children and 10 specimens from human immunodeficiency virus-infected patients were collected between 1999-2010 and screened using microscopy. Forty-eight Cryptosporidium oocyst-positive isolates were identified and analysed using a generic TaqMan assay targeting the 18S rRNA to detect Cryptosporidium species and two other TaqMan assays to identify Cryptosporidium hominis and Cryptosporidium parvum. The 18S rRNA assay detected Cryptosporidium species in all 48 of the stool specimens. The C. parvum TaqMan assay correctly identified five/48 stool samples, while 37/48 stool specimens were correctly amplified in the C. hominis TaqMan assay. The results obtained in this study support previous findings showing that C. hominis infections are more prevalent than C. parvum infections in Brazil and they demonstrate that the TaqMan RT-PCR procedure is a simple, fast and valuable tool for the detection and differentiation of Cryptosporidium species.