The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

BackgroundIntestinal parasitic infections are considered a serious public health problem and widely distributed worldwide, mainly in urban and rural environments of tropical and subtropical countries. Globally, soil-transmitted helminths and protozoa are the most common intestinal parasites. Blastocystis sp. is a highly prevalent suspected pathogenic protozoan, and considered an unusual protist due to its significant genetic diversity and host plasticity.Methodology/main findingsA total of 294 stool samples were collected from inhabitants of three rural valleys in Rio de Janeiro, Brazil. The stool samples were evaluated by parasitological methods, fecal culture, nested PCR and PCR/Sequencing. Overall prevalence by parasitological analyses was 64.3% (189 out of 294 cases). Blastocystis sp. (55.8%) was the most prevalent, followed by Endolimax nana (18.7%), Entamoeba histolytica complex (7.1%), hookworm infection (7.1%), Entomoeba coli (5.8%), Giardia intestinalis (4.1%), Iodamoeba butchilii (1.0%), Trichuris trichiura (1.0%), Pentatrichomonas hominis (0.7%), Enterobius vermicularis (0.7%), Ascaris lumbricoides (0.7%) and Strongyloides stercoralis (0.7%). Prevalence of IPIs was significantly different by gender. Phylogenetic analysis of Blastocystis sp. and BLAST search revealed five different subtypes: ST3 (34.0%), ST1 (27.0%), ST2 (27.0%), ST4 (3.5%), ST8 (7.0%) and a non-identified subtype.Conclusions/significanceOur findings demonstrate that intestinal parasite infection rates in rural areas of the Sumidouro municipality of Rio de Janeiro, Brazil are still high and remain a challenge to public health. Moreover, our data reveals significant genetic heterogeneity of Blastocystis sp. subtypes and a possible novel subtype, whose confirmation will require additional data. Our study contributes to the understanding of potential routes of transmission, epidemiology, and genetic diversity of Blastocystis sp. in rural areas both at a regional and global scale.

Anisakid nematode larvae from Trichiurus lepturus off coast of Rio de Janeiro were studied using light, laser confocal and scanning electron microscopy, in addition to a molecular approach. Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2), partial 28S (LSU) and internal transcribed spacers (ITS-1, 5.8S, ITS-2) of ribosomal DNA were amplified using the polymerase chain reaction and sequenced to evaluate the phylogenetic relationships between the nematode taxa. The morphological and genetic profiles confirmed that, of the 1,030 larvae collected from the 64 fish examined, 398 were analysed, of which 361 were Hysterothylacium sp. and 37 were Anisakis typica. Larvae of Hysterothylacium sp. were not identified to the species level due to the absence of similar sequences for adult parasites; however, the ITS sequence clustered in the phylogenetic tree with sequences of H. deardorffoverstreetorum, whereas an mtDNA cox-2 and LSU concatenated phylogenetic analysis demonstrated the presence of two clades, both of them under the same name as the larval H. deardorffoverstreetorum. Data on the occurrence of parasites during the winter and summer months were compared using the t-test. The greatest prevalence and intensity of infection were recorded for larval Hysterothylacium, with a prevalence of 51.56% and an intensity of up to 55 parasites per fish. The larval Anisakis exhibit a higher abundance and intensity of infection in the winter months, and those of Hysterothylacium during the summer. However, the t-test indicated no significant differences between the abundance and intensity of infection recorded during the months of collection for either of these larval nematodes. All sequences generated in this study were deposited in GenBank.

The aim of the present study was to evaluate polymerase chain reaction (PCR) as an alternative tool for diagnosing schistosomiasis in individuals with low-level parasite burden from areas of low endemicity or under occasional risk of infection by Schistosoma mansoni. A total of 102 samples were tested in this study using 2 PCR assays utilizing distinct primer pairs. One of the primer pairs was targeted to a highly repeated 121-base pair sequence of S. mansoni, and the other was targeted to Schistosoma 28S rDNA. The samples were divided into 4 groups according to parasite burden of the individual as follows: 16 individuals with schistosomiasis excreting less than 10 eggs per gram of feces (EPG), 18 individuals excreting higher than 10 EPG, 22 individuals with reactive IgG-ELISA against S. mansoni soluble membrane antigen and negative coproscopy, and 46 controls samples including 25 individuals with other intestinal parasites and 21 individuals with negative parasitologic examination. The results obtained with stool samples from individuals with schistosomiasis showed a high sensitivity for PCR as S. mansoni DNA was detected in 91% (31/34) of the samples analyzed. No amplification was observed in 3 stool samples from individuals excreting below 10 EPG. The specificity of the test for both pairs of primers was 100%. In the group of seropositive individuals, S. mansoni DNA was detected in 59% (13/22) of fecal samples, corroborating the serologic results. Overall, PCR can be an important tool for detecting S. mansoni infection in individuals excreting few eggs in feces. Moreover, the determination of the infection through the detection of S. mansoni DNA in stool samples from seropositive individuals represents a new means of confirming the results of IgG-ELISA for schistosomiasis. Therefore, studies in this direction should be encouraged and extended.

The enteric protist Blastocystis is one of the most frequently reported parasites infecting both humans and many other animal hosts worldwide. A remarkable genetic diversity has been observed in the species, with 17 different subtypes (STs) on a molecular phylogeny based on small subunit RNA genes (SSU rDNA). Nonetheless, information regarding its distribution, diversity and zoonotic potential remains still scarce, especially in groups other than primates. In Brazil, only a few surveys limited to human isolates have so far been conducted on Blastocystis STs. The aim of this study is to determine the occurrence of Blastocystis subtypes in non-human vertebrate and invertebrate animal groups in different areas of the state of Rio de Janeiro, Brazil. A total of 334 stool samples were collected from animals representing 28 different genera. Blastocystis cultivated samples were subtyped using nuclear small subunit ribosomal DNA (SSU rDNA) sequencing. Phylogenetic analyses and BLAST searches revealed six subtypes: ST5 (28.8%), ST2 (21.1%), ST1 and ST8 (19.2%), ST3 (7.7%) and ST4 (3.8%). Our findings indicate a considerable overlap between STs in humans and other animals. This highlights the importance of investigating a range of hosts for Blastocystis to understand the eco-epidemiological aspects of the parasite and its host specificity.

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.

Background Blastocystis is a cosmopolitan protist parasite found in the human gastrointestinal tract and is highly prevalent in developing countries. Recent molecular studies have revealed extensive genetic diversity, which has been classified into different subtypes (STs) based on sequence analysis of small subunit ribosomal RNA gene. Blastocystis is one of the most common fecal parasites in Brazil, but the diversity of subtypes remains unknown in the country. This study aimed to determine the distribution of Blastocystis STs in an urban community in Duque de Caxias, Rio de Janeiro, Brazil.MethodsA total of 64 stool samples positive for Blastocystis in Pavlova’s medium were subtyped by PCR and sequenced using primers targeting the small subunit rRNA gene, in addition to phylogenetic analysis and subtype-specific PCR using sequence-tagged-site (STS) primers.Results Endolimax nana (14%), Entamoeba complex (10.5%), Taenia sp. (0.6%), Trichuris trichiura (1.3%) and Enterobius vermicularis (1.3%) were detected in Blastocystis-positive samples. Of the 64 samples tested by PCR/DNA sequencing, 55 were identified as ST1 (42%), ST3 (49%), ST2 (7%) and ST4 (2%), and the presence of mixed ST (ST1 + ST3) infection was detected in nine samples (14%).ConclusionsDNA sequencing and phylogenetic analysis of Brazilian Blastocystis isolates identified four different subtypes. To our knowledge, this study provided the first genetic characterization of Blastocystis subtypes in an urban area of Rio de Janeiro, Brazil. We also identified ST4 for the first time in Brazil. Further studies are necessary to determine the distribution of STs across human populations in Rio de Janeiro.

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.