Two cold shock domain containing proteins trigger the development of infectious Trypanosoma brucei.

Cold shock proteins are members of a family of DNA- and RNA-binding proteins with one or more evolutionarily conserved cold shock domain (CSD). These proteins have a wide variety of biological functions, including DNA-damage repair, mRNA stability, and regulation of transcription, splicing and translation. We previously identified two CSD containing proteins, CSD1 and CSD2, in the protozoan parasite Trypanosoma brucei to be required for RBP6-driven metacyclic production, albeit at different steps of the developmental program. During metacyclogenesis T. brucei undergoes major morphological and metabolic changes that culminate in the establishment of quiescent metacyclic parasites and the acquisition of mammalian infectivity. To investigate the specific role of CSD1 and CSD2 in this process, we ectopically expressed CSD1 or CSD2 in non-infectious procyclic parasites and discovered that each protein is sufficient to produce infectious metacyclic parasites in 24 hours. Domain truncation assays determined that the N-terminal domain, but not the C-terminal domain, of CSD1 and CSD2 was required for metacyclic development. Furthermore, conserved amino acid residues in the CSD of CSD1 and CSD2, known to be important for binding nucleic acids, were found to be necessary for metacyclic production. Using single-end enhanced crosslinking and immunoprecipitation (seCLIP) we identified the specific binding motif of CSD1 and CSD2 as "ANACAU" and the bound mRNAs were enriched for biological processes, including lipid metabolism, microtubule-based movement and nucleocytoplasmic transport that are likely involved in the transition to bloodstream form-like cells.

Evaluation of a New Immunochromatographic Test Using Recombinant Antigen B8/1 for Diagnosis of Cystic Echinococcosis.

Diagnosis of cystic echinococcosis (CE) is based on the identification of the cyst(s) by imaging, using immunodiagnostic tests mainly as complementary tools in clinical settings. Among the antigens used for immunodiagnosis, previous studies described a good performance of the recombinant antigen B8/1 (rAgB) in an enzyme-linked immunosorbent assay (ELISA) format; however, in remote parts of areas where the disease is endemic, the implementation of an ELISA is difficult, so a more simple, rapid, and reliable method such as the immunochromatographic test (ICT) is required. In this study, using a set of 50 serum samples from patients with surgically confirmed CE, we compared the performance of an ICT and that of an ELISA using the rAgB. The overall sensitivities of ICT and ELISA were not statistically different (78% versus 72%; P = 0.36). The overall agreement between both tests was moderate (κ = 0.41; P < 0.01). Concordance between ICT and ELISA was substantial or almost perfect for patients with liver involvement (κ = 0.65; P < 0.001) and patients with more than one hydatid cyst (κ = 0.82; P < 0.001), respectively. Moreover, specificity analysis using a total of 88 serum samples from healthy individuals (n = 20) and patients (n = 68) with other parasitic infections revealed that ICT had a specificity of 89.8%. ICT and ELISA had similar performance for the detection of specific antibodies to E. granulosus, and ICT had a high specificity, opening the possibility of using ICT as a screening tool in rural settings.

Genotypic relationships between Taenia saginata, Taenia asiatica and their hybrids.

Partial sequences of the DNA polymerase delta (pold) gene from Taenia saginata-like adult worms were sequenced. Phylogenetic analysis revealed that pold gene sequences were clearly divided into two clades, differing from each other in five to seven nucleotides. There is little doubt that T. saginata and Taenia asiatica were once separated into two distinct taxa as has been concluded in previous studies. On the other hand, most of the adult worms, which were identified as T. asiatica using mitochondrial DNA, were homozygous for an allele that originated from the allele of T. saginata via single nucleotide substitution. These results indicate that most of the adult worms, which had been called T. asiatica, are not actually 'pure T. asiatica' but instead originated from the hybridization of 'pure T. saginata' and 'pure T. asiatica'.

Detection of human taeniases in Tibetan endemic areas, China.

Detection of taeniasis carriers of Taenia solium is essential for control of cysticercosis in humans and pigs. In the current study, we assessed the positive detection rate of a self-detection tool, stool microscopy with direct smear and coproPCR for taeniasis carriers in endemic Tibetan areas of northwest Sichuan. The self-detection tool through questioning about a history of proglottid expulsion within the previous one year showed an overall positive detection rate of more than 80% for Taenia saginata, T. solium and T. asiatica. The positive detection rate was similar for T. saginata and T. solium. In 132 taeniid tapeworm carriers, 68 (51·5%) were detected by microscopy and 92 (69·7%) were diagnosed by coproPCR. A combination of microscopy and coproPCR increased the positive detection rate to 77·3%. There remained 10 cases (7·6%) coproPCR negative but microscopy positive. Due to the high cost and complicated process, coproPCR is required for the identification of Taenia species only when necessary, though it had a significant higher positive detection rate than microscopy. Combined use of self-detection and stool microscopy are recommended in community-based mass screening for taeniases in this Tibetan area or in other situation-similar endemic regions.

Serological and molecular tools to detect neurologic parasitic zoonoses in rural Cameroon.

Parasitic helminthiases, such as toxocariasis, cysticercosis and paragonimiasis are a public health threat, since they can affect the brain leading to neurological disorders. Epilepsy and paragonimiasis are common in southwestern Cameroon. We reviewed the literature for studies using antigens to diagnose toxocariasis, cysticercosis, and paragonimiasis. Serology revealed that 61 (36.3%), 26 (15.5%) and 2 (1.2%) of 168 persons examined [78 males (15.2 +/- 8.2 years old), 90 females (12.9 +/- 5.9 years old), 143 persons < 20 years old] had antibody responses to toxocariasis, paragonimiasis and cysticercosis, respectively. Of the 14 people with epilepsy, 5 were seropositive for Toxocara antigens and 1 was positive for both Toxocara and Paragonimus antigens. Two children were serologically confirmed to have cysticercosis. Serologic screening for cysticercosis may be feasible to detect asymptomatic cysticercosis in children in endemic areas leading to early treatment. The causative Paragonimus species was confirmed to be P. africanus by molecular sequencing. Education, screening and confirmation test for these diseases may be needed for control in Cameroon.

Identification of positive and negative regulators in the stepwise developmental progression towards infectivity in Trypanosoma brucei.

Trypanosoma brucei is a protozoan parasite that causes important human and livestock diseases in sub-Saharan Africa. By overexpressing a single RNA-binding protein, RBP6, in non-infectious procyclics trypanosomes, we previously recapitulated in vitro the events occurring in the tsetse fly vector, namely the development of epimastigotes and infectious, quiescent metacyclic parasites. To identify genes involved in this developmental progression, we individually targeted 86 transcripts by RNAi in the RBP6 overexpression cell line and assessed the loss-of-function phenotypes on repositioning the kinetoplast, an organelle that contains the mitochondrial genome, the expression of BARP or brucei alanine rich protein, a marker for epimastigotes, and metacyclic variant surface glycoprotein. This screen identified 22 genes that positively or negatively regulate the stepwise progression towards infectivity at different stages. Two previously uncharacterized putative nucleic acid binding proteins emerged as potent regulators, namely the cold shock domain-containing proteins CSD1 and CSD2. RNA-Seq data from a selected group of cell lines further revealed that the components of gene expression regulatory networks identified in this study affected the abundance of a subset of transcripts in very similar fashion. Finally, our data suggest a considerable overlap between the genes that regulate the formation of stumpy bloodstream form trypanosomes and the genes that govern the development of metacyclic form parasites.

Evaluation of a loop-mediated isothermal amplification method using fecal specimens for differential detection of Taenia species from humans.

We compared the performance of loop-mediated isothermal amplification (LAMP) with that of a multiplex PCR method for differential detection of human Taenia parasites in fecal specimens from taeniasis patients. The LAMP method, with no false positives, showed a higher sensitivity (88.4%) than the multiplex PCR (37.2%). Thus, it is expected that the LAMP method has a high value for molecular diagnosis of taeniasis.

Loop-mediated isothermal amplification method for differentiation and rapid detection of Taenia species.

Rapid detection and differentiation of Taenia species are required for the control and prevention of taeniasis and cysticercosis in areas where these diseases are endemic. Because of the lower sensitivity and specificity of the conventional diagnosis based on microscopical examination, molecular tools are more reliable for differential diagnosis of these diseases. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) assay for differential diagnosis of infections with Taenia species with cathepsin L-like cysteine peptidase (clp) and cytochrome c oxidase subunit 1 (cox1) genes. LAMP with primer sets to the cox1 gene could differentiate between three species, and LAMP with primer sets to the clp gene could differentiate Taenia solium from Taenia saginata/Taenia asiatica. Restriction enzyme digestion of the LAMP products from primer set Tsag-clp allowed the differentiation of Taenia saginata from Taenia asiatica. We demonstrated the high specificity of LAMP by testing known parasite DNA samples extracted from proglottids (n = 100) and cysticerci (n = 68). LAMP could detect one copy of the target gene or five eggs of T. asiatica and T. saginata per gram of feces, showing sensitivity similar to that of PCR methods. Furthermore, LAMP could detect parasite DNA in all taeniid egg-positive fecal samples (n = 6). Due to the rapid, simple, specific, and sensitive detection of Taenia species, the LAMP assays are valuable tools which might be easily applicable for the control and prevention of taeniasis and cysticercosis in countries where these diseases are endemic.

Seroprevalence and risk factors of human cysticercosis and taeniasis prevalence in a highly endemic area of epilepsy in Bangoua, west Cameroon.

Cysticercosis caused by the larvae of Taenia solium is a serious and emerging threat to public health in the endemic areas as well as in the non-endemic areas.　Neurocysticercosis, an affection of the central nervous system is a leading cause of epilepsy in endemic areas. This study was carried out to investigate human cysticercosis, taeniasis and risk factors, and also their association with epilepsy in Bangoua, west Cameroon where epilepsy is highly prevalent. Out of 384 people investigated, 12 (3.1%) exhibited antibody response against low molecular weight antigens of T. solium by ELISA. Immunoblot revealed that six persons (1.6%) were seropositive with the same antigens. Among 61 epileptic patients, only one was seropositive by immunoblot and the study did not find any statistically significant difference (P>0.05) in seropositivity to T. solium between epileptic persons (1/61, 1.6%) and non-epileptic group (5/323, 1.5%). In addition, cysticercosis was associated with households eating pork meat from pigs slaughtered at home, but not with other factors. The risk factors including pig farming, the consumption of pork meat, vegetables, and non-drinkable water were attenuated by the relatively good hygiene and pig husbandry practices of the population. No egg of Taenia was found in stool by microscopic examination. All data obtained in this study suggested that cysticercosis might not be the principal causative agent of epilepsy in this area.

Simple Identification of Human Taenia Species by Multiplex Loop-Mediated Isothermal Amplification in Combination with Dot Enzyme-Linked Immunosorbent Assay.

For differential detection of Taenia solium, Taenia saginata, and Taenia asiatica, loop-mediated isothermal amplification (LAMP) assay targeting the cytochrome c oxidase subunit 1 gene has been recently developed and shown to be sensitive, specific, and effective. However, to achieve differential identification, one specimen requires three reaction mixtures containing a primer set of each Taenia species separately, which is complex and time consuming and increases the risk of cross-contamination. In this study, we developed a simple differential identification of human Taenia species using multiplex LAMP (mLAMP) in combination with dot enzyme-linked immunosorbent assay (dot-ELISA). Forward inner primers of T. solium, T. saginata, and T. asiatica labeled with fluorescein isothiocyanate (FITC), digoxigenin (DIG), and tetramethylrhodamine (TAMRA), respectively, and biotin-labeled backward inner primers were used in mLAMP. The mLAMP assay succeeded in specific amplification of each respective target gene in a single tube. Furthermore, the mLAMP product from each species was easily distinguished by dot-ELISA with an antibody specific for FITC, DIG, or TAMRA. The mLAMP assay in combination with dot-ELISA will make identification of human Taenia species simpler, easier, and more practical.

Cryptic diversity in hymenolepidid tapeworms infecting humans.

An adult hymenolepidid tapeworm was recovered from a 52-year-old Tibetan woman during a routine epidemiological survey for human taeniasis/cysticercosis in Sichuan, China. Phylogenetic analyses based on sequences of nuclear 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit 1 showed that the human isolate is distinct from Hymenolepis diminuta and Hymenolepis nana, the common parasites causing human hymenolepiasis. Proglottids of the human isolate were unfortunately unsuitable for morphological identification. However, the resultant phylogeny demonstrated the human isolate to be a sister species to Hymenolepis hibernia from Apodemus mice in Eurasia. The present data clearly indicate that hymenolepidid tapeworms causing human infections are not restricted to only H. diminuta and H. nana.

The present situation of human taeniases and cysticercosis in Asia.

Three human Taenia species, Taenia solium, Taenia saginata, and Taenia asiatica, are endemic in rural regions of Asia, with these species sympatrically distributed in parts of Thailand and China. The pork tapeworm, T. solium, is the most pathogenic to humans, causing cysticercosis and neurocysticercosis (NCC). Due to its public health impact, T. solium taeniasis/cysticercosis is one of 17 neglected tropical diseases (NTDs) prioritized by the World Health Organization. In this review, the current status of taeniasis and NCC in several Asian countries will be presented as will possible prevention and control strategies. Among the known T. solium endemic areas in Asia, Bali, Indonesia is of high importance due to the risk of tourists becoming infected. Therefore, special attention will be given to ongoing control initiatives in Bali.

Loop-mediated isothermal amplification method for a differential identification of human Taenia tapeworms.

Loop-mediated isothermal amplification (LAMP), which employs a Bst DNA polymerase with strand-displacement activity and four primers (two inner primers and two outer primers) recognizing six distinct regions on the target DNA, is a highly sensitive, specific, simple, and rapid nucleotide amplification method. Moreover, because the Bst DNA polymerase resists much DNA polymerase inhibitors present in biological specimens, the LAMP method is suitable for the detection of infectious agents from clinical material such as fecal samples. Here, we describe the LAMP method which can differentially detect and identify human Taenia tapeworms, Taenia solium, T. saginata, and T. asiatica, using DNA specimens prepared from parasite tissue and human fecal sample.

Recent hybridization between Taenia asiatica and Taenia saginata.

Five Taenia tapeworms collected from humans in Tibetan Plateau, Sichuan, China, where three species of human Taenia are sympatrically endemic, were examined for the mitochondrial cox1 gene and two nuclear genes, ef1 and elp. Phylogenetic analyses of these genes revealed that two adult worms showed nuclear-mitochondrial discordance, suggesting that they originated from hybridization between Taenia saginata and Taenia asiatica. One of two worms had T. asiatica-type mtDNA, whereas another worm had T. saginata-type mtDNA, indicating that reciprocal hybridization between T. saginata and T. asiatica could occur. The worm having T. asiatica-type mtDNA was heterozygous at both nuclear loci with T. saginata-type alleles and T. asiatica-type alleles. In another worm, the ef1 locus was heterozygous with a T. saginata-type alleles and T. asiatica-type alleles, while the elp locus was homozygous with T. saginata-type alleles. Self-fertilization is the main reproductive method of the genus Taenia. Since self-fertilization represents a type of inbreeding, each locus in the offspring would become homozygous over generations with genetic drift. The fact that some nuclear loci are still heterozygous means that hybridization might have occurred recently. Hybridization between T. asiatica and T. saginata is probably an ongoing event in many areas in which they are sympatrically endemic.

A loop-mediated isothermal amplification method for a differential identification of Taenia tapeworms from human: application to a field survey.

In this study, we applied a loop-mediated isothermal amplification method for identification of human Taenia tapeworms in Tibetan communities in Sichuan, China. Out of 51 proglottids recovered from 35 carriers, 9, 1, and 41 samples were identified as Taenia solium, Taenia asiatica and Taenia saginata, respectively. Same results were obtained afterwards in the laboratory, except one sample. These results demonstrated that the LAMP method enabled rapid identification of parasites in the field surveys, which suggested that this method would contribute to the control of Taenia infections in endemic areas.

The first workshop towards the control of cestode zoonoses in Asia and Africa.

The first workshop towards the control of cestode zoonoses in Asia and Africa was held in Asahikawa Medical University, Japan on 15 and 16 Feb 2011. This meeting was fully supported by the Asian Science and Technology Strategic Cooperation Promotion Programs sponsored by the Special Coordination Funds for Promoting Science and Technology, the Ministry of Education Japan (MEXT) for 3 years from 2010 to Akira Ito. A total of 24 researchers from 9 countries joined together and discussed the present situation and problems towards the control of cestode zoonoses. As the meeting was simultaneously for the establishment of joint international, either bilateral or multilateral collaboration projects, the main purposes were directed to 1) how to detect taeniasis/cysticercosis infected patients, 2) how to differentiate Taenia solium from two other human Taenia species, T. saginata and T. asiatica, 3) how to evaluate T. asiatica based on the evidence of hybrid and hybrid-derived adult tapeworms from Thailand and China, 4) how to evaluate T. solium and T. hyaenae and other Taenia species from the wild animals in Ethiopia, and 5) how to detect echinococcosis patients and 6) how to differentiate Echinococcus species worldwide. Such important topics are summarized in this meeting report.

State-of-the-art Echinococcus and Taenia: phylogenetic taxonomy of human-pathogenic tapeworms and its application to molecular diagnosis.

The taxonomy of tapeworms belonging to the family Taeniidae has been controversial because of the paucity of adult phenotypic characters and the great plasticity of larvae in intermediate hosts. The family consists of the medically important two genera Echinococcus and Taenia, which are closely related to each other. Cladistic approaches using the molecular data of DNA and the numerical data of morphologic characters are clarifying phylogenetic relationships among the members of these genera. The nucleotide data of worldwide taeniid parasites accumulated in public DNA databases may provide a basis for the development of molecular diagnostic tools, and make it possible to identify the parasites, at least the human Taenia spp. by non-morphologists. Furthermore, the detection of intraspecific genetic variations prompts evolutionary and ecological studies to address fundamental questions of parasite distributional patterns. Here, we introduce the recent advances of taeniid phylogeny and its application to molecular diagnosis.

Serological studies of neurologic helminthic infections in rural areas of southwest cameroon: toxocariasis, cysticercosis and paragonimiasis.

BACKGROUND: Both epilepsy and paragonimiasis had been known to be endemic in Southwest Cameroon. A total of 188 people (168 and 20 with and without symptoms confirmed by clinicians, respectively, 84.6% under 20 years old) were selected on a voluntary basis. Among 14 people (8.3%) with history of epilepsy, only one suffered from paragonimiasis. Therefore, we challenged to check antibody responses to highly specific diagnostic recombinant antigens for two other helminthic diseases, cysticercosis and toxocariasis, expected to be involved in neurological diseases. Soil-transmitted helminthic infections were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Fecal samples were collected exclusively from the 168 people. Eggs of Ascaris lumbricoides, Trichuris trichiura and hookworms were found from 56 (33.3%), 72 (42.8%), and 19 (11.3%) persons, respectively. Serology revealed that 61 (36.3%), 25 (14.9%) and 2 (1.2%) of 168 persons showed specific antibody responses to toxocariasis, paragonimiasis and cysticercosis, respectively. By contrast, 20 people without any symptoms as well as additional 20 people from Japan showed no antibody responses. Among the 14 persons with epilepsy, 5 persons were seropositive to the antigen specific to Toxocara, and one of them was simultaneously positive to the antigens of Paragonimus. The fact that 2 children with no history of epilepsy were serologically confirmed to have cysticercosis strongly suggests that serological survey for cysticercosis in children is expected to be useful for early detection of asymptomatic cysticercosis in endemic areas. CONCLUSIONS/SIGNIFICANCE: Among persons surveyed, toxocariasis was more common than paragonimiasis, but cysticercosis was very rare. However, the fact that 2 children were serologically confirmed to have cysticercosis was very important, since it strongly suggests that serology for cysticercosis is useful and feasible for detection of asymptomatic cysticercotic children in endemic areas for the early treatment.

Paragonimiasis in Cameroon: molecular identification, serodiagnosis and clinical manifestations.

Paragonimiasis is a common parasitic zoonosis in Cameroon and neighbouring countries in Western Africa. Serum, sputum and faecal samples were collected in an endemic area of South West Province, Cameroon, after administration of a questionnaire to identify individuals with appropriate symptoms and histories. Microscopic examination revealed eggs in sputum from 16 people, but none in any faecal sample. These 16 were among the 25 and 26 people, respectively, positive by ELISA and by immunoblot using Paragonimus africanus crude antigens. Copro-DNA detection was attempted using 23 faecal samples (18 from sputum egg-negative and five from sputum egg-positive individuals). Copro-DNA was detected in four of the five sputum egg-positive individuals. These results strongly suggest that: (1) serology is much more sensitive than sputum examination for diagnosis of paragonimiasis; and (2) a copro-DNA test may be more sensitive than a microscopic search for eggs in faeces. Molecular sequence data from ITS2 and cox1 genes confirmed that adult worms experimentally raised in cats were P. africanus and that eggs from sputum or other worm products from human faeces also belonged to this species. Based on these results, 26 of 168 persons (15.5%) were diagnosed as suffering from paragonimiasis.