Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale.

Population genetic studies are efficient for inferring the invasion history based on a comparison of native and invasive populations, especially when conducted at species scale. An expected outcome in invasive populations is variability loss, and this is especially true in self-fertilizing species. We here focus on the self-fertilizing Pseudosuccinea columella, an invasive hermaphroditic freshwater snail that has greatly expanded its geographic distribution and that acts as intermediate host of Fasciola hepatica, the causative agent of human and veterinary fasciolosis. We evaluated the distribution of genetic diversity at the largest geographic scale analysed to date in this species by surveying 80 populations collected during 16 years from 14 countries, using eight nuclear microsatellites and two mitochondrial genes. As expected, populations from North America, the putative origin area, were strongly structured by selfing and history and harboured much more genetic variability than invasive populations. We found high selfing rates (when it was possible to infer it), none-to-low genetic variability and strong population structure in most invasive populations. Strikingly, we found a unique genotype/haplotype in populations from eight invaded regions sampled all over the world. Moreover, snail populations resistant to infection by the parasite are genetically distinct from susceptible populations. Our results are compatible with repeated introductions in South America and flash worldwide invasion by this unique genotype/haplotype. Our study illustrates the population genetic consequences of biological invasion in a highly selfing species at very large geographic scale. We discuss how such a large-scale flash invasion may affect the spread of fasciolosis.

Synthetic peptides for the immunodiagnosis of hepatitis A virus infection.

VP1, VP2 and VP3 molecules of hepatitis A virus are exposed capsid proteins that have shown to be antigenic and are used for diagnosis in recombinant-antigen commercial kits. In this study, we developed a sequence analysis in order to predict diagnostic peptide epitopes, followed by their spot synthesis on functionalized cellulose paper (Pepscan). This paper with synthetic peptides was tested against a sera pool of hepatitis A patients. Two peptide sequences, that have shown an antigenic recognition, were selected for greater scale synthesis on resin. A dimeric form of one of these peptides (IMT-1996), located in the C-Terminus region of protein VP1, was antigenic with a recognition frequency of 87-100% of anti-IgG antibodies and 100% of anti-IgM antibodies employing the immunological assays MABA and ELISA. We propose peptide IMT-1996, with less than twenty residues, as a cheaper alternative for prevalence studies and diagnosis of hepatitis A infection.

Trypanosoma cruzi genotyping supports a common source of infection in a school-related oral outbreak of acute Chagas disease in Venezuela.

Trypanosoma cruzi I, a discrete typing unit (DTU) found in human infections in Venezuela and other countries of the northern region of South America and in Central America, has been recently classified into five intra-DTU genotypes (Ia, Ib, Ic, Id, Ie) based on sequence polymorphisms found in the spliced leader intergenic region. In this paper we report the genotype identification of T. cruzi human isolates from one outbreak of acute orally acquired Chagas disease that occurred in a non-endemic region of Venezuela and from T. cruzi triatomine and rat isolates captured at a guava juice preparation site which was identified as the presumptive source of infection. The genotyping of all these isolates as TcId supports the view of a common source of infection in this oral Chagas disease outbreak through the ingestion of guava juice. Implications for clinical manifestations and dynamics of transmission cycles are discussed.

Oral transmission of Chagas disease: typing of Trypanosoma cruzi from five outbreaks occurred in Venezuela shows multiclonal and common infections in patients, vectors and reservoirs.

In Venezuela six episodes of oral transmission of Chagas disease (OChD) have been described, being the one reported in 2007 with a total of 103 people infected the largest worldwide. This work shows the use of three molecular markers (mini-exon gene and domains 24Sα and 18S of the ribosomal RNA) to characterize the infecting Trypanosoma cruzi strain of patients, reservoirs and vectors involved in five of the six OChD outbreaks. For this, 28 T. cruzi isolates were characterized by PCR, and the products of these reactions cloned and sequenced to reveal the existence of different TcI SL-IR genotypes. We also describe a new PCR assay able to discriminate between TcIb and TcId parasite populations. In summary, we have identified mostly parasites with the TcId haplotype and multiclonal populations with predominance of haplotype TcId (65.2%). Additionally, populations of haplotypes TcIb, TcIa and mixtures (TcId+TcIb, TcId+TcIa, TcIb+TcIa) are recurrent in samples obtained from children. The analysis of the SL-IR motif showed two clones depicting a different motif that could be an evidence for a possible hybrid haplotype between TcIa and TcIb (haplotype TcIa/Ib). Interestingly, in a single patient haplotype differences between T.cruzi isolates obtained pre and post-treatment were found. In conclusion, our findings show that in order to understand the pathogenic mechanisms involved in the orally acquired Chagas disease there is a need to join efforts to study T. cruzi haplotypes, their tissue tropisms and their susceptibility to chemoteraphy.

Detection of the Sm31 antigen in sera of Schistosoma mansoni- infected patients from a low endemic area.

Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14-18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113-122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83.3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis.

Distribution of Lymnaeidae (Mollusca: Pulmonata), intermediate snail hosts of Fasciola hepatica in Venezuela.

An extensive malacological survey was carried out between 2005-2009 in order to clarify the exact number of lymnaeid species which may be intermediate hosts of Fasciola hepatica in Venezuela. Four species were discovered during this survey, including two local species: Lymnaea cubensis and Lymnaea cousini and two exotic species: Lymnaea truncatula and Lymnaea columella. The most common local species was L. cubensis which was found at 16 out of the 298 sampling sites. This species has a large distribution area throughout the Northern part of Venezuela and was encountered from sea level to an altitude of 1,802 m in state of Trujillo. The second local species L. cousini was collected at only two sites of the Andean Region at altitudes of 3,550 m and 4,040 m, respectively. The European L. truncatula was found at 24 sites all located in the states of Mérida and Táchira at an altitude varying between 1,540-4,000 m. The respective distribution areas of L. cubensis and L. truncatula do not appear to overlap, but more detailed malacological surveys are needed. The fourth lymnaeid species, L. columella was collected in a canal from Mérida at an altitude of 1,929 m and in an irrigation canal from the state of Guárico, at an altitude of 63 m. The role of these four lymnaeid species in the transmission of fascioliasis in Venezuela is discussed.

Distribution of Lymnaeidae (Mollusca: Pulmonata), intermediate snail hosts of Fasciola hepatica in Venezuela

An extensive malacological survey was carried out between 2005-2009 in order to clarify the exact number of lymnaeid species which may be intermediate hosts of Fasciola hepatica in Venezuela. Four species were discovered during this survey, including two local species: Lymnaea cubensis and Lymnaea cousini and two exotic species: Lymnaea truncatula and Lymnaea columella. The most common local species was L. cubensis which was found at 16 out of the 298 sampling sites. This species has a large distribution area throughout the Northern part of Venezuela and was encountered from sea level to an altitude of 1,802 m in state of Trujillo. The second local species L. cousini was collected at only two sites of the Andean Region at altitudes of 3,550 m and 4,040 m, respectively. The European L. truncatula was found at 24 sites all located in the states of Mérida and Táchira at an altitude varying between 1,540-4,000 m. The respective distribution areas of L. cubensis and L. truncatula do not appear to overlap, but more detailed malacological surveys are needed. The fourth lymnaeid species, L. columella was collected in a canal from Mérida at an altitude of 1,929 m and in an irrigation canal from the state of Guárico, at an altitude of 63 m. The role of these four lymnaeid species in the transmission of fascioliasis in Venezuela is discussed.

Detection of schistosomiasis cases in low-transmission areas based on coprologic and serologic criteria The Venezuelan experience.

Low and very-low intensities of infection hinder the diagnosis of schistosomiasis. Therefore, new parameters should be established in order to more accurately identify active cases and true infection prevalence, for the adequate implementation of a control program. After the survey and analysis of the epidemiological characteristics of five Venezuelan communities, we propose three criteria for the definition of a "schistosomiasis case", based on different diagnostic methods: stool examination, ELISA-soluble egg antigen with sodium metaperiodate (SMP-ELISA), alkaline phosphatase immunoassay (APIA) and the circumoval precipitin test (COPT). Briefly, criterion I: persons with Schistosoma mansoni eggs in stools; criterion II: persons without eggs in stools, with positive COPT, without previous antischistosome chemotherapy in the last year; and criterion III: persons without eggs in stools, with negative COPT, with two positive immunoenzymatic tests (SMP-ELISA and APIA), and with no previous chemotherapy. The incorporation of serological tests to epidemiologic surveillance in areas of low-transmission tries to compensate the underestimation of prevalence based only on parasitological diagnosis.

Schistosoma: cross-reactivity and antigenic community among different species.

It is not unusual to find common molecules among different species of the genus Schistosoma. When those molecules are antigenic, they may be used in immunodiagnosis and vaccines, but they could also be applied to taxonomic and evolutionary studies. To study cross-reactivity and antigenic community among different species of schistosomes, plasmas from laboratory animals infected with Schistosoma bovis, S. guineensis, S. rodhaini, S. haematobium, and four strains of S. mansoni were evaluated with a crude extract of adult worms of S. mansoni by Western blot. Using the multiple antigen blot assay, plasmas from these infected animals were exposed to a selected group of synthetic peptides from Sm28GST, Sm28TPI, Sm elastase, Sm97, Sm32, Sm31, and Sm Cathepsin L. The results presented herein demonstrate differential cross-reactivity and antigenic community among the Mansoni and Haematobium groups of schistosomes, which is of relevance as an additional new tool for phylogenetic studies of schistosomes as well as for diagnosis and vaccine purposes.

Lymnaea cousini Jousseaume, 1887 (Gastropoda: Lymnaeidae): first record for Venezuela.

Lymnaea cousini Jousseaume, 1887 was collected in Mucubaji, Merida State, Venezuela, from a permanent pond located at a very high altitude (3760 m). Identification of the collected specimens was made by comparison with the original description of the shell by Jousseaume and the description of the renal organ and reproductive system of topotypic specimens by Paraense.

Lymnaea cousini Jousseaume, 1887 (Gastropoda: Lymnaeidae): first record for Venezuela

Lymnaea cousini Jousseaume, 1887 was collected in Mucubaji, Merida State, Venezuela, from a permanent pond located at a very high altitude (3760 m). Identification of the collected specimens was made by comparison with the original description of the shell by Jousseaume and the description of the renal organ and reproductive system of topotypic specimens by Paraense.

Importance of including Blomia tropicalis in the routine diagnosis of Venezuelan patients with persistent allergic symptoms.

BACKGROUND: Blomia tropicalis is a common mite found in the house dust of many tropical countries including Venezuela. The prevalence of skin test and specific serum immunoglobulin (Ig)E reactivity to B. tropicalis in Venezuela has not been previously evaluated. METHODS: In the present study we evaluated the skin reactivity by skin prick test and specific IgE by a multiple antigen blot assay, against B. tropicalis and Dermatophagoides pteronyssinus, in a group of 115 subjects who attended the Allergy Clinic of the Institute of Biomedicine, Caracas, Venezuela, and we studied possible cross reactions between similar proteins of these two mites. RESULTS: One hundred and six patients with persistent allergic respiratory symptoms showed a positive skin prick test to at least one of the mite extracts, with the frequency of positive reactions to B. tropicalis being as high as to D. pteronyssinus. Twelve patients reacted only to D. pteronyssinus and 13 different patients only to B. tropicalis. Specific IgE to each of the mite extracts was found with similar frequency, and the results coincided with the skin test reactivity. CONCLUSIONS: The study indicated the importance of including B. tropicalis in routine diagnostic testing in tropical and sub-tropical situations.

Immunodiagnosis of parasitic diseases with synthetic peptides.

Parasitic diseases remain as a major public health problem worldwide, not only based on their historically high morbidity and mortality rates, but also because risk factors associated with their transmission are increasing. Laboratory diagnosis and particularly immunodiagnosis is a basic tool for the demonstration, clinical management and control of these infections. Classically, the serological tests for the detection of antibodies or antigens are based on the use of crude and purified antigens. Synthetic peptides have opened a new field and perspectives, as the source of pure epitopes and molecules for diagnosis of malaria, Chagas' disease, leishmaniasis, schistosomiasis, hidatidosis, cysticercosis and fasciolosis based on the detection of antibodies and circulating antigens. Herein, are critically reviewed the relevant advances and applications of the synthetic peptides on immunodiagnosis of parasitic diseases. A variety of sequences, constructs (monomers, polymers, MAPs), immunological methods and samples have been used, demonstrating their diagnostic potential. However, in most parasitic infections it is necessary to use more than a single peptide in order to avoid the genetic restriction against certain epitopes, as well as to test them in well characteized groups of patients, in order to confirm their sensitivity and specificity. The concept of multidiagnosis with synthetic peptides, using a novel multi-dot blot assay is introduced. Finally, the chemical imitation of antigens, offers a tremendous posibilities in the diagnosis of parasitic infections in developing countries since this strategy is cheaper, simpler, reproducible, useful for large scale testing and in most cases, specific and sensitive.

Schistosomiasis mansoni in areas of low transmission: epidemiological characterization of Venezuelan foci.

Severe schistosomiasis is a rare event in Venezuela nowadays, after a successful national campaign by the Schistosomiasis Control Program. Unfortunately, this program has practically disappeared, and snail surveillance in field is not a priority, anymore. Thus, schistosomiasis has become a neglected disease in this country. However, surveys in different populations from the endemic area have shown particular epidemiological features described herein. In five communities we evaluated 2,175 persons and searched for the presence of Biomphalaria glabrata snails. Some markers were used for classifying schistosomiasis foci: mean age of the persons with Schistosoma mansoni eggs in the stools, serological tests, presence of B. glabrata snails, and intensity of infection. Places without B. glabrata snails and with few schistosomiasis cases were defined as "past transmission sites"; a site with abundant snails but few cases was defined as "potential risk"; "new transmission" foci were characterized by the presence of infected snails and young people passing eggs in the stools. A "re-emergent" focus has shared these last features, showing in addition a place where schistosomiasis had been reported before. Recent evidences of active transmission with the increasing dispersion of B. glabrata snails, point out the necessity for the re-establishment of the Schistosomiasis Control Program in Venezuela.

Antigenic community between Schistosoma mansoni and Biomphalaria glabrata: on the search of candidate antigens for vaccines.

We have previously confirmed the presence of common antigens between Schistosoma mansoni and its vector, Biomphalaria glabrata. Cross-reactive antigens may be important as possible candidates for vaccine and diagnosis of schistosomiasis. Sera from outbred mice immunized with a soluble Biomphalaria glabrata antigen (SBgA) of non-infected B. glabrata snails recognized molecules of SBgA itself and S. mansoni AWA by Western blot. Recognition of several molecules of the SBgA were inhibited by pre-incubation with AWA (16, 30, 36, 60 and 155 kDa). The only specific molecule of AWA, inhibited by SBgA, was a 120 kDa protein. In order to determine which epitopes of SBgA were glycoproteins, the antigen was treated with sodium metaperiodate and compared with non-treated antigen. Molecules of 140, 60 and 24 kDa in the SBgA appear to be glycoproteins. Possible protective effects of the SBgA were evaluated immunizing outbred mice in two different experiments using Freund's Adjuvant. In the first one (12 mice/group), we obtained a significant level of protection (46%) in the total worm load, with a high variability in worm recovery. In the second experiment (22 mice/group), no significant protection was observed, neither in worm load nor in egg production per female. Our results suggest that SBgA constitutes a rich source of candidate antigens for diagnosis and prophylactic studies.

Schistosomiasis mansoni in low transmission areas: abdominal ultrasound.

In endemic areas with low prevalence and low intensity of infection, the diagnosis of hepatic pathology due to the Schistosoma mansoni infection is very difficult. In order to establish the hepatic morbidity, a double-blind study was achieved in Venezuelan endemic areas, with one group of patients with schistosomiasis and the other one of non-infected people, that were evaluated clinically and by abdominal ultrasound using the Cairo classification. Schistosomiasis diagnosis was established based on parasitologic and serological tests. The increase of the hepatic size at midclavicular and midsternal lines (in hepatometry) and the hard liver consistency were the clinical parameters able to differentiate infected persons from non infected ones, as well as the presence of left lobe hepatomegaly detected by abdominal ultrasound. The periportal thickening, especially the mild form, was frequent in all age groups in both infected and uninfected patients. There was not correlation between the intensity of infection and ultrasound under the current circumstances. Our data suggest that in Venezuela, a low endemic area of transmission of schistosomiasis, the hepatic morbidity is mild and uncommon. The Cairo classification seems to overestimate the prevalence of periportal pathology. The specificity of the method must be improved, especially for the recognition of precocious pathology. Other causes of hepatopathies must be investigated.

Laboratory diagnosis of Schistosomiasis in areas of low transmission: a review of a line of research.

After 57 years of successful control of schistosomiasis in Venezuela, the prevalence and intensity of infection have declined. Approximately 80% of the individuals eliminate less than 100 eggs/g of stools, therefore morbidity is mild and the majority are asymptomatic. The sensitivity of Kato-Katz decreases to approximately 60%. Available serological methods for the detection of circulating antigens only reach a 70% of sensitivity. Tests based on the detection of antibodies by immunoenzymatic assays have been improved. The circumoval precipitine test has shown a high sensitivity (97%), specificity (100%), and correlation with oviposition, being considered the best confirmatory diagnostic test. Additionally to the classical immunoenzymatic assays, the development of the alkaline phosphatase immunoassay, allowed to reach a 100% specificity with an 89% sensitivity. Recently, we have developed a modified ELISA in which the soluble egg antigen is treated with sodium metaperiodate (SMP-ELISA) in order to eliminate the glycosilated epitopes responsible for the false positive reactions. The specificity and sensitivity reaches 97% and 99%, respectively. Synthetic peptides from the excretory-secretory enzymes, cathepsin B (Sm31) legumain (Sm32) and cathepsin D (Sm45), have been synthesized. The combination of two peptides derived from the Sm31 have been evaluated, reaching a sensitivity of 96% when analyzed independently and with a 100% specificity. Antibodies raised in rabbits against peptides derived from the Sm31 and Sm32 are currently evaluated in two different antigen-capture-based assays. The development of a simple, cheap and reliable test that correlates with parasite activity is a major goal.

Laboratory diagnosis of Schistosomiasis in areas of low transmission. A review of a line of research

After 57 years of successful control of schistosomiasis in Venezuela, the prevalence and intensity of infection have declined. Approximately 80 percent of the individuals eliminate less than 100 eggs/g of stools, therefore morbidity is mild and the majority are asymptomatic. The sensitivity of Kato-Katz decreases to approximately 60 percent. Available serological methods for the detection of circulating antigens only reach a 70 percent of sensitivity. Tests based on the detection of antibodies by immunoenzymatic assays have been improved. The circumoval precipitine test has shown a high sensitivity (97 percent), specificity (100 percent), and correlation with oviposition, being considered the best confirmatory diagnostic test. Additionally to the classical immunoenzymatic assays, the development of the alkaline phosphatase immunoassay, allowed to reach a 100 percent specificity with an 89 percent sensitivity. Recently, we have developed a modified ELISA in which the soluble egg antigen is treated with sodium metaperiodate (SMP-ELISA) in order to eliminate the glycosilated epitopes responsible for the false positive reactions. The specificity and sensitivity reaches 97 percent and 99 percent, respectively. Synthetic peptides from the excretory-secretory enzymes, cathepsin B (Sm31) legumain (Sm32) and cathepsin D (Sm45), have been synthesized. The combination of two peptides derived from the Sm31 have been evaluated, reaching a sensitivity of 96 percent when analyzed independently and with a 100 percent specificity. Antibodies raised in rabbits against peptides derived from the Sm31 and Sm32 are currently evaluated in two different antigen-capture-based assays. The development of a simple, cheap and reliable test that correlates with parasite activity is a major goal

Schistosomiasis mansoni in low transmission areas. Abdominal ultrasound

In endemic areas with low prevalence and low intensity of infection, the diagnosis of hepatic pathology due to the Schistosoma mansoni infection is very difficult. In order to establish the hepatic morbidity, a double-blind study was achieved in Venezuelan endemic areas, with one group of patients with schistosomiasis and the other one of non-infected people, that were evaluated clinically and by abdominal ultrasound using the Cairo classification. Schistosomiasis diagnosis was established based on parasitologic and serological tests. The increase of the hepatic size at midclavicular and midsternal lines (in hepatometry) and the hard liver consistency were the clinical parameters able to differentiate infected persons from non infected ones, as well as the presence of left lobe hepatomegaly detected by abdominal ultrasound. The periportal thickening, especially the mild form, was frequent in all age groups in both infected and uninfected patients. There was not correlation between the intensity of infection and ultrasound under the current circumstances. Our data suggest that in Venezuela, a low endemic area of transmission of schistosomiasis, the hepatic morbidity is mild and uncommon. The Cairo classification seems to overestimate the prevalence of periportal pathology. The specificity of the method must be improved, especially for the recognition of precocious pathology. Other causes of hepatopathies must be investigated

Schistosomiasis mansoni in areas of low transmission. Epidemiological characterization of Venezuelan foci

Severe schistosomiasis is a rare event in Venezuela nowadays, after a successful national campaign by the Schistosomiasis Control Program. Unfortunately, this program has practically disappeared, and snail surveillance in field is not a priority, anymore. Thus, schistosomiasis has become a neglected disease in this country. However, surveys in different populations from the endemic area have shown particular epidemiological features described herein. In five communities we evaluated 2,175 persons and searched for the presence of Biomphalaria glabrata snails. Some markers were used for classifying schistosomiasis foci: mean age of the persons with Schistosoma mansoni eggs in the stools, serological tests, presence of B. glabrata snails, and intensity of infection. Places without B. glabrata snails and with few schistosomiasis cases were defined as "past transmission sites"; a site with abundant snails but few cases was defined as "potential risk"; "new transmission" foci were characterized by the presence of infected snails and young people passing eggs in the stools. A "re-emergent" focus has shared these last features, showing in addition a place where schistosomiasis had been reported before. Recent evidences of active transmission with the increasing dispersion of B. glabrata snails, point out the necessity for the re-establishment of the Schistosomiasis Control Program in Venezuela