Monica Pajuelo

COMPARISON OF THE PEPTIDASE ACTIVITY IN THE ONCOSPHERE EXCRETORY/SECRETORY PRODUCTS OF TAENIA SOLIUM AND TAENIA SAGINATA

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.

Burden of Norovirus and Rotavirus in Children After Rotavirus Vaccine Introduction, Cochabamba, Bolivia

The effectiveness of rotavirus vaccine in the field may set the stage for a changing landscape of diarrheal illness affecting children worldwide. Norovirus and rotavirus are the two major viral enteropathogens of childhood. This study describes the prevalence of norovirus and rotavirus 2 years after widespread rotavirus vaccination in Cochabamba, Bolivia. Stool samples from hospitalized children with acute gastroenteritis (AGE) and outpatients aged 5-24 months without AGE were recruited from an urban hospital serving Bolivias third largest city. Both viruses were genotyped, and norovirus GII.4 was further sequenced. Norovirus was found much more frequently than rotavirus. Norovirus was detected in 69/201 (34.3%) of specimens from children with AGE and 13/71 (18.3%) of those without diarrhea. Rotavirus was detected in 38/201 (18.9%) of diarrheal specimens and 3/71 (4.2%) of non-diarrheal specimens. Norovirus GII was identified in 97.8% of norovirus-positive samples; GII.4 was the most common genotype (71.4% of typed specimens). Rotavirus G3P[8] was the most prevalent rotavirus genotype (44.0% of typed specimens) and G2P[4] was second most prevalent (16.0% of typed specimens). This community is likely part of a trend toward norovirus predominance over rotavirus in children after widespread vaccination against rotavirus.

Taenia solium infection in Peru: a collaboration between Peace Corps Volunteers and researchers in a community based study.

Background Neurocysticercosis is a leading cause of seizures and epilepsy in most of the world, and it occurs when Taenia solium larval cysts infect the central nervous system. T. solium tapeworm infection is endemic in much of Peru, but there are scarce data on the prevalence in many rural highland communities where it is likely to be hyper-endemic. Peace Corps Volunteers live and work in these communities; however, to our knowledge, they have not been used to facilitate public health research. Materials and Methods We utilized Peace Corps Volunteers to estimate the prevalence of T. solium tapeworm infection in seven rural communities in northern Peru. A convenience non-random sampling frame was used. Peace Corps Volunteers facilitated the collection of stool samples (N = 2,328), which were analyzed by sedimentation and microscopy. Niclosamide treatment and purgation preceded species identification, which was done by PCR-REA. Results Taenia sp. egg-positive stool samples were found in three of the seven communities we surveyed. The overall prevalence of Taenia sp. egg positivity was 2.1% (49/2,328) (95% CI = 1.6–2.8%) with prevalence up to 4.3% (42/977) (95% CI = 3.1–5.8%) by community. All 34 of the specimens tested by PCR-REA were T. solium. The overall prevalence of T. solium tapeworm infection was 1.5% (34/2,328) (95% CI = 1.0–2.0%). Prevalence up to 2.9% (28/977) (95% CI = 1.9–4.1%) by community was observed. Conclusion/Significance This study recorded high T. solium tapeworm prevalence, and identified hyper-endemic rural communities. It demonstrates that synergy between researchers and Peace Corps Volunteers can be an effective means to conducting large-scale, community-based studies in remote areas of Peru.

Identification and Characterization of Microsatellite Markers Derived from the Whole Genome Analysis of Taenia solium

Background Infections with Taenia solium are the most common cause of adult acquired seizures worldwide, and are the leading cause of epilepsy in developing countries. A better understanding of the genetic diversity of T. solium will improve parasite diagnostics and transmission pathways in endemic areas thereby facilitating the design of future control measures and interventions. Microsatellite markers are useful genome features, which enable strain typing and identification in complex pathogen genomes. Here we describe microsatellite identification and characterization in T. solium, providing information that will assist in global efforts to control this important pathogen. Methods For genome sequencing, T. solium cysts and proglottids were collected from Huancayo and Puno in Peru, respectively. Using next generation sequencing (NGS) and de novo assembly, we assembled two draft genomes and one hybrid genome. Microsatellite sequences were identified and 36 of them were selected for further analysis. Twenty T. solium isolates were collected from Tumbes in the northern region, and twenty from Puno in the southern region of Peru. The size-polymorphism of the selected microsatellites was determined with multi-capillary electrophoresis. We analyzed the association between microsatellite polymorphism and the geographic origin of the samples. Results The predicted size of the hybrid (proglottid genome combined with cyst genome) T. solium genome was 111 MB with a GC content of 42.54%. A total of 7,979 contigs (>1,000 nt) were obtained. We identified 9,129 microsatellites in the Puno-proglottid genome and 9,936 in the Huancayo-cyst genome, with 5 or more repeats, ranging from mono- to hexa-nucleotide. Seven microsatellites were polymorphic and 29 were monomorphic within the analyzed isolates. T. solium tapeworms were classified into two genetic groups that correlated with the North/South geographic origin of the parasites. Conclusions/Significance The availability of draft genomes for T. solium represents a significant step towards the understanding the biology of the parasite. We report here a set of T. solium polymorphic microsatellite markers that appear promising for genetic epidemiology studies.

The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs.

Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection.

Automatic pneumonia detection based on ultrasound video analysis

Pneumonia is a disease which causes high mortality in children under five years old, particularly in developing countries. This paper proposes a novel application of ultrasound video analysis for the detection of pneumonia. This application is based on the processing of small video chunks, in which an image processing algorithm analyzes each frame to get some overall video statistics. Then, based on these quantities, the likeness of presence of pneumonia in the video is determined. The algorithm exploits different geometrical properties of typical anatomical and pathological features that commonly appear in lung sonography and which are already clinically typified in the literature. Our technique has been tested on different transverse thoracic scanning protocols and probes maneuvers, thus, under a variety of clinical and usage protocols. Then, it can be targeted towards screening applications. We present encouraging results (AUC measure between 0.7851 and 0.9177) based on the analysis of 346 videos with an average duration of eight seconds. The analyzed videos were taken from children who were between three and five years old. Finally, our algorithm can be used directly as a classifier, but we detail how its performance may be enhanced if used as a first stage of a larger pipeline of other complementary pneumonia detection processes.Pneumonia is a disease which causes high mortality in children under five years old, particularly in developing countries. This paper proposes a novel application of ultrasound video analysis for the detection of pneumonia. This application is based on the processing of small video chunks, in which an image processing algorithm analyzes each frame to get some overall video statistics. Then, based on these quantities, the likeness of presence of pneumonia in the video is determined. The algorithm exploits different geometrical properties of typical anatomical and pathological features that commonly appear in lung sonography and which are already clinically typified in the literature. Our technique has been tested on different transverse thoracic scanning protocols and probes maneuvers, thus, under a variety of clinical and usage protocols. Then, it can be targeted towards screening applications. We present encouraging results (AUC measure between 0.7851 and 0.9177) based on the analysis of 346 videos with an average duration of eight seconds. The analyzed videos were taken from children who were between three and five years old. Finally, our algorithm can be used directly as a classifier, but we detail how its performance may be enhanced if used as a first stage of a larger pipeline of other complementary pneumonia detection processes.

Automatic detection of pneumonia analyzing ultrasound digital images

Pneumonia is one of the major causes of child mortality. Unfortunately, in developing countries there is a lack of infrastructure and medical experts in rural areas to provide the required diagnostics opportunely. Lung ultrasound echography has proved to be an important tool to detect lung consolidates as evidence of pneumonia. This paper presents a method for automatic diagnostics of pneumonia using ultrasound imaging of the lungs. The approach presented here is based on the analysis of patterns present in rectangular segments from the ultrasound digital images. Specific features from the characteristic vectors were obtained and classified with standard neural networks. A training and testing set of positive and negative vectors were compiled. Vectors obtained from a single patient were included only in the testing or in the training set, but never in both. Our approach was able to correctly classify vectors with evidence of pneumonia, with 91.5% sensitivity and 100% specificity.

Filtering of the skin portion on lung ultrasound digital images to facilitate automatic diagnostics of pneumonia

Pneumonia is one of the major causes of child mortality, but it is curable if one can achieves early diagnostics. Unfortunately, in developing countries there is a lack of infrastructure and medical experts in rural areas to provide the required diagnostics opportunely. Lung ultrasound echography has proved to be an important tool to detect lung consolidates as evidence of pneumonia. The use of ultrasound to detect pneumonia is limited by the image analysis for interpretation, which is carried by human experts. Pattern recognition and image analysis is a potential tool to facilitate recognition of pneumonia consolidates in absence of medical experts for automatic diagnostics. To perform an automatic analysis of lung ultrasound images for pneumonia detection, the noise introduced by the image portion of the skin, notably complicates the processing and interpretation. This paper presents a methodology to recognize and eliminate the portion of the skin in lung ultrasound images.

A novel enolase from Taenia solium metacestodes and its evaluation as an immunodiagnostic antigen for porcine cysticercosis

Cysticercosis is a worldwide parasitic disease of humans and pigs principally caused by infection with the larvae of the pork tapeworm Taenia solium. Through the use of the recently-made-available T. solium genome, we identified a gene within a novel 1448 bp ORF that theoretically encodes for a 433 amino acid-long protein and predicted to be an α-enolase closely related to enolases of other flatworms. Additional bioinformatic analyses revealed a putative plasminogen-binding region on this protein, suggesting a potential role for this protein in pathogenesis. On this basis, we isolated the mRNA encoding for this presumptive enolase from T. solium metacestodes and reverse-transcribed it into cDNA before subsequently cloning and expressing it in both E. coli (rEnoTs) and insect cells (rEnoTsBac), in a 6xHis tagged manner. The molecular weights of these two recombinant proteins were ∼48 and ∼50 kDa, respectively, with the differences likely attributable to differential glycosylation. We used spectrophotometric assays to confirm the enolase nature of rEnoTs as well as to measure its enzymatic activity. The resulting estimates of specific activity (60.000 U/mg) and Km (0.091 mM) are quite similar to the catalytic characteristics of enolases of other flatworms. rEnoTs also exhibited high immunogenicity, eliciting a strong polyclonal antibody response in immunized rabbits. We subsequently employed rEnoTsBac for use in an ELISA aimed at discriminating between healthy pigs and those infected with T. solium. This diagnostic assay exhibited a sensitivity of 88.4% (95% CI, 74.92%-96.11%) and a specificity of 83.7% (95% CI: 69.29%-93.19%). In conclusión, this study reports on and enzymatically characterizes a novel enolase from T. solium metacestode, and shows a potential use as an immunodiagnostic for porcine cysticercosis.

Molecular Detection of Taeniid Eggs in Beetles Collected in an Area Endemic for Taenia solium

The aim of this study was to demonstrate the presence of Taenia solium eggs in beetles collected from sources within the natural environment through molecular techniques. Fifty-four pools of beetles were collected in three villages in Piura, Peru. DNA was extracted using the FastDNA spin kit for soil. Molecular identification of Taenia species was then performed through partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Finally, positive samples were sequenced to determine the tapeworm species. Seven positive samples were obtained through polymerase chain reaction amplification. Sequencing confirmed that two samples were from T. solium and three samples were from Taenia hydatigena. The other two samples could not be specifically identified. Our findings demonstrate that dung beetles ingest T. solium and T. hydatigena eggs under natural conditions and suggest that beetles may play a role in the dynamics of transmission of these cestodes.