Identification of human intestinal parasites affecting an asymptomatic peri-urban Argentinian population using multi-parallel quantitative real-time polymerase chain reaction

Background: In resource-limited countries, stool microscopy is the diagnostic test of choice for intestinal parasites (soil-transmitted helminths and/or intestinal protozoa). However, sensitivity and specificity is low. Improved diagnosis of intestinal parasites is especially important for accurate measurements of prevalence and intensity of infections in endemic areas. Methods: The study was carried out in Orán, Argentina. A total of 99 stool samples from a local surveillance campaign were analyzed by concentration microscopy and McMaster egg counting technique compared to the analysis by multi-parallel quantitative real-time polymerase chain reaction (qPCR). This study compared the performance of qPCR assay and stool microscopy for 8 common intestinal parasites that infect humans including the helminths Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, and the protozoa Giardia lamblia, Cryptosporidium parvum/hominis, and Entamoeba histolytica, and investigated the prevalence of polyparasitism in an endemic area. Results: qPCR showed higher detection rates for all parasites as compared to stool microscopy except T. trichiura. Species-specific primers and probes were able to distinguish between A. duodenale (19.1 %) and N. americanus (36.4 %) infections. There were 48.6 % of subjects co-infected with both hookworms, and a significant increase in hookworm DNA for A. duodenale versus N. americanus (119.6 fg/μL: 0.63 fg/μL, P∈<∈0.001) respectively. qPCR outperformed microscopy by the largest margin in G. lamblia infections (63.6 % versus 8.1 %, P∈<∈0.05). Polyparasitism was detected more often by qPCR compared to microscopy (64.7 % versus 24.2 %, P∈<∈0.05). Conclusions: Multi-parallel qPCR is a quantitative molecular diagnostic method for common intestinal parasites in an endemic area that has improved diagnostic accuracy compared to stool microscopy. This first time use of multi-parallel qPCR in Argentina has demonstrated the high prevalence of intestinal parasites in a peri-urban area. These results will contribute to more accurate epidemiological survey, refined treatment strategies on a public scale, and better health outcomes in endemic settings.Fil: Cimino, Rubén Oscar. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jeun, Rebecca. Baylor College Of Medicine; Estados UnidosFil: Juarez, Marisa. Universidad Nacional de Salta; ArgentinaFil: Cajal, Pamela S.. Universidad Nacional de Salta; ArgentinaFil: Vargas Flores, Paola Andrea. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Echazú, Adriana. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bryan, Patricia E.. Baylor College Of Medicine; Estados UnidosFil: Nasser, Julio Rubén. Universidad Nacional de Salta; ArgentinaFil: Krolewiecki, Alejandro Javier. Universidad Nacional de Salta; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mejia, Rojelio. Baylor College Of Medicine; Estados Unidos. Universidad Nacional de Salta; Argentin

Parasitic Infections Represent a Significant Health Threat Among Recent Immigrants in Chicago

Parasitic infections are likely under-recognized among immigrant populations in the USA. We conducted a cross-sectional study to evaluate if such infections have health impacts among recent immigrants in Chicago and to identify predictive factors for parasitic infections. A total of 133 recent immigrants were enrolled, filling out a standardized medical questionnaire and providing blood and stool samples. Appriximately 12% of subjects (15/125) who provided a blood or stool sample for testing were found to have evidence of current or prior infection with a pathogenic parasite, of which Toxocara spp. (8 subjects, 6.4%) and Strongyloides stercoralis (5 subjects, 4%) were most commonly identified. Parasitic infection was more likely among subjects who had immigrated within the previous 2 years and those with a self-reported history of worms in the stool. The most useful surrogate markers identified for parasitic infections were an elevated immunoglobulin E level (seen in 46.7% (7/15) of subjects with parasitic infections and 20% (22/110) of uninfected individuals, p = 0.04) and the presence of Blastocystis hominis cysts on Ova & Parasite exam (detected in 38.5% (5/13) of subjects with parasitic infections who provided a stool sample and 5.1% (5/98) of uninfected subjects, p = 0.002). Our study found that parasitic infections may be common in recent US immigrants, which highlights an important health disparity among a vulnerable population that merits further study. Additionally, clinical risk factors, symptoms, and laboratory findings traditionally thought to be associated with parasites were commonly found but not predictive of infection in this study population

Diagnostic Accuracy of Five Serologic Tests for Strongyloides stercoralis Infection

Background:The diagnosis of Strongyloides stercoralis (S. stercoralis) infection is hampered by the suboptimal sensitivity of fecal-based tests. Serological methods are believed to be more sensitive, although assessing their accuracy is difficult because of the lack of sensitivity of a fecal-based reference ("gold") standard.Methods:The sensitivity and specificity of 5 serologic tests for S. stercoralis (in-house IFAT, NIE-ELISA and NIE-LIPS and the commercially available Bordier-ELISA and IVD-ELISA) were assessed on 399 cryopreserved serum samples. Accuracy was measured using fecal results as the primary reference standard, but also using a composite reference standard (based on a combination of tests).Results:According to the latter standard, the most sensitive test was IFAT, with 94.6% sensitivity (91.2-96.9), followed by IVD-ELISA (92.3%, 87.7-96.9). The most specific test was NIE-LIPS, with specificity 99.6% (98.9-100), followed by IVD-ELISA (97.4%, 95.5-99.3). NIE-LIPS did not cross-react with any of the specimens from subjects with other parasitic infections. NIE-LIPS and the two commercial ELISAs approach 100% specificity at a cut off level that maintains ≥70% sensitivity.Conclusions:NIE-LIPS is the most accurate serologic test for the diagnosis of S. stercoralis infection. IFAT and each of the ELISA tests are sufficiently accurate, above a given cut off, for diagnosis, prevalence studies and inclusion in clinical trials.Fil: Bisoffi, Zeno. Sacro Cuore Hospital; ItaliaFil: Buonfrate, Dora. Sacro Cuore Hospital; ItaliaFil: Sequi, Marco. Istituto Di Ricerche Farmacologiche Mario Negri; ItaliaFil: Mejia, Rojelio. National Institute Of Allergy And Infectious Diseases; Estados UnidosFil: Cimino, Rubén Oscar. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Krolewiecki, Alejandro Javier. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Albonico, Marco. Sacro Cuore Hospital; ItaliaFil: Gobbo, Maria. Sacro Cuore Hospital; ItaliaFil: Bonafini, Stefania. Sacro Cuore Hospital; ItaliaFil: Angheben, Andrea. Sacro Cuore Hospital; ItaliaFil: Requena-Mendez, Ana. Universidad de Barcelona; EspañaFil: Muñoz, José. Universidad de Barcelona; EspañaFil: Nutman, Thomas B.. National Institute Of Allergy And Infectious Diseases; Estados Unido

The hookworm Ancylostoma ceylanicum intestinal transcriptome provides a platform for selecting drug and vaccine candidates

BACKGROUND: The intestine of hookworms contains enzymes and proteins involved in the blood-feeding process of the parasite and is therefore a promising source of possible vaccine antigens. One such antigen, the hemoglobin-digesting intestinal aspartic protease known as Na-APR-1 from the human hookworm Necator americanus, is currently a lead candidate antigen in clinical trials, as is Na-GST-1 a heme-detoxifying glutathione S-transferase. METHODS: In order to discover additional hookworm vaccine antigens, messenger RNA was obtained from the intestine of male hookworms, Ancylostoma ceylanicum, maintained in hamsters. RNA-seq was performed using Illumina high-throughput sequencing technology. The genes expressed in the hookworm intestine were compared with those expressed in the whole worm and those genes overexpressed in the parasite intestine transcriptome were further analyzed. RESULTS: Among the lead transcripts identified were genes encoding for proteolytic enzymes including an A. ceylanicum APR-1, but the most common proteases were cysteine-, serine-, and metallo-proteases. Also in abundance were specific transporters of key breakdown metabolites, including amino acids, glucose, lipids, ions and water; detoxifying and heme-binding glutathione S-transferases; a family of cysteine-rich/antigen 5/pathogenesis-related 1 proteins (CAP) previously found in high abundance in parasitic nematodes; C-type lectins; and heat shock proteins. These candidates will be ranked for downstream antigen target selection based on key criteria including abundance, uniqueness in the parasite versus the vertebrate host, as well as solubility and yield of expression. CONCLUSION: The intestinal transcriptome of A. ceylanicum provides useful information for the identification of proteins involved in the blood-feeding process, representing a first step towards a reverse vaccinology approach to a human hookworm vaccine. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13071-016-1795-8) contains supplementary material, which is available to authorized users

A Novel, Species-Specific, Real-Time PCR Assay for the Detection of the Emerging Zoonotic Parasite Ancylostoma Ceylanicum in Human Stool

Historically, Ancylostoma ceylanicum has been viewed as an uncommon cause of human hookworm infection, with minimal public health importance. However, recent reports have indicated that this zoonotic hookworm causes a much greater incidence of infection within certain human populations than was previously believed. Current methods for the species-level detection of A. ceylanicum rely on techniques that involve conventional PCR accompanied by restriction enzyme digestions. These PCR-based assays are not only labo- rious but they lack sensitivity as they target suboptimal regions on the DNA. As efforts aimed at the eradication of hookworm disease have grown substantially over the last decade, the need for sensitive and specific tools to monitor and evaluate programmatic successes has correspondingly escalated. Since a growing body of evidence suggests that patient responses to drug treatment can vary based upon the species of hookworm that is causing infection, accurate species-level diagnostics are advantageous. Accordingly, the novel real-time PCR-based assay described here provides a sensitive, species-specific diag- nostic tool that will facilitate the accurate mapping of disease endemicity and will aid in the evaluation of progress of programmatic deworming efforts

Immunization with the recombinant antigen Ss-IR induces protective immunity to infection with Strongyloides stercoralis in mice.

Human intestinal infections with the nematode Strongyloides stercoralis remain a significant problem worldwide and a vaccine would be a useful addition to the tools available to prevent and control this infection. The goal of this study was to test single antigens for their efficacy in a vaccine against S. stercoralis larvae in mice. Alum was used as the adjuvant in these studies and antigens selected for analysis were either recognized by protective human IgG (Ss-TMY-1, Ss-EAT-6, and Ss-LEC-5) or were known to be highly immunogenic in humans (Ss-NIE-1 and Ss-IR). Only mice immunized with the Ss-IR antigen demonstrated a significant decrease of approximately 80% in the survival of larval parasites in the challenge infection. Antibodies, recovered from mice with protective immunity to S. stercoralis after immunization with Ss-IR, were used to locate the antigen in the larvae. Confocal microscopy revealed that IgG from mice immunized with Ss-IR bound to the surface of the parasites and observations by electron microscopy indicated that IgG bound to granules in the glandular esophagus. Serum collected from mice immunized with Ss-IR passively transferred immunity to naïve mice. These studies demonstrate that Ss-IR, in combination with alum, induces high levels of protective immunity through an antibody dependent mechanism and may therefore be suitable for further development as a vaccine against human strongyloidiasis

Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection.

Background: The diagnosis of Strongyloides stercoralis (S. stercoralis) infection is hampered by the suboptimal sensitivity of fecal-based tests. Serological methods are believed to be more sensitive, although assessing their accuracy is difficult because of the lack of sensitivity of a fecal-based reference ('gold') standard. Methods: The sensitivity and specificity of 5 serologic tests for S. stercoralis (in-house IFAT, NIE-ELISA and NIE-LIPS and the commercially available Bordier-ELISA and IVD-ELISA) were assessed on 399 cryopreserved serum samples. Accuracy was measured using fecal results as the primary reference standard, but also using a composite reference standard (based on a combination of tests). Results: According to the latter standard, the most sensitive test was IFAT, with 94.6% sensitivity (91.2-96.9), followed by IVD-ELISA (92.3%, 87.7-96.9). The most specific test was NIE-LIPS, with specificity 99.6% (98.9-100), followed by IVD-ELISA (97.4%, 95.5-99.3). NIE-LIPS did not cross-react with any of the specimens from subjects with other parasitic infections. NIE-LIPS and the two commercial ELISAs approach 100% specificity at a cut off level that maintains ≥70% sensitivity. Conclusions: NIE-LIPS is the most accurate serologic test for the diagnosis of S. stercoralis infection. IFAT and each of the ELISA tests are sufficiently accurate, above a given cut off, for diagnosis, prevalence studies and inclusion in clinical trials

Accuracy of Five Serologic Tests for the Follow up of Strongyloides stercoralis Infection

BACKGROUND: Traditional faecal-based methods have poor sensitivity for the detection of S. stercoralis, therefore are inadequate for post-treatment evaluation of infected patients who should be carefully monitored to exclude the persistence of the infection. In a previous study, we demonstrated high accuracy of five serology tests for the screening and diagnosis of strongyloidiasis. Aim of this study is to evaluate the performance of the same five tests for the follow up of patients infected with S. stercoralis. METHODS: Retrospective study on anonymized, cryo-preserved samples available at the Centre for Tropical Diseases (Negrar, Verona, Italy). Samples were collected before and from 3 to 12 months after treatment. The samples were tested with two commercially-available ELISA tests (IVD, Bordier), two techniques based on a recombinant antigen (NIE-ELISA and NIE-LIPS) and one in-house IFAT. The results of each test were evaluated both in relation to the results of fecal examination and to those of a composite reference standard (classifying as positive a sample with positive stools and/or at least three positive serology tests). The associations between the independent variables age and time and the dependent variable value of serological test (for all five tests), were analyzed by linear mixed-effects regression model. RESULTS: A high proportion of samples demonstrated for each test a seroreversion or a relevant decline (optical density/relative light units halved or decrease of at least two titers for IFAT) at follow up, results confirmed by the linear mixed effects model that showed a trend to seroreversion over time for all tests. In particular, IVD-ELISA (almost 90% samples demonstrated relevant decline) and IFAT (almost 87%) had the best performance. Considering only samples with a complete negativization, NIE-ELISA showed the best performance (72.5% seroreversion). CONCLUSIONS: Serology is useful for the follow up of patients infected with S. stercoralis and determining test of cure

Impact of intestinal parasites on microbiota and cobalamin gene sequences: A pilot study

Background: Approximately 30% of children worldwide are infected with gastrointestinal parasites. Depending on the species, parasites can disrupt intestinal bacterial microbiota affecting essential vitamin biosynthesis. Methods: Stool samples were collected from 37 asymptomatic children from a previous cross-sectional Argentinian study. A multi-parallel real-time quantitative PCR was implemented for Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, Cryptosporidium spp., Entamoeba histolytica and Giardia duodenalis. In addition, whole-genome sequencing analysis was conducted for bacterial microbiota on all samples and analyzed using Livermore Metagenomic Analysis Toolkit and DIAMOND software. Separate analyses were carried out for uninfected, Giardia-only, Giardia + helminth co-infections, and helminth-only groups. Results: For Giardia-only infected children compared to uninfected children, DNA sequencing data showed a decrease in microbiota biodiversity that correlated with increasing Giardia burden and was statistically significant using Shannon's alpha diversity (Giardia-only > 1 fg/μl 2.346; non-infected group 3.253, P = 0.0317). An increase in diversity was observed for helminth-only infections with a decrease in diversity for Giardia + helminth co-infections (P = 0.00178). In Giardia-only infections, microbiome taxonomy changed from Firmicutes towards increasing proportions of Prevotella, with the degree of change related to the intensity of infection compared to uninfected (P = 0.0317). The abundance of Prevotella bacteria was decreased in the helminths-only group but increased for Giardia + helminth co-infections (P = 0.0262). Metagenomic analysis determined cobalamin synthesis was decreased in the Giardia > 1 fg/μl group compared to both the Giardia < 1 fg/μl and the uninfected group (P = 0.0369). Giardia + helminth group also had a decrease in cobalamin CbiM genes from helminth-only infections (P = 0.000754). Conclusion: The study results may provide evidence for an effect of parasitic infections enabling the permissive growth of anaerobic bacteria such as Prevotella, suggesting an altered capacity of vitamin B12 (cobalamin) biosynthesis and potential impact on growth and development in children.Fil: Mejia, Rojelio. Baylor College of Medicine; Estados Unidos. Universidad Nacional de Salta; ArgentinaFil: Damania, Ashish. Baylor College of Medicine; Estados UnidosFil: Jeun, Rebecca. Baylor College of Medicine; Estados UnidosFil: Bryan, Patricia E.. Baylor College of Medicine; Estados UnidosFil: Vargas, Paola. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Juarez, Marisa del Valle. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Cajal, Silvana Pamela. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Nasser, Julio Rubén. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; ArgentinaFil: Krolewiecki, Alejandro Javier. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; ArgentinaFil: Lefoulon, Emilie. New England Biolabs; Estados UnidosFil: Long, Courtney. New England Biolabs; Estados UnidosFil: Drake, Evan. New England Biolabs; Estados UnidosFil: Cimino, Rubén Oscar. Universidad Nacional de Salta. Sede Regional Orán. Instituto de Investigación de Enfermedades Tropicales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta; ArgentinaFil: Slatko, Barton. New England Biolabs; Estados Unido