A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/µL for DNA samples diluted in water, 10 fg/µL for Fasciola/snail DNA scramble, and 100 fg/µL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Demographic Characteristics and Low Iron Status Markers Are Associated with Hemoglobin Levels and Anemia among Children Living at High Elevation in Cusco, Peru.

Anemia is a complex condition associated with diet, chronic infections, and blood loss. Children living at high altitudes have higher absolute hemoglobin levels due to hypoxemia. However, they are exposed to repeated infections and dietary limitations. We conducted a cross-sectional study to identify factors affecting the hemoglobin concentration in children living in high-altitude rural communities in the Anta province of Peru. All children 3-16 years of age attending public schools were invited to participate. We enrolled children 3-16 years old in schools and visited their homes to collect demographic, socioeconomic, medical history, and anthropometric data. Children provided blood and stool samples for complete blood counts, iron status markers, and helminth infection testing. Among the 2,000 children enrolled, the mean age was 9.9 (±3.4) years, 1,004 (50.2%) were female, and the median residence altitude was 3,398 (interquartile range 3,35-3,497) meters. The mean hemoglobin level was 15 (±1.15) mg/dL; 320 (16%) had anemia as defined by WHO. Children with anemia were more likely to have lower serum iron levels (odds ratio [OR] 2.8 [95% CI 2.2-3.6], P <0.001) and serum transferrin saturation (OR 2.8 [95% CI 2-3.9], P <0.001). Younger age (OR 0.85 [95% CI 0.82-0.89], P <0.001), stunting (OR 0.68 [95% CI 0.59-0.79], P <0.001), education of the mother (OR 0.94 [95% CI 0.91-0.98], P <0.005), and low eosinophils (OR 0.49 [95% CI 0.26-0.9], P = 0.022) were associated with anemia. Helminth infections were not associated with anemia. Anemia among children at high altitude is multifactorial, but iron deficiency is a contributing factor. Further studies are needed to evaluate iron status and anemia in children living at high altitudes.

Plasma cytokines during acute human fascioliasis.

Fascioliasis is a foodborne trematode endemic worldwide. Children under 15 years have the highest prevalence of infection. We hypothesized that acute fascioliasis would be associated with more pronounced cytokine changes than in chronic disease or no helminth infections. To test this hypothesis, 33 children who lived in the Peruvian highlands were classified into 3 groups: acute fascioliasis, chronic fascioliasis, and no helminth infection. Type Th1, Th2, and Th17 cytokines were measured in plasma by cytometric bead array. Children with acute infection had higher levels of IL-5 and IL-17 compared with controls (p < 0.001 and p < 0.007, respectively). The increased IL-5 plasma concentration in children with acute infection was associated with the eosinophilia found in that group.

A Comparison of the Risk for Chronic Fascioliasis between Children 3 to 5 Years and Children 6 to 12 Years of Age in the Cusco Region of Peru.

School-age children bear the highest burden of fascioliasis in endemic countries. Few studies have addressed Fasciola in preschool children. We performed a secondary data analysis using two Fasciola databases from Cusco, Peru, comparing preschoolers with elementary school children. We included 2,630 children, 50% were female, the median age was 8.4 years (interquartile range [IQR] 6.1-10.5), and 15% (396/2,630) were < 5 years of age. Children < 5 years were less likely to be infected with Fasciola hepatica (P = 0.008) and Hymenolepis nana (P < 0.001) and more likely to have anemia (P < 0.001) and a lower median height for age Z (HAZ) score (P = 0.002). Fascioliasis was less common in younger children, but this group may be at higher risk for chronic complications caused by fascioliasis.

Triclabendazole Treatment Failure for Fasciola hepatica Infection among Preschool and School-Age Children, Cusco, Peru1.

We conducted a retrospective cohort study of children who had chronic fascioliasis in the highlands of Peru to determine triclabendazole treatment efficacy. Children passing Fasciola eggs in stool were offered directly observed triclabendazole treatment (>1 doses of 10 mg/kg). Parasitologic cure was evaluated by using microscopy of stool 1-4 months after each treatment. A total of 146 children who had chronic fascioliasis participated in the study; 53% were female, and the mean ± SD age was 10.4 ± 3.1 years. After the first treatment, 55% of the children achieved parasitologic cure. Cure rates decreased after the second (38%), third (30%), and fourth (23%) treatments; 17 children (11.6%) did not achieve cure after 4 treatments. Higher baseline egg counts and lower socioeconomic status were associated with triclabendazole treatment failure. Decreased triclabendazole efficacy in disease-endemic communities threatens control efforts. Further research on triclabendazole resistance and new drugs to overcome it are urgently needed.

Comparison of Liver Condemnation and Bile Microscopy As Tools to Estimate Fasciola hepatica Prevalence and Burden in the Anta Province of Cusco in Peru.

Fascioliasis is a zoonotic infection linked to significant economic losses in the livestock industry. Infection prevalence and estimated financial burden vary across locations owing to different diagnostic tests used. The accuracy of liver condemnation to estimate the prevalence and costs of fascioliasis has seldom been evaluated. We performed a pilot study to determine the prevalence and burden of Fasciola hepatica infection among cattle slaughtered at the municipal abattoir in the Anta province of the Cusco highlands in Peru. We compared liver condemnation with bile microscopy for the diagnosis of infection and prediction of carcass weight. Data were collected from 2009 slaughtered cattle for 1 year. The overall prevalence of Fasciola infection by bile microscopy was 62.5% (1247/2009). A higher prevalence was observed after the rainy season from March to August than from September to February (p < 0.01). Fascioliasis prevalence during the first 6 months was 77.4% (714/923), combining the results of condemnation and microscopy. Bile microscopy diagnosed more infections than liver condemnation (62.7% (579/923) versus 55.4% (511/923), McNemar test p < 0.01). The agreement of the bile microscopy testing with liver condemnation was fair (κ = 0.247). Animal age, gender, breed, and liver condemnation predicted carcass weight [F (df 4, 704) = 61.1, p < 0.001]. Liver condemnation and bile microscopy are complementary tools for evaluation of the prevalence and burden of fascioliasis in livestock. Large scale studies are warranted to confirm our results.

Stunting in pre-school and school-age children in the Peruvian highlands and its association with Fasciola infection and demographic factors.

Fascioliasis is a zoonotic trematode infection that is endemic in the highlands of Peru. Chronic fascioliasis can be asymptomatic and remain undiagnosed for years. Chronic malnutrition in children, as manifested by stunting, leads to delayed cognitive development and lost productivity. We hypothesized that fascioliasis is among the factors associated with stunting in children from endemic areas. We conducted a cross-sectional study among children attending pre-school and school in 26 communities in the Anta province in the Cusco region of Peru. We conducted interviews to collect information on demographic, socioeconomic, and medical history. Blood was collected and tested for complete cell count and FAS2 ELISA for Fasciola antibodies. Three stool samples per participant were tested for parasites by Kato-Katz and Lumbreras rapid sedimentation methods. Chronic fascioliasis was determined by the presence of ova in stool. Children's height, weight, and age were recorded and used to calculate height for age Z scores (HAZ). Three thousand children participated in the study. Nine percent (264) of children had at least one positive test for Fasciola infection, 6% (164) had chronic fascioliasis, and 3% (102) had only positive antibody tests. The median HAZ was -1.41 (IQR: -2.03 to -0.81) and was similar in males and females. Twenty six percent (776) of children had stunting with HAZ < -2. Children with chronic fascioliasis had a lower median HAZ than children without Fasciola (-1.54 vs. -1.4, p = 0.014). History of treatment for malnutrition, history of treatment for anemia, having other helminths in stool, lower socioeconomic score, living at a higher elevation, and fewer years of schooling of both parents were associated with a lower HAZ score. In a multiple regression analysis, older age and a lower socioeconomic score were associated with a lower HAZ score. While fascioliasis and other helminths were associated with lower HAZ, they were not independent of the socioeconomic score.

Fasciola hepatica Infection Risk for Adult Household Members Living with Children with Fascioliasis in Cusco, Peru.

Fasciola hepatica is highly prevalent in the highlands of Peru. School-age children have the greatest risk of infection. Mass treatment of at-risk populations has been proposed to control the infection and prevent complications. However, the decreasing effectiveness of triclabendazole raises concerns regarding this strategy. Previous studies reported aggregation of Fasciola infection among family members. This study aimed to determine the risk of fascioliasis among household members living with Fasciola-infected children identified through school-based testing. We conducted a cross-sectional study including adult members of households where children with and without fascioliasis were identified. Demographic, epidemiological, and socioeconomic information was collected. One blood sample was drawn to test for Fasciola antibodies, and three stool samples were collected for microscopy for Fasciola ova. We tested 326 adults from 213 households. Of these adult subjects, chronic fascioliasis (24 of 326, 7.4%) was the most common helminth infection. Thirty-nine subjects (12.7%) tested positive for Fasciola antibodies. Combining microscopy and serum antibody tests, 13.2% (43 of 326) had evidence of Fasciola infection. One third (104 of 326, 31.9%) of the participants lived with at least one child infected with Fasciola hepatica. Adults with fascioliasis were four times more likely to live with an infected child. Poverty and diet were associated with increased risk of Fasciola infection. Adults with fascioliasis were significantly more likely to live with Fasciola-infected children.

Regulatory T cell expansion resolves after effective strongyloidiasis treatment in subjects with HTLV-1 co-infection.

BACKGROUND: Regulatory T-cells (Tregs) are increased in patients with HTLV-1/Strongyloides stercoralis co-infection, and they may modify otherwise protective antigen-specific cytokine production. We hypothesized that effective anti-helminthic treatment would decrease Tregs and restore antigen-specific cytokine responses. METHODS/RESULTS: We enrolled 19 patients with Strongyloides larvae in their stool by Baerman's test. Six were positive and 13 negative for antibody to HTLV-1 by ELISA, with positive tests confirmed by immunoblot. Before treatment, co-infected subjects had higher Tregs percentages and lower antigen-stimulated IL-5 levels compared to subjects with Strongyloides without HTLV-1. All patients were treated with ivermectin. After effective treatment, Tregs percentages decreased in patients with HTLV-1; however, antigen-specific IL-5 production remained blunted in co-infected subjects. CONCLUSION: These results suggest that treating strongyloidiasis infection decreases circulating Tregs, but antigen-specific cytokine remains altered. This may reflect blunting of sensitization by Tregs.

Regulatory T cells and IgE expression in duodenal mucosa of Strongyloides stercoralis and human T lymphotropic virus type 1 co-infected patients.

BACKGROUND: Strongyloides stercoralis is an intestinal nematode unique in its ability to replicate in the human host, allowing ongoing cycles of autoinfection, persisting for decades within the same host. Although usually asymptomatic, overwhelming infections can occur in Strongyloides and HTLV-1 co-infected individuals (SS/HTLV-1). Regulatory T cells (Tregs) are able to blunt specific Th2 responses necessary to control the parasite. We previously reported that peripheral blood Tregs are increased in SS/HTLV-1 and correlate with low Th2 responses. We hypothesized that Tregs are also increased at the site of infection in duodenal mucosa. METHODS: Paraffin embedded duodenal biopsies were obtained from 10 SS/HTLV-1 patients, 3 controls with non-parasitic chronic duodenitis, and 2 healthy controls. Immunohistochemistry was performed using monoclonal antibodies against human CD3, CD8, IgE and FoxP3. The number of cells were counted using a conventional light microscope. The number of CD3+, CD8+, FoxP3+ and IgE positive cells per 0.35 mm2 was measured using ImagePro Plus software comparing areas adjacent or distant from parasite material. RESULTS: In patients with SS/HTLV-1, T lymphocyte counts and CD8+ cells were lower in areas adjacent to the parasite compared to non-adjacent areas (CD3+: adjacent: 6.5 [Interquartile range (IQR: 2.8-12.3)]; non-adjacent: 24.5 [IQR: 20.9-34.4]; Mann-Whitney p = 0.0003; CD8+: adjacent: 4.5 [IQR: 2.3-11.8]; non-adjacent: 21 [IQR: 15.3-42.9]; Mann-Whitney p = 0.0011). Tregs cells in the intestines (FoxP3+ expressing cells) were increased in patients with SS/HTLV-1 compared with patients with chronic duodenitis (SS/HTLV-1: 1.5 [IQR: 0.7-2.3]; duodenitis controls: 0 [range 0-0.7]; healthy controls: 0; Mann-Whitney p = 0.034). There was also a trend towards fewer eosinophils adjacent to the parasites. Among SS/HTLV-1 patients the number of IgE expressing cells was increased for in areas not adjacent to the parasite compared to non-adjacent areas (ANOVA, p = 0.001). CONCLUSIONS: Our data shows increased Treg cell numbers localized adjacent to the parasites in the duodenum SS/HTLV-1 patients. In addition, other T lymphocytes and IgE expressing cells were decreased adjacent to the parasites, suggesting an important role for Tregs in down-regulating local parasite effector responses.

Socioeconomic Factors Associated with Fasciola hepatica Infection Among Children from 26 Communities of the Cusco Region of Peru.

Fasciola hepatica is the most widely distributed trematode-affecting humans. The Andes Mountains are highly endemic for fascioliasis. We report results of a cross-sectional study evaluating the epidemiology of Fasciola among children in 26 agricultural communities in the Cusco region of Peru. Children 3 to 16 years old were enrolled in preschools and schools. Blood from participants was tested for complete blood counts, transaminases, and Fasciola antibodies. Stool samples were tested for Fasciola and other parasites. A total of 2,515 children were included in the analysis and the mean age was 9.6 years (±3.6). Ten percent (253) of the children had at least one positive test for Fasciola, 6% had chronic infection, and 0.4% acute infection. The rest of the subjects had only antibodies against Fasciola. The prevalence of infection varied from 0% to 20% between communities. Children with evidence of Fasciola exposure were older, lived at higher altitudes, and had a lower socioeconomic status than children without infection. The logistic regression analysis showed that children from Ancahuasi district, older children, and children with higher measures of poverty were more likely to have Fasciola exposure. Fascioliasis is common in the Cusco region and associated with poverty. However, the distribution varies markedly between communities.

Regulatory T-Cell Dynamics in Cutaneous and Mucocutaneous Leishmaniasis due to Leishmania braziliensis.

To evaluate the dynamics of regulatory T cells (Tregs) during tegumentary leishmaniasis, we assessed peripheral blood and biopsies from 54 patients. Patients with cutaneous leishmaniasis (CL) had a decreased proportion of Tregs in the peripheral blood, but the proportion was higher in the biopsies of lesions. During treatment of CL, circulating Tregs increased reaching normal proportions, whereas antigen-specific interferon-γ responses diminished. By contrast, circulating Tregs from mucosal leishmaniasis patients failed to normalize during treatment. C-C chemokine receptor type 5 was expressed on a large proportion of Tregs at the site of infection. These results demonstrate increased Tregs at the site of infection, possibly homing from the peripheral circulation.

Killing of Cryptosporidium sporozoites by Lactoferrin.

Intestinal infection caused by Cryptosporidium is a major contributor to diarrhea morbidity and mortality in young children around the world. Current treatments for children suffering from cryptosporidiosis are suboptimal. Lactoferrin is a glycoprotein found in breast milk. It has showed bacteriostatic and antimicrobial activity in the intestine. However, the effects of lactoferrin on the intestinal parasite Cryptosporidium have not been reported. In this study, we investigated the anticryptosporidial activity of human lactoferrin on different stages of Cryptosporidium. Physiologic concentrations of lactoferrin killed Cryptosporidium parvum sporozoites, but had no significant effect on oocysts viability or parasite intracellular development. Since sporozoites are essential for the infection process, our data reinforce the importance of breastfeeding and point to the potential of lactoferrin as a novel therapeutic agent for cryptosporidiosis.

Prevalence and Risk Factors for Human Cystic Echinococcosis in the Cusco Region of the Peruvian Highlands Diagnosed Using Focused Abdominal Ultrasound.

AbstractLatin America is among the highly endemic regions for cystic echinococcosis (CE). In Peru, an estimated 1,139 disability-adjusted life years are lost annually from surgical treatment of CE. This is comparable with the combined total for Argentina, Brazil, Uruguay, and Chile. The prevalence of human infection has been investigated in the central Peruvian Andes, but there are no community-based screening data from other regions of Peru. We carried out a population survey in January 2015 using abdominal ultrasound to estimate the prevalence of abdominal CE in the Canas and Canchis provinces, in the Cusco region of Peru. Among 1,351 subjects screened, 41 (3%) had CE. There was significant variation between communities with similar socioeconomic features in a small geographical area. A history of CE was reported by 4.1% of the screened subjects, among whom 30.3% still had CE on ultrasound. Among patients reporting previous CE treatment, 14.9% had CE in active stages. Limited education, community of residence, and knowing people with CE in the community were associated with CE. These results demonstrate a significant burden of CE in the region and suggest the need for further investigations, control activities, and optimization of clinical management for CE in this area.

Hymenolepis nana Impact Among Children in the Highlands of Cusco, Peru: An Emerging Neglected Parasite Infection.

Hymenolepis nana is the most common cestode infection in the world. However, limited information is available regarding its impact on affected populations. We studied the epidemiology and symptoms associated with hymenolepiasis among children 3-16 years old in 16 rural communities of the highlands of the Cusco region in Peru. Information on demographics, socioeconomic status, symptoms as reported by parents, and parasitological testing was obtained from the database of an ongoing Fasciola hepatica epidemiologic study. A total of 1,230 children were included in the study. Forty-five percent were infected with at least one pathogenic intestinal parasite. Giardia spp. (22.9%) was the most common, followed by Hymenolepis (17.4%), Fasciola (14.1%), Ascaris lumbricoides (6.1%), and Strongyloides stercoralis (2%). The prevalence of Hymenolepis infection varied by community, by other parasitic infections, and by socioeconomic status. However, only years of education of the mother, use of well water, and age less than 10 years were associated with Hymenolepis infection in the multivariate analysis. Hymenolepis nana infection was associated with diarrhea, jaundice, headaches, fever, and fatigue. Children with > 500 eggs/g of stool were more likely to have symptoms of weight loss, jaundice, diarrhea, and fever. Hymenolepis nana infection and age were the only factors retained in the multivariate analysis modeling diarrhea. Hymenolepiasis is a common gastrointestinal helminth in the Cusco region and is associated with significant morbidity in children in rural communities. The impact caused by the emergence of Hymenolepis as a prevalent intestinal parasite deserves closer scrutiny.

[Massive hookworm infection as a cause of intestinal bleeding and severe anemia]. / Infección masiva por Ancylostoma duodenale como causa de hemorragia intestinal y anemia severa.

Overt gastrointestinal bleeding caused by hookworm infection is rarely reported. We present a 34 year old male with lower gastrointestinal bleeding with evidence of massive hookworm infection on colonoscopy and discuss the need to consider hookworm infection as a possible etiology of gastrointestinal bleed in endemic areas.

Treatment Failure after Multiple Courses of Triclabendazole among Patients with Fascioliasis in Cusco, Peru: A Case Series.

Triclabendazole is reported to be highly effective in treatment of human fascioliasis. We present 7 of 19 selected cases of human fascioliasis referred to our center in the Cusco region of Peru that failed to respond to triclabendazole. These were mostly symptomatic adults of both sexes that continued passing Fasciola eggs in the stool despite multiple treatments with 2 doses of triclabendazole at 10 mg/kg per dose. We documented the presence of eggs by rapid sedimentation and Kato Katz tests after each treatment course. We found that repeated triclabendazole courses were not effective against fascioliasis in this group of people. These findings suggest that resistance to triclabendazole may be an emerging problem in the Andes.

Fascioliasis and eosinophilia in the highlands of Cuzco, Peru and their association with water and socioeconomic factors.

There are limited data about the epidemiology of fascioliasis in Cuzco, Peru. We studied children 3-12 years old from six communities in the highlands of Cuzco to evaluate the epidemiology of fascioliasis; 227 children were included, one-half were female, the mean age was 7.5 (±2.6) years, and 46.2±% had one or more parasites, including Fasciola (9.7%), Ascaris (12.8%), Hymenolepis (9.3%), Trichuris (1.3%), hookworm (1.8%), Strongyloides (0.9%), and Giardia (27.8%). Fasciola was associated with the number of siblings in the household, drinking untreated water, and giardiasis. Eosinophilia was encountered in 21% of children and more common in those drinking untreated water at home and those infected with a parasite, but the differences were not significant. Eating water plants was not associated with Fasciola or eosinophilia. Fascioliasis and eosinophilia were common in the highlands of Cuzco. Fascioliasis was associated with socioeconomic factors and drinking water.

CD4+ T cell subsets and Tax expression in HTLV-1 associated diseases.

Human T lymphotropic virus type 1 (HTLV-1) infection displays variable clinical manifestations. These include inflammatory diseases such as HTLV-1 associated myelopathy (HAM) or immunosuppressive conditions such as Strongyloides stercoralis hyperinfection. The viral protein, Tax causes activation and proliferation of T cells. We hypothesize that the expression of Tax in T cell subsets characterizes the clinical manifestations of HTLV-1. To test this hypothesis, we measured T helper 1 effector cells and regulatory T cells (Tregs) among Tax expressing lymphocytes from peripheral blood mononuclear cells (PBMCs) of 32 HTLV-1 infected patients with HAM, with S. stercoralis co-infection or with asymptomatic infection. We observed increased ratios of Th1/Treg among Tax expressing lymphocytes in HAM patients. These data suggest that the expression of Tax among the different target cells may explain the variable presentation of HTLV-1.

Acute mountain sickness impact among travelers to Cusco, Peru.

BACKGROUND: Increasing numbers of travelers are visiting high altitude locations in the Andes. The epidemiology of acute mountain sickness (AMS) among tourists to high altitude in South America is not well understood. METHODS: A cross-sectional study to evaluate the epidemiology, pre-travel preparation, and impact of AMS among travelers to Cusco, Peru (3,400 m) was performed at Cusco's International Airport during June 2010. Foreign travelers, 18 years or older, staying 15 days or less, departing Cusco were invited to participate. Demographic, itinerary, and behavioral data were collected. The Lake Louise Clinical score (LLCS) was used to assess AMS symptoms. RESULTS: In total, 991 travelers participated, median age 32 years (interquartile range 25-49), 55.5% female, 86.7% tourists, mostly from the United States (48.2%) and England (8.1%). Most (76.7%) flew from sea level to Cusco and 30.5% visited high altitude in the previous 2 months. Only 29.1% received AMS advice from a physician, 19% recalled advice on acetazolamide. Coca leaf products (62.8%) were used more often than acetazolamide (16.6%) for prevention. AMS was reported by 48.5% and 17.1% had severe AMS. One in five travelers with AMS altered their travel plans. Travelers older than 60 years, with recent high altitude exposure, who visited lower cities in their itinerary, or used acetazolamide were less likely to have AMS. Using coca leaf products was associated with increased AMS frequency. CONCLUSIONS: AMS was common and adversely impacted plans of one in five travelers. Acetazolamide was associated with decreased AMS but was prescribed infrequently. Other preventive measures were not associated with a decrease in AMS in this population. Pre-travel preparation was suboptimal.