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Neurocysticercosis (NCC) is a helminth infection affecting the central nervous system caused by the larval stage (cysticercus) of Taenia solium. Since vascular alteration and blood–brain barrier (BBB) disruption contribute to NCC pathology, it is postulated that angiogenesis could contribute to the pathology of this disease. This study used a rat model for NCC and evaluated the expression of two angiogenic factors called vascular endothelial growth factor (VEGF-A) and fibroblast growth factor (FGF2). Also, two markers for BBB disruption, the endothelial barrier antigen and immunoglobulin G, were evaluated using immunohistochemical and immunofluorescence techniques. Brain vasculature changes, BBB disruption, and overexpression of angiogenesis markers surrounding viable cysts were observed. Both VEGF-A and FGF2 were overexpressed in the tissue surrounding the cysticerci, and VEGF-A was overexpressed in astrocytes. Vessels showed decreased immunoreactivity to endothelial barrier antigen marker and an extensive staining for IgG was found in the tissues surrounding the cysts. Additionally, an endothelial cell tube formation assay using human umbilical vein endothelial cells showed that excretory and secretory antigens of T. solium cysticerci induce the formation of these tubes. This in vitro model supports the hypothesis that angiogenesis in NCC might be caused by the parasite itself, as opposed to the host inflammatory responses alone. In conclusion, brain vasculature changes, BBB disruption, and overexpression of angiogenesis markers surrounding viable cysts were observed. This study also demonstrates that cysticerci excretory-secretory processes alone can stimulate angiogenesis. © 2018 Wiley Periodicals, Inc.

Neurocysticercosis is a parasitic brain disease caused by the larval form (Cysticercus cellulosae) of the Taenia solium and is the leading cause of preventable epilepsy world-wide. However, the pathophysiology and relation to the wide range of clinical features remains poorly understood. Axonal swelling is emerging as an important early pathological finding in multiple neurodegenerative diseases and causes of brain injury, but has not been well-described in neurocysticercosis. Histological analysis was performed on human, rat and porcine NCC brain specimens to identify axonal pathology. Rat infection was successfully carried out via two routes of inoculation: direct intracranial injection and oral feeding. Extensive axonal swellings, in the form of spheroids, were observed in both humans and rats and to a lesser extent in pigs with NCC. Spheroids demonstrated increased immunoreactivity to amyloid precursor protein and neurofilament indicating probable impairment of axonal transport. These novel findings demonstrate that spheroids are present in NCC which is conserved across species. Not only is this an important contribution towards understanding that pathogenesis of NCC, but it also provides a model to analyze the association of spheroids with specific clinical features and to investigate the reversibility of spheroid formation with antihelmintic treatment.

Although the lifecycle of Schistosoma spp. and pathophysiology of schistosomiasis have been established, the mechanism by which cercariae find their host is not well understood. Speculatively, host infection by random and accidental host contact is not as biologically plausible as a biochemical mechanism of mammalian attraction. A few studies have indicated that biochemical cues and temperature gradients may play a role in host identification, attraction and attachment triggers. This study aimed to elucidate these mechanisms more specifically through evaluation of biochemical, age and temperature influences leading to Schistosoma mansoni cercariae attraction and attachment behaviors. Oleic acid, a common unsaturated free fatty acid in the outer layer of human skin, was tested for cercariae attraction across biologically relevant concentrations. Influence of media type (beeswax, nail varnish and agar), age-dependent behavior variability and environmentally appropriate temperatures (22 and 30 °C) were also evaluated. Results indicated that oleic acid at concentrations of 0.3, 0.9 and 1.8 g/mL in beeswax significantly increased median attachment to media (median attachment of 7.50%, 4.20% and 3.71%, respectively, P0.001), compared with plain beeswax, with maximal attachment of 30.30% at 0.3g/mL of oleic acid. In media containing 0.3 g/mL of oleic acid, cercarial attachment was highest for freshly emerged cercariae to 5h post-emergence, with a significant decrease in attachment behavior at 10h post-emergence (P0.01). Aquatic temperature at which cercariae were exposed to media did not yield significant results (P value0.05). Biochemical, age and environmental factors influencing cercarial host attraction and attachment behavior have been elucidated by this study. This information will inform further development of devices for environmental surveillance and potentially improve cercarial exposure prevention strategies.

The guinea pig (Cavia porcellus) is a natural reservoir for Trypanosoma cruzi but has seldom been used as an experimental infection model. We developed a guinea pig infection model for acute and chronic Chagas disease. Seventy-two guinea pigs were inoculated intradermally with 10(4) trypomastigotes of T. cruzi strain Y (experimental group); 18 guinea pigs were used as control group. Eight animals from the experimental group and two from the control group were sacrificed 5, 15, 20, 25, 40, 55, 115, 165, and 365 days after inoculation. During the acute phase (15 to 55 days), we observed parasitemia (with a peak on day 20) and positive IgM and IgG Western blots with anti-shed acute-phase antigen bands. The cardiac tissue showed vasculitis, necrosis (on days 40 to 55), moderate to severe inflammation, and abundant amastigote nests. Smaller numbers of amastigote nests were also present in kidney, brain, and other organs. In the early chronic phase (115 to 165 days), parasitemia disappeared and anti-T. cruzi IgG antibodies were still detectable. In cardiac tissue, the number of amastigote nests and the grade of inflammation decreased. In the chronic phase (365 days), the cardiac tissue showed vasculitis and fibrosis; detectable parasite DNA was associated with higher grades of inflammation. The experimental T. cruzi infection model in guinea pigs shows kinetics and pathologic changes similar to those of the human disease.

Giardia intestinalis is a common gastrointestinal protozoan worldwide, but its effects on childhood growth in developing countries are not clearly understood. The authors aimed to describe its effects on child growth. They followed 220 Peruvian children daily for diarrhea, weekly for stool samples, and monthly for anthropometry. The authors modeled the effect of nutritional status on the risk of Giardia infection and the risk of diarrhea attributable to Giardia using negative binomial regression. They modeled the effects of Giardia infection on growth using linear regression, with 85% of children becoming infected with Giardia and 87% of these becoming reinfected. In multivariable analysis, the risk of Giardia infection did not vary with weight for age (relative risk = 1.00, 95% confidence interval: 0.89, 1.12) or height for age (relative risk = 0.92, 95% confidence interval: 0.82, 1.04). Giardiasis did not affect growth at 1 or 2 months following the first infection at any age interval. The longitudinal prevalence of Giardia between 6 and 24 months of age was not associated with height gain in that interval (p = 0.981). Giardia was not associated with an increased risk of diarrhea at any age interval. Study results question the importance of Giardia as a childhood pathogen in developing countries where giardiasis is hyperendemic.

The biological basis for the specificity of host infectivity patterns of Cryptosporidium spp., in particular C. hominis and C. parvum, has yet to be fully elucidated. Comparison of the C. parvum and C. hominis P23 and GP900 predicted amino acid sequences revealed 3 differences in P23 and 4 and 17 differences in GP900 domains 1 and 5, respectively. Using monoclonal antibodies developed against the surface (glyco)proteins P23 and GP900 of the C. parvum Iowa isolate, solubilized glycoprotein from three C. hominis isolates was screened for reactivity using Western immunoblots. One of ten P23 MAbs and three of 21 GP900 MAbs were not reactive with any of the three C. hominis isolates. The non-reactive P23 MAb binds to a peptide epitope, while the non-reactive GP900 MAbs bind to either carbohydrate/carbohydrate-dependent or peptide epitopes of C. parvum. These results demonstrate phenotypic differences between C. hominis and C. parvum within two (glyco)proteins that are involved in parasite gliding motility and attachment/invasion.

Taeniasis due to Taenia solium is a disease with important public health consequences, since the larval stage is not exclusive to the animal intermediate, the pig, but also infects humans, causing neurocysticercosis. Early diagnosis and treatment of T. solium tapeworm carriers is important to prevent human cysticercosis. Current diagnosis based on microscopic observation of eggs lacks both sensitivity and specificity. In the present study, a nested-PCR assay targeting the Tso31 gene was developed for the specific diagnosis of taeniasis due to T. solium . Initial specificity and sensitivity testing was performed using stored known T. solium -positive and -negative samples. The assay was further analyzed under field conditions by conducting a case-control study of pretreatment stool samples collected from a population in an area of endemicity. Using the archived samples, the assay showed 97% (31/32) sensitivity and 100% (123/123) specificity. Under field conditions, the assay had 100% sensitivity and specificity using microscopy/enzyme-linked immunosorbent assay coproantigen testing as the gold standards. The Tso31 nested PCR described here might be a useful tool for the early diagnosis and prevention of taeniasis/cysticercosis.

Objective. Chagas disease, a disease caused by Trypanosoma cruzi, disproportionately affects poor people throughout Latin America. In Mexico, assessments of officially reported burden have not been previously reported. To evaluate discontinuity between surveillance data and data from other sources, we used data from the Mexican Ministry of Health to describe the distribution of reported Chagas disease over time in Mexico and compare it with estimates from the literature. Methods. We summarized age and sex differences for Chagas cases and mortality for 1995–2013 and 1982–2010, respectively. We examined the spatial distribution of Chagas disease over time with respect to disease burden. We further compared officially reported figures with estimates from the literature. Results. Among 6,494 officially reported cases, rates of Chagas disease were highest in adults aged 25–44 years (47.3%). Mortality was highest in adults aged ≥45 years (423/495, 85.5%). The data indicated increasing temporal trends for incidence and mortality. The greatest burden occurred in southern states, with increasing spatial distribution over time. Fewer than 900 cases and 40 deaths were officially reported annually, in contrast to estimates from the literature of approximately 69,000 new cases and 25,000 deaths annually. Conclusion. While increasing trends in officially reported data have been observed, large discrepancies in case estimates compromise our understanding of Chagas disease epidemiology. Reported cases based on current practices are not enough to correctly assess the Chagas disease burden and spatial distribution in Mexico. Understanding the true epidemiology of this disease will lead to more focused and successful control and prevention strategies to decrease disease burden.

Cryptosporidium species are ubiquitous in the environment and are frequently detected in the stools of children who live where sanitation conditions are poor. To better characterize the immune response to these parasites, we monitored immunoglobulin G (IgG) antibody levels in a cohort of children from Lima, Peru. Two new enzyme-linked immunosorbent assays based on the C. parvum (bovine, subtype IIa) Iowa strain 17-kDa and 27-kDa antigens were used to measure IgG antibody levels in longitudinal serum samples. Antibody responses were detected during infections with C. parvum , C. felis , and C. meleagridis and with four different subtypes of C. hominis . We also noted that the magnitude of the antibody response was related to the number of previous infections and that older children generally had higher levels of antibodies to the two C. parvum antigens. Antibody responses were not associated with infections with either Cyclospora sp. or Giardia sp. We believe the antibody assays will be important tools for monitoring the success of future public health interventions.

Summary Background Inadequate water and sanitation adversely affect the health of children in developing countries. We aimed to assess the effects of water and sanitation on childhood health in a birth cohort of Peruvian children. Methods We followed up children once a day for diarrhoea and once a month for anthropometry, and obtained data for household water and sanitation at baseline. Findings At 24 months of age, children with the worst conditions for water source, water storage, and sanitation were 1·0 cm (95% CI 0·1–0·8) shorter and had 54% (−1 to 240) more diarrhoeal episodes than did those with the best conditions. Children from households with small storage containers had 28% (1–63) more diarrhoeal episodes than did children from households with large containers. Lack of adequate sewage disposal explained a height deficit of 0·9 cm (0·2–1·7) at 24 months of age. Better water source alone did not accomplish full health benefits. In 24-month-old children from households with a water connection, those in households without adequate sewage disposal and with small storage containers were 1·8 cm (0·1–3·6) shorter than children in households with sewage and with large storage containers. Interpretation Our findings show that nutritional status is a useful endpoint for water and sanitation interventions and underscores the need to improve sanitation in developing countries. Improved and more reliable water sources should discourage water storage at risk of becoming contaminated, decrease diarrhoeal incidence, and improve linear growth in children.

Detection and enumeration of Cryptosporidium parvum in both treated and untreated waters are important to facilitate prevention of future cryptosporidiosis incidents. Immunomagnetic separation (IMS)-fluorescent antibody (FA) detection and IMS-PCR detection efficiencies were evaluated in two natural waters seeded with nominal seed doses of 5, 10, and 15 oocysts. IMS-FA detected oocysts at concentrations at or below the three nominal oocyst seed doses, illustrating that IMS-FA is sensitive enough to detect low oocyst numbers. However, the species of the oocysts could not be determined with this technique. IMS-PCR, targeting the 18S rRNA gene in this study, yielded positive amplification for 17 of the 18 seeded water samples, and the amplicons were subjected to restriction fragment length polymorphism digestion and DNA sequencing for species identification. Interestingly, the two unseeded, natural water samples were also PCR positive; one amplicon was the same base pair size as the C. parvum amplicon, and the other amplicon was larger. These two amplified products were determined to be derived from DNA of Cryptosporidium muris and a dinoflagellate. These IMS-PCR results illustrate that (i) IMS-PCR is able to detect low oocyst numbers in natural waters, (ii) PCR amplification alone is not confirmatory for detection of target DNA when environmental samples are used, (ii) PCR primers, especially those designed against the rRNA gene region, need to be evaluated for specificity with organisms closely related to the target organism, and (iv) environmental amplicons should be subjected to appropriate species-specific confirmatory techniques.

We compared the epidemiologic characteristics of cyclosporiasis and cryptosporidiosis in data from a cohort study of diarrhea in a periurban community near Lima, Peru. Children had an average of 0.20 episodes of cyclosporiasis/year and 0.22 episodes of cryptosporidiosis/year of follow-up. The incidence of cryptosporidiosis peaked at 0.42 for 1-year-old children and declined to 0.06 episodes/child-year for 5- to 9-year-old children. In contrast, the incidence of cyclosporiasis was fairly constant among 1- to 9-year-old children (0.21 to 0.28 episodes/child-year). Likelihood of diarrhea decreased significantly with each episode of cyclosporiasis; for cryptosporidiosis, this trend was not statistically significant. Both infections were more frequent during the warm season (December to May) than the cooler season (June to November). Cryptosporidiosis was more frequent in children from houses without a latrine or toilet. Cyclosporiasis was associated with ownership of domestic animals, especially birds, guinea pigs, and rabbits.

Background. Longitudinal investigations into the origins of adult essential hypertension have found elevated blood pressure in children to accurately track into adulthood, however the direct causes of essential hypertension in adolescence and adulthood remains unclear. Methods. We revisited 152 Peruvian adolescents from a birth cohort tracked from 0 to 30 months of age, and evaluated growth via monthly anthropometric measurements between 1995 and 1998, and obtained anthropometric and blood pressure measurements 11–14 years later. We used multivariable regression models to study the effects of infantile and childhood growth trends on blood pressure and central obesity in early adolescence. Results. In regression models adjusted for interim changes in weight and height, each 0.1 SD increase in weight for length from 0 to 5 months of age, and 1 SD increase from 6 to 30 months of age, was associated with decreased adolescent systolic blood pressure by 1.3 mm Hg (95% CI −2.4 to −0.1) and 2.5 mm Hg (95% CI −4.9 to 0.0), and decreased waist circumference by 0.6 (95% CI −1.1 to 0.0) and 1.2 cm (95% CI −2.3 to −0.1), respectively. Growth in infancy and early childhood was not significantly associated with adolescent waist-to-hip ratio. Conclusions. Rapid compensatory growth in early life has been posited to increase the risk of long-term cardiovascular morbidities such that nutritional interventions may do more harm than good. However, we found increased weight growth during infancy and early childhood to be associated with decreased systolic blood pressure and central adiposity in adolescence.

Cyclospora cayetanensis is an apicomplexan protozoan parasite which has emerged as an important cause of epidemic and endemic diarrhea. Water-borne as well as food-borne outbreaks have occurred, including a large number of U.S. cases associated with raspberries imported from Guatemala. Molecular markers exist for tracing the epidemiology of many of the bacterial pathogens associated with water-borne or food-borne diarrhea, such as serotyping and pulsed-field electrophoresis. However, there are currently no molecular markers available for C. cayetanensis . The intervening transcribed spacer (ITS) regions between the small- and large-subunit rRNA genes demonstrate much greater sequence variability than the small-subunit rRNA sequence itself and have been useful for the molecular typing of other organisms. Thus, ITS1 variability might allow the identification of different genotypes of C. cayetanensis . In order to determine the degree of ITS1 variability among C. cayetanensis isolates, the ITS1 sequences of C. cayetanensis isolates from a variety of sources, including raspberry-associated cases, cases from Guatemala, and pooled and individual isolates from Peru, were obtained. The ITS1 sequences of all five raspberry-associated isolates were identical, consistent with their origin from a single source. In contrast, one of the two Guatemala isolates and two Peruvian isolates contained multiple ITS1 sequences. These multiple sequences could represent multiple clones from a single clinical source or, more likely, variability of the ITS1 region within the genome of a single clone.

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis , a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID 50 ) and a minimal infective dose (MID) for E. intestinalis . The TCID 50 is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID 50 have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25°C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log 10 reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.

The infectivity of three Cryptosporidium parvum isolates (Iowa [calf], UCP [calf], and TAMU [horse]) of the C genotype was investigated in healthy adults. After exposure, volunteers recorded the number and form of stools passed and symptoms experienced. Oocyst excretion was assessed by immunofluorescence. The ID50 differed among isolates: Iowa, 87 (SE, 19; 95% confidence interval [CI], 48.67-126); UCP, 1042 (SE, 1000; 95% CI, 0-3004); and TAMU, 9 oocysts (SE, 2.34; 95% CI, 4.46-13.65); TAMU versus Iowa, P=.002 or UCP, P=.019. Isolates also differed significantly (P=.045) in attack rate between TAMU (86%) and Iowa (52%) or UCP (59%). A trend toward a longer duration of diarrhea was seen for the TAMU (94.5 h) versus UCP (81.6 h) and Iowa (64.2 h) isolates. C. parvum isolates of the C genotype differ in their infectivity for humans.

A 50% infectious dose (ID50) of 132 Cryptosporidium parvum oocysts was previously determined in serologically negative individuals (ELISA). In this study, 17 healthy adults with pre-existing anti-C. parvum serum IgG were challenged with 500-50,000 oocysts. Infection and diarrhea were associated with the higher challenge doses. The ID50 was 1,880 oocysts, > 20-fold higher than in seronegative volunteers. Fecal oocysts were detected in only seven (53.8%) of 13 individuals with clinical cryptosporidiosis, indicating that the host response may effectively decrease the number of oocysts produced. Subjects with the highest absorbances prior to challenge had little to no increase in IgG following challenge, whereas volunteers with lower reactivities showed significant postchallenge increases. This suggests that an upper limit of serum IgG was present in some subjects, while others were further stimulated by an additional exposure. These data indicate that prior exposure to C. parvum provides protection from infection and illness at low oocyst doses.

Background: Longitudinal investigations into the origins of adult essential hypertension have found elevated blood pressure in children to accurately track into adulthood, however the direct causes of essential hypertension in adolescence and adulthood remains unclear. Methods: Cohort study, we revisited 152 Peruvian adolescents from a birth cohort evaluated with monthly anthropometric measurements between 1995 and 1998, and obtained anthropometric and blood pressure measurements 11 to 14 years later. We used multivariable regression models to study the effects of infantile and childhood growth trends on blood pressure and central obesity in early adolescence. Results: In regression models adjusted for childhood growth, each 0.1 SD increase in weight for length from 0 to 5 months of age, and 1 SD increase from 6 to 30 months of age, predicted decreased adolescent systolic blood pressure by 1.3 mm Hg (95% CI -2.4 to -0.1) and 2.5 mm Hg (95% CI -4.9 to 0.0), and decreased waist circumference by 0.6 (95% CI -1.1 to 0.0) and 1.2 cm (95% CI -2.3 to -0.1) respectively. Growth in infancy and early childhood was not a significant predictor of adolescent waist-to-hip ratio. Conclusions: Rapid compensatory growth in early life has been posited to increase the risk of long-term cardiovascular morbidities such that nutritional interventions may do more harm than good. However, we found increased weight growth during infancy and early childhood to predict decreases in adolescent systolic blood pressure and central adiposity.

Using an indirect immunofluorescence assay on stool samples, we found a 6.4% prevalence of cryptosporidiosis among 403 children less than five years of age with acute diarrhea in Mexico City over a one-year period. The prevalence was highest (11.4%) during the rainy summer months. Most Cryptosporidium parvum cases occurred in infants less than one year of age. Cryptosporidium parvum was more common in malnourished children (P < 0.05) and in nonbreast-fed infants less than six months of age (P < 0.01). Neither dwelling characteristics nor the presence of domestic animals at home were associated with C. parvum infection. Enteropathogenic bacteria were found in 26.8% of the children; Escherichia coli, Salmonella, and Shigella being the most frequently identified. None of 100 serum samples tested showed antibodies against human immunodeficiency virus. When compared with immunofluorescence, the acid-fast technique showed a sensitivity of 76.9% and a specificity of 98.9%, with a predictive positive value of 83.3%. It was concluded that 1) monoclonal antibody-based immunofluorescence is a better diagnostic tool than the acid-fast technique, 2) the prevalence of cryptosporidiosis in this population was similar to that of other developing countries, 3) clinical manifestations were nonspecific, and 4) C. parvum was more common in malnourished children and in nonbreast-fed infants.

This study investigated whether a child's first infection with Cryptosporidium parvum had an acute effect on weight gain. Specifically, the authors compared monthly rates of weight gain between C. parvum-infected and noninfected children. Over a 2-year period (1989-1991), a cohort of Peruvian children aged 0-3 months at recruitment were followed twice weekly for assessment of daily diarrheal status, weekly for C. parvum stool examinations, and monthly for anthropometric measurements. Data on 207 children permitted the authors to examine the effect of C. parvum infection on weight gain. During the 2-year study period, 45% (94/207) of the children became infected with C. parvum for the first time. Weight gain intervals in 57 of the 94 infected children met criteria for analysis. Of these, 63 percent (36/57) were asymptomatic (i.e., had no diarrhea). On average, children with symptomatic cryptosporidiosis gained (i.e., grew) 342 g less (95% confidence interval 167-517) during the first month of infection than did children without diarrhea who were not yet infected. The effect of asymptomatic cryptosporidiosis was less severe: On average, children with asymptomatic infection gained 162 g less (95% confidence interval 27-297) during the first month of infection than did children without diarrhea who were not yet infected. Symptomatic cryptosporidiosis retarded weight gain more than did asymptomatic cryptosporidiosis, but the latter was twice as common. Since asymptomatic cryptosporidiosis is more prevalent, it may have more of an overall adverse effect on child growth in the community than symptomatic cryptosporidiosis.During September 1989-November 1991 in Peru, 207 infants, 0-3 months old at enrollment and living in a periurban shanty town in Lima, were followed to determine whether a child's first infection with Cryptosporidium parvum had an adverse effect on weight gain during the first month of infection and whether poor nutritional status was a risk factor for C. parvum. Infection with C. parvum occurred at least once in 45% (97) of the infants. Median age at onset of a first C. parvum infection was 16 months. The growth interval of only 57 (61%) of C. parvum infected children was evaluated. 36 (63%) children were asymptomatic. The remaining 21 infected children had diarrhea. Children with cryptosporidiosis, whether symptomatic or asymptomatic, gained less weight during the first month of infection than children without cryptosporidiosis. Children with symptomatic cryptosporidiosis gained 184 g less weight during the first month of infection than did children with asymptomatic cryptosporidiosis. Children with symptomatic cryptosporidiosis gained 342 g less during the first month of infection than did children who were not infected with C. parvum and did not have diarrhea. Children with asymptomatic cryptosporidiosis gained 162 g less during the first month of infection than did children who were not infected with C. parvum and did not have diarrhea. The adverse effect of C. parvum on weight gain was strongest in children younger than 12 months. Neither wasting nor underweight was a significant risk factor for C. parvum infection. Stunting tended to increase the risk of C. parvum infection but the effect was not statistically significant. These findings show that C. parvum infection has an adverse effect on growth. Asymptomatic cryptosporidiosis is of special concern, since it was more prevalent and thus probably would have more of an overall adverse effect on child growth in the community.

Background Interruption of vector-borne transmission of Trypanosoma cruzi remains an unrealized objective in many Latin American countries. The task of vector control is complicated by the emergence of vector insects in urban areas. Methods Utilizing data from a large-scale vector control program in Arequipa, Peru, we explored the spatial patterns of infestation by Triatoma infestans in an urban and peri-urban landscape. Multilevel logistic regression was utilized to assess the associations between household infestation and household- and locality-level socio-environmental measures. Results Of 37,229 households inspected for infestation, 6,982 (18.8%; 95% CI: 18.4 – 19.2%) were infested by T. infestans. Eighty clusters of infestation were identified, ranging in area from 0.1 to 68.7 hectares and containing as few as one and as many as 1,139 infested households. Spatial dependence between infested households was significant at distances up to 2,000 meters. Household T. infestans infestation was associated with household- and locality-level factors, including housing density, elevation, land surface temperature, and locality type. Conclusions High levels of T. infestans infestation, characterized by spatial heterogeneity, were found across extensive urban and peri-urban areas prior to vector control. Several environmental and social factors, which may directly or indirectly influence the biology and behavior of T. infestans, were associated with infestation. Spatial clustering of infestation in the urban context may both challenge and inform surveillance and control of vector reemergence after insecticide intervention.

The objective of this study was to determine if human genotypes of Giardia lamblia could be found in canine companion animals from urban and peri-urban environments in Tucson, Arizona. Canine fecal samples collected from the Humane Society of Southern Arizona between July 2006 and April 2009 were screened for G. lamblia infection using immunofluorescent microscopy and confirmed by polymerase chain reaction (PCR). Of the 672 samples screened, 196 were found positive by IFA and 185 of those positive were successfully amplified through PCR. Sequencing analysis showed samples were primarily of the C or D genotypes (n = 154), or showing a mix of the C and D genotypes (n = 10). One sample showed a mixed infection of a human genotype (A) and a dog-specific genotype (C). These data are consistent with previous studies showing dog specific genotypes to be dominant in environments where dog-to-dog transmission is likely to occur, and provides further evidence that multiple genes should be targeted for more accurate genotype characterization.

Cryptosporidium parvum arginine aminopeptidase (RAP) was studied during in vitro excystation. Specific RAP inhibitors were identified by using C. parvum extracts. Amastatin, a series of alpha-aminoboronic acids, and the chelating agents EDTA and 1,10-phenanthrolene, but not endoproteinase inhibitors, blocked enzymatic activity. RAP inhibitors found to be effective in soluble enzymatic assays were then studied for their effect on in vitro excystation. 1,10-Phenanthrolene, amastatin, and H-boronorleucine (pinacol) inhibited excystation by 84, 57, and 61%, respectively, compared with solvent-treated control oocysts. Sporozoites remained viable within the oocyst as determined by propidium iodide and fluorescein diacetate dye uptake, suggesting that alpha-aminoboronic acids were not directly lethal to the parasite.

The molecular-based magnet test for malaria is shown to be more sensitive than the thin blood film test. The globally used thin blood film test is less sensitive because it uses preparation steps that result in the reduction of the absolute number of diagnostically pertinent erythrocytes. Several reports of diagnostic error with the thin film test and the thick film test have appeared in the literature. In marked contrast to the commonly accepted tests, the magnet test concurrently partitions and concentrates the infected erythrocytes present in the initial sample. The magnetic test permits a brief and sensitive microscopic-based enumeration of the malaria-infected erythrocytes in the enriched sample. Diagnostically pertinent hemozoin is simply identified through two of its specific molecular properties: paramagnetism and birefringence. The former property mediates the capture and enrichment of malaria-infected erythrocytes within the magnetic flux and the latter property manifests the characteristic birefringence demonstrated by polarized light.

When exposed to 254-nm UV, spores of Encephalitozoon intestinalis , Encephalitozoon cuniculi , and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m 2 , respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.

While childhood malnutrition is associated with increased morbidity and mortality, less well understood is how early childhood growth influences height and body composition later in life. We revisited 152 Peruvian children who participated in a birth cohort study between 1995 and 1998, and obtained anthropometric and bioimpedance measurements 11 to 14 years later. We used multivariable regression models to study the effects of childhood anthropometric indices on height and body composition in early adolescence. Each standard deviation decrease in length-for-age at birth was associated with a decrease in adolescent height-for-age of 0.7 SD in both boys and girls (all p

Objectives. To improve our understanding of climate variability and diarrheal disease at the community level and inform predictions for future climate change scenarios, we examined whether the El Niño climate pattern is associated with increased rates of diarrhea among Peruvian children. Methods. We analyzed daily surveillance data for 367 children aged 0 to 12 years from 2 cohorts in a peri-urban shantytown in Lima, Peru, 1995 through 1998. We stratified diarrheal incidence by 6-month age categories, season, and El Niño, and modeled between-subject heterogeneity with random effects Poisson models. Results. Spring diarrheal incidence increased by 55% during El Niño compared with before El Niño. This increase was most acute among children older than 60 months, for whom the risk of a diarrheal episode during the El Niño spring was nearly 100% greater (relative risk = 1.96; 95% confidence interval = 1.24, 3.09). Conclusions. El Niño–associated climate variability affects community rates of diarrhea, particularly during the cooler seasons and among older children. Public health officials should develop preventive strategies for future El Niño episodes to mitigate the increased risk of diarrheal disease in vulnerable communities.

Control of cryptosporidiosis is currently hampered by the absence of drugs or vaccines proven consistently effective against Cryptosporidium parvum. On the basis of observations that anti-C. parvum antibody has therapeutic effect against cryptosporidiosis, cows were immunized with C. parvum to produce hyperimmune colostral antibody. An antibody-rich fraction was prepared and differentiated from control (nonhyperimmune) antibody by enzyme-linked immunosorbent assay, immunofluorescence assay, immunoelectron microscopy, and in vitro neutralizing titer against DEAE-cellulose-isolated C. parvum sporozoites. Oocyst, purified sporozoite, and merozoite antigens recognized by hyperimmune antibody were defined by Western blot (immunoblot). Hyperimmune antibody recognized antigens common to oocysts, sporozoites, and merozoites, as well as stage-specific antigens. Upon incubation with hyperimmune antibody, sporozoites underwent distinct morphologic changes characterized by progressive formation and eventual release of membranous sporozoite surface antigen-antibody complexes, similar to the malaria circumsporozoite precipitate reaction. The infectivity of sporozoites having undergone this reaction was neutralized. The reaction was minimal or absent on sporozoites incubated with control antibody. To determine therapeutic effect in vivo, persistent C. parvum infection was established in adult severe combined immune-deficient (SCID) mice by oral inoculation with 10(7) oocysts. At 5 weeks postinfection, infected mice were treated for 10 days with hyperimmune or control antibody by inclusion in drinking water and daily gavage. Fecal oocyst shedding and infection scores in the gastrointestinal tract and gall bladder/common bile duct in hyperimmune antibody-treated mice were significantly lower than those in the control antibody-treated mice. Hyperimmune bovine antibody prepared against C. parvum may provide a first-generation therapy for control of cryptosporidiosis. Additionally, the defined antigens can be evaluated as subunit immunogens to produce better-characterized polyclonal antibody for control of cryptosporidiosis or as targets for monoclonal antibody-based immunotherapy.

Combinations of 10 Cryptosporidium parvum oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.

To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment.

Background The history of Chagas disease control in Peru and many other nations is marked by scattered and poorly documented vector control campaigns. The complexities of human migration and sporadic control campaigns complicate evaluation of the burden of Chagas disease and dynamics of Trypanosoma cruzi transmission. Methodology/Principal Findings We conducted a cross-sectional serological and entomological study to evaluate temporal and spatial patterns of T. cruzi transmission in a peri-rural region of La Joya, Peru. We use a multivariate catalytic model and Bayesian methods to estimate incidence of infection over time and thereby elucidate the complex history of transmission in the area. Of 1,333 study participants, 101 (7.6%; 95% CI: 6.2–9.0%) were confirmed T. cruzi seropositive. Spatial clustering of parasitic infection was found in vector insects, but not in human cases. Expanded catalytic models suggest that transmission was interrupted in the study area in 1996 (95% credible interval: 1991–2000), with a resultant decline in the average annual incidence of infection from 0.9% (95% credible interval: 0.6–1.3%) to 0.1% (95% credible interval: 0.005–0.3%). Through a search of archival newspaper reports, we uncovered documentation of a 1995 vector control campaign, and thereby independently validated the model estimates. Conclusions/Significance High levels of T. cruzi transmission had been ongoing in peri-rural La Joya prior to interruption of parasite transmission through a little-documented vector control campaign in 1995. Despite the efficacy of the 1995 control campaign, T. cruzi was rapidly reemerging in vector populations in La Joya, emphasizing the need for continuing surveillance and control at the rural-urban interface., Author Summary The historically rural problem of Chagas disease is increasing in urban areas in Latin America. Peri-rural development may play a critical role in the urbanization of Chagas disease and other parasitic infections. We conducted a cross-sectional study in an urbanizing rural area in southern Peru, and we encountered a complex history of Chagas disease in this peri-rural environment. Specifically, we discovered: (1) long-standing parasite transmission leading to substantial burden of infection; (2) interruption in parasite transmission resulting from an undocumented insecticide application campaign; (3) relatively rapid re-emergence of parasite-infected vector insects resulting from an unsustained control campaign; (4) extensive migration among peri-rural inhabitants. Long-standing parasite infection in peri-rural areas with highly mobile populations provides a plausible mechanism for the expansion of parasite transmission to nearby urban centers. Lack of commitment to control campaigns in peri-rural areas may have unforeseen and undesired consequences for nearby urban centers. Novel methods and perspectives are needed to address the complexities of human migration and erratic interventions.

Background. Giardia lamblia is ubiquitous in multiple communities of nonindustrialized nations. Genotypes A1, A2, and B (Nash groups 1, 2, and 3, respectively) are found in humans, whereas genotypes C and D are typically found in dogs. However, genotypes A and B have occasionally been identified in dogs. Methods. Fecal Giardia isolates from 22 families and their dogs, living in Pampas de San Juan, were collected over 7 weeks in 2002 and 6 weeks in 2003. Samples were genotyped, followed by sequencing and haplotyping of many of these isolates by using loci on chromosomes 3 and 5. Results. Human infections were all caused by isolates of genotypes A2 and B. Human coinfections with genotypes A2 and B were common, and the reassortment pattern of different subtypes of A2 isolates supports prior observations that suggested recombination among genotype A2 isolates. All dogs had genotypes C and/or D, with one exception of a dog with a mixed B/D genotype infection. Conclusions. In a region of high endemicity where infected dogs and humans constantly commingle, different genotypes of Giardia are almost always found in dogs and humans, suggesting that zoonotic transmission is very uncommon.

Oocysts of a Cryptosporidium isolate from guinea pigs were not infectious for adult mice, but were infectious for two of three newborn calves and for suckling mice. However, oocysts isolated from calves or mice infected with guinea pig Cryptosporidium were not infectious for guinea pigs. Four isolates of C. parvum from calves were incapable of infecting weanling guinea pigs. Microscopic examination of tissue from the colon and cecum of suckling guinea pigs inoculated with C. parvum revealed sparse infection of some pups. These host range studies and previously described differences in 125I-labeled oocyst surface protein profiles between Cryptosporidium sp. from guinea pigs and C. parvum suggest they are distinct species. We propose the name Cryptosporidium wrairi be retained. Studies with monoclonal antibodies indicate that C. wrairi and C. parvum are antigenically related.

Background. We conducted a study of congenital Trypanosoma cruzi infection in Santa Cruz, Bolivia. Our objective was to apply new tools to identify weak points in current screening algorithms, and find ways to improve them. Methods. Women presenting for delivery were screened by rapid and conventional serological tests. For infants of infected mothers, blood specimens obtained on days 0, 7, 21, 30, 90, 180, and 270 were concentrated and examined microscopically; serological tests were performed for the day 90, 180, and 270 specimens. Maternal and infant specimens, including umbilical tissue, were tested by polymerase chain reaction (PCR) targeting the kinetoplast minicircle and by quantitative PCR. Results. Of 530 women, 154 (29%) were seropositive. Ten infants had congenital T. cruzi infection. Only 4 infants had positive results of microscopy evaluation in the first month, and none had positive cord blood microscopy results. PCR results were positive for 6 (67%) of 9 cord blood and 7 (87.5%) of 8 umbilical tissue specimens. PCR-positive women were more likely to transmit T. cruzi than were seropositive women with negative PCR results ( ). Parasite loads determined by quantitative PCR were higher for mothers of infected infants P ! .05 than for seropositive mothers of uninfected infants ( ). Despite intensive efforts, only 58% of at-risk infants P ! .01 had a month 9 specimen collected. Conclusions. On the basis of the low sensitivity of microscopy in cord blood and high rate of loss to followup, we estimate that current screening programs miss one-half of all infected infants. Molecular techniques may improve early detection.

LP-BM5 murine leukemia virus infection caused alterations in splenic T cell subsets in adult C57BL/6 female mice. Prolonged infection resulted in increased immunosuppression and a concomitant decreased resistance to Cryptosporidium parvum infection. Significant Cryptosporidium colonization of the intestinal villi was seen 10 days after oral challenge in mice infected with murine retrovirus for 3 months but not in non-virally infected controls. Parasite numbers per villus of retrovirally infected mice were 20-fold higher than in controls, which showed only occasional parasites. Feces from most virally infected mice but none from controls contained oocysts. Cryptosporidium infection in mice after 4 and 5 months of retroviral infection was accompanied by severe immunosuppression and parasite levels 50-5000 times higher than in controls. A high level of infection persisted 21 days after Cryptosporidium challenge in virally infected mice, while controls cleared their transient and marginal infection. These results further characterize LP-BM5 infection as a murine model of retrovirally induced acquired immune deficiency.

Nested PCR was performed on individually isolated Giardia lamblia cysts in replicates of 50 for sets of 1, 2, 3, 4, 5, 7, and 10 cysts. Amplification ranged from 80% for 1 cyst to 100% for 10 cysts. The results suggest that nested PCR is well adapted for G. lamblia single-cyst detection.

SummaryGiardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is an enteric protozoan parasite with two nuclei, and it might be one of the earliest branching eukaryotes [1]. However, the discovery of at least rudimentary forms of certain features, such as Golgi and mitochondria, has refuted the proposal that its emergence from the eukaryotic lineage predated the development of certain eukaryotic features. The recent recognition of many of the genes known to be required for meiosis in the genome has also cast doubt on the idea that Giardia is primitively asexual, but so far there has been no direct evidence of sexual reproduction in Giardia, and population data have suggested clonal reproduction. We did a multilocus sequence evaluation of the genotype A2 reference strain, JH, and five genotype A2 isolates from a highly endemic area in Peru. Loci from different chromosomes yielded significantly different phylogenetic trees, indicating that they do not share the same evolutionary history; within individual loci, tests for recombination yielded significant statistical support for meiotic recombination. These observations provide genetic data supportive of sexual reproduction in Giardia.

Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.

For most of human evolution, man lived in widely dispersed, small nomadic groups that subsisted on hunting and gathering. While this lifestyle undoubtedly exposed individuals to the risk of occasional zoonotic disease transmission, population size likely minimized the effect of exposure to such diseases. The Neolithic period, which saw the advent of the agricultural revolution and the eventual domestication of certain animal species, not only brought about a rapid increase and concentration in human population but also fostered an environment in which substantial increases in infectious and nutritional diseases could rapidly spread (Armelagos, 1991). In addition, man’s descent from the trees, subsequent eccrine evolution, and the development of agriculture forced him to become “water-bound,” thereby putting him in contact with a group of infectious agents that can be described as “water associated” (Desowitz, 1981). It is within this context that many zoonotic diseases, including those of nematode origin that are discussed in this chapter, may have gained a foothold in human populations. When one thinks about food-borne nematode infections, the first parasites that usually come to mind are Trichinella and Anisakis. While these two are certainly important in the overall context of food-borne infections caused by nematodes, since they are directly transmitted via infected food to susceptible individuals, one also has to consider other less frequently encountered nematode infections and infections caused by rather common nematodes, but which are transferred to humans via indirect food-borne routes. The overall goal of this chapter is to provide a rather broad, general perspective of the important nematode parasites that may find their way into our food chain, either directly or indirectly, and thus may serve as a potential source of infection and disease.

Cyclospora infecting humans emerged as a pathogen in the late 1970s and has since been largely associated with disease of children in the tropics, travelers and expatriates to developing countries, and the immunocompromised. It has gained recent notoriety because of food-borne disease outbreaks that have sparked much interest in trying to better define the epidemiology of this most intriguing parasite. It is clear from studies performed on Cyclospora that much further research is required to better understand certain facets of this parsite’s lifecycle, interactions of the parasite with its host and interactions of the parasite with the environment.

List of contributors Preface 1: Epidemiology 1. Epidemiology and zoonotic potential of Giardia infections R.C. Andrew Thompson 2. Entamoeba histolytica and Entamoeba dispar, the non-identical twins C. Graham Clark 3. Epidemiology and strain variation of Cryptosporidium Rachel M. Chalmers and David P. Casemore 4. Cyclospora cayetanensis: An emergent and still perplexing coccidian parasite Charles R. Sterling and Ynes R. Ortega 2: Host parasite interactions 5. Antigenic variation of the VSP genes of Giardia lamblia Rodney D. Adam and Theodore E. Nash 6. Pathogenesis and immunity to Entamoeba histolytica Jessica L. Tarleton and William A. Petri Jr. 7. Innate and T cell-mediated immune responses in cryptosporidiosis Carol R. Wyatt and Vincent McDonald 3: Treatment and Control 8. Rationale approaches to treating Cryptosporidium, Cyclospora, Giardia and Entamoeba Jan R. Mead and Pablo Okhuysen 9. Inactivation and removal of enteric protozoa in water Frank W. Schaefer, III, Marilyn M. Marshall and Jennifer L. Clancy 10. Monitoring of Giardia and Cryptosporidium in water in the UK and US Jennifer L. Clancy and Paul R. Hunter 4: Genomics 11.Entanioeba histolytica genome James J. McCoy and Barbara J. Mann 12. Ciyptosporidium parvum genomics: Impact on research and control Guan Zhu and Mitchell S. Abrahamsen Index

Publisher Summary This chapter reviews a Cryptosporidium parvum volunteer study in the context of infectivity and immunity. Cryptosporidium infections are widespread in most societies worldwide. Transmission is principally through drinking and recreational water supplies; however, it can also be contracted through person-to-person and animal-to-person contact. Recent advances in molecular techniques have identified two distinct Cryptosporidium genotypes associated with different transmission cycles. Genotype 1 is associated with human infections, whereas genotype 2 is zoonotic in nature and passed readily between humans as well as a variety of animal species. Because of the constraints in animal models, human subjects have been studied to understand many basic aspects of the infection and the disease. In 1993, Cryptosporidium volunteer study was carried out to better recognize the infectivity of the parasite, the illness attack rate, the natural history of the disease, and the host immune response. Healthy volunteers between the ages of 18 and 50 years were challenged with a single oocyst dose of known concentration, and they were monitored for six weeks. Four geographically-diverse, genotype 2 isolates-Iowa, TAMU, UCP, and Moredun-have been studied in volunteers who were seronegative by ELISA prior to challenge. The results showed that several parameters, including illness attack rate, and the severity of symptoms also vary among isolates, but do not directly associate with infectivity (ID50). Increase in the Reactivity Index(RI) can be associated with oocyst shedding.