Charles R. Sterling

The Infectivity of Cryptosporidium parvum in Healthy Volunteers

BACKGROUND Small numbers of Cryptosporidium parvum oocysts can contaminate even treated drinking water, and ingestion of oocysts can cause diarrheal disease in normal as well as immunocompromised hosts. Since the number of organisms necessary to cause infection in humans is unknown, we performed a study to determine the infective dose of the parasite in healthy adults. METHODS After providing informed consent, 29 healthy volunteers without evidence of previous C. parvum infection, as determined by the absence of anti-cryptosporidium-specific antibodies, were given a single dose of 30 to 1 million C. parvum oocysts obtained from a calf. They were then monitored for oocyst excretion and clinical illness for eight weeks. Household contacts were monitored for secondary spread. RESULTS Of the 16 subjects who received an intended dose of 300 or more oocysts, 14 (88 percent) became infected. After a dose of 30 oocysts, one of five subjects (20 percent) became infected, whereas at a dose of 1000 or more oocysts, seven of seven became infected. The median infective dose, calculated by linear regression, was 132 oocysts. Of the 18 subjects who excreted oocysts after the challenge dose, 11 had enteric symptoms and 7 (39 percent) had clinical cryptosporidiosis, consisting of diarrhea plus at least one other enteric symptom. All recovered, and there were no secondary cases of diarrhea among household contacts. CONCLUSIONS In healthy adults with no serologic evidence of past infection with C. parvum, a low dose of C. parvum oocysts is sufficient to cause infection.

Virulence of Three Distinct Cryptosporidium parvum Isolates for Healthy Adults

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.

Isolation of Cryptosporidium oocysts and sporozoites using discontinuous sucrose and isopycnic Percoll gradients.

Techniques for the large-scale isolation of Cryptosporidium oocysts and sporozoites, obtained from the feces of experimentally infected Holstein calves, were developed employing discontinuous sucrose gradients and isopycnic Percoll gradients. The oocyst recovery method utilized 2 sequential discontinuous sucrose gradients followed by 1 Percoll gradient. Recovered oocysts were essentially free of debris and bacteria and represented 34% of the original oocyst suspension. Sporozoites were recovered from excystation mixtures on a single Percoll gradient. Sixty-three percent of the original sporozoites were recovered with 2.2% contamination by intact oocysts and virtually no oocyst walls.

Effect of water and sanitation on childhood health in a poor Peruvian peri-urban community

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.

Cryptosporidium Species and Subtypes and Clinical Manifestations in Children, Peru

One-sentence summary for table of contents: Different genotypes and subtypes are linked to different clinical manifestations.

Population Genetics Provides Evidence for Recombination in Giardia

Giardia lamblia (syn. Giardia intestinalis, Giardia duodenalis) is an enteric protozoan parasite with two nuclei, and it might be one of the earliest branching eukaryotes. 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.

OCCURRENCE OF CRYPTOSPORIDIUM OOCYSTS IN SEWAGE EFFLUENTS AND SELECTED SURFACE WATERS

An existing method for the detection of Cryptosporidium oocysts in water was modified to investigate oocyst prevalence in large volumes of water. Surface waters and sewage effluents were filtered, eluted from the filter, and concentrated using centrifugation. The resultant pellet was then homogenized, sonicated, and placed on a sucrose gradient to separate oocysts from the sediment. The uppermost gradient layer was then examined by immunofluorescence using a labeled monoclonal antibody. Using this technique, average numbers of oocysts detected in raw and treated sewage were 5.18 X 10(3) and 1.30 X 10(3)/L, respectively. Filtered sewage effluents had significantly lower numbers of oocysts (10.0/L). These data show that sand filtration may reduce the concentrations of this parasite in waste waters. Highly variable oocyst numbers were encountered in surface waters. Since Cryptosporidium oocysts are frequently present in environmental waters, they could be responsible for waterborne outbreaks of disease.

Epidemiologic differences between cyclosporiasis and cryptosporidiosis in Peruvian children.

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.

ANTIGENS OF CRYPTOSPORIDIUM SPOROZOITES RECOGNIZED BY IMMUNE SERA OF INFECTED ANIMALS AND HUMANS

The humoral response of humans, calves, and horses to Cryptosporidium sporozoite antigens was evaluated using a western blot technique. Sera from calves, humans, and horses were obtained at various times following the detection of infection. Sera were reacted with detergent-solubilized, sodium dodecyl sulfate polyacrylamide gel-electrophoresed (SDS-PAGE) sporozoite antigens. The number of antigens recognized by immune sera from humans and animals increased with time postinfection. A 20-kDa antigen appears to be a major sporozoite surface determinant labeled via membrane protein biotinylation and recognized by mouse monoclonal antibodies using indirect immunofluorescence and western blotting. Detectable recognition of the 20-kDa band occurred in 3-wk postinfection (PI) sera from all species tested. Reactivity to the 20-kDa band diminished significantly in sera 5 mo PI or longer from infected humans with no known recurrence of cryptosporidial diarrhea. In contrast, 12-mo PI sera from an individual constantly exposed to oocysts under working conditions was as strongly reactive as the 3-wk convalescent sera. Serum reactivity to the 20-kDa antigen appears to be a good indicator of exposure to Cryptosporidium.

Congenital Trypanosoma cruzi Transmission in Santa Cruz, Bolivia

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 (P < .05). Parasite loads determined by quantitative PCR were higher for mothers of infected infants than for seropositive mothers of uninfected infants P < .01). Despite intensive efforts, only 58% of at-risk infants 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 follow-up, we estimate that current screening programs miss one-half of all infected infants. Molecular techniques may improve early detection.