Recent diarrhea is associated with elevated salivary IgG responses to Cryptosporidium in residents of an eastern Massachusetts community.

BACKGROUND: Serological data suggest that Cryptosporidium infections are common but underreported. The invasiveness of blood sampling limits the application of serology in epidemiological surveillance. We pilot-tested a non-invasive salivary anti-Cryptosporidium antibody assay in a community survey involving children and adults. MATERIALS AND METHODS: Families with children were recruited in a Massachusetts community in July; symptoms data were collected at 3 monthly follow-up mail surveys. One saliva sample per person (n = 349) was collected via mail, with the last survey in October. Samples were analyzed for IgG and IgA responses to a recombinant C. hominis gp15 sporozoite protein using a time-resolved fluorometric immunoassay. Log-transformed assay results were regressed on age using penalized B-splines to account for the strong age-dependence of antibody reactions. Positive responses were defined as fluorescence values above the upper 99% prediction limit. RESULTS: Forty-seven (13.5%) individuals had diarrhea without concurrent respiratory symptoms during the 3-month-long follow-up; eight of them had these symptoms during the month prior to saliva sampling. Two individuals had positive IgG responses: an adult who had diarrhea during the prior month and a child who had episodes of diarrhea during each survey month (Fisher's exact test for an association between diarrhea and IgG response: p = 0.0005 for symptoms during the prior month and p = 0.02 for symptoms during the entire follow-up period). The child also had a positive IgA response, along with two asymptomatic individuals (an association between diarrhea and IgA was not significant). CONCLUSION: These results suggest that the salivary IgG specific to Cryptosporidium antigens warrants further evaluation as a potential indicator of recent infections.

Seasonality in six enterically transmitted diseases and ambient temperature.

We propose an analytical and conceptual framework for a systematic and comprehensive assessment of disease seasonality to detect changes and to quantify and compare temporal patterns. To demonstrate the proposed technique, we examined seasonal patterns of six enterically transmitted reportable diseases (EDs) in Massachusetts collected over a 10-year period (1992-2001). We quantified the timing and intensity of seasonal peaks of ED incidence and examined the synchronization in timing of these peaks with respect to ambient temperature. All EDs, except hepatitis A, exhibited well-defined seasonal patterns which clustered into two groups. The peak in daily incidence of Campylobacter and Salmonella closely followed the peak in ambient temperature with the lag of 2-14 days. Cryptosporidium, Shigella, and Giardia exhibited significant delays relative to the peak in temperature (approximately 40 days, P<0.02). The proposed approach provides a detailed quantification of seasonality that enabled us to detect significant differences in the seasonal peaks of enteric infections which would have been lost in an analysis using monthly or weekly cumulative information. This highly relevant to disease surveillance approach can be used to generate and test hypotheses related to disease seasonality and potential routes of transmission with respect to environmental factors.

The seasonality of human campylobacter infection and Campylobacter isolates from fresh, retail chicken in Wales.

Seasonal peaks in both human campylobacter infections and poultry isolates have been observed in several European countries but remain unexplained. We compared weekly data on human campylobacter infections with thermophilic Campylobacter isolation rates from fresh, retail chicken samples (n = 514) purchased weekly in Wales between January and December 2002. Human isolates (n = 2631) peaked between weeks 22 and 25 (early June) and chicken isolates (n = 364) between weeks 24 and 26 (late June). In the absence of a temporal association, we postulate that the seasonal rise in humans is not caused by a rise in isolation rates in poultry but that both are more likely to be associated with a common, but as yet unidentified, environmental source.

Host cell apoptosis impairs Cryptosporidium parvum development in vitro.

The absence of a self-sustaining in vitro propagation method for Cryptosporidium parvum is a major obstacle for research on this parasite. Conventional cell monolayers are unsuitable for long-term parasite propagation because the level of infection decreases over time and few oocysts, if any, are produced. The interaction between parasite and host cell was studied to identify factors limiting parasite development in vitro. Loss of substrate adherence and death of parasitized host cells was observed in 2 epithelial cell lines. Nuclear morphology, DNA laddering, annexin V binding, and terminal deoxytransferase-mediated dUTP nick end labeling indicated that host cell death occurred by apoptosis. At 6 hr postinfection, only a minority of infected cells remained in the monolayer, and few survived the initial phase of parasite development without losing adherence. Treatment of infected monolayers with caspase inhibitors drastically reduced cell detachment but failed to increase the number of parasites in monolayers. In contrast, cell cultures grown on laminin-coated plates showed a higher proportion of infected cells. These observations indicate that cell detachment and apoptosis in C. parvum-infected cell culture negatively affect parasite survival in vitro.

Presence of double-stranded RNAs in human and calf isolates of Cryptosporidium parvum.

We examined the occurrence of 2 virus-like double-stranded (ds)RNAs in human and calf isolates of Cryptosporidium parvum senso latu and other microorganisms, including 7 other members of the genus. A total of 32 isolates of C. parium, 16 from humans (5 from acquired immune deficiency syndrome patients) and 16 from calves, were analyzed. Ethidium bromide staining, or Northern blot analysis, or reverse transcription/polymerase chain reaction, or all 3 methods, revealed that both genotype 1 and genotype 2 isolates of C. parvum possessed these dsRNAs. No other Cryptosporidium spp. or other organisms examined possessed these dsRNAs. Comparison analysis of partial cDNA sequences of dsRNAs from human and calf isolates revealed a high degree of similarity (>92% and >93% identical nucleotides for large and small dsRNAs, respectively). Slight, consistent differences in nucleotide sequences could be seen at select sites and were associated with an isolate being either genotype 1 or 2. Because of the widespread distribution of the dsRNAs, the similarity of these molecules between isolates, and high host specificity, these nucleic acids may prove to represent species-specific molecular markers for C. parvum. Evidence also suggests that the dsRNA can be utilized for molecular genotyping of C. parvum.

Rifabutin but not clarithromycin prevents cryptosporidiosis in persons with advanced HIV infection.

BACKGROUND: Macrolides have been reported to be effective for the prevention of cryptosporidiosis in persons with HIV infection. OBJECTIVE: To evaluate the efficacy of clarithromycin and rifabutin for the prevention of cryptosporidiosis in persons with advanced HIV infection. DESIGN: Cross-protocol analysis involving 2288 individuals with a history of a CD4 cell counts of < or = 100 x 10(6) cell/l who were enrolled in two prospective clinical trials to prevent Mycobacterium avium complex (MAC) infection and cytomegalovirus (CMV) end-organ disease. INTERVENTIONS: Clarithromycin 500 mg twice daily, rifabutin 300-450 mg daily, the combination of the two or no MAC prophylaxis. MAIN OUTCOME MEASUREMENT: Laboratory-confirmed cryptosporidiosis. Subjects were analyzed in an intent-to-treat and as-treated manner using time-to-event analyses (Cox proportional hazards models). RESULTS: The median length of follow up was 463 days. The median CD4 count at entry was 29 x 10(6) cell/l (range 0-182). There were 60 cases of cryptosporidiosis during the prospective observational period, with an event rate of 2.2 per 100 person-years. In the intent-to-treat [relative risk (RR) 0.50; 95% confidence interval (CI) 0.26-0.96; P = 0.041 and as-treated (RR 0.42; 95% CI 0.20-0.91; P = 0.03) analyses, rifabutin alone was significantly associated with a lower rate of cryptosporidiosis. Clarithromycin alone was not protective in similar analyses (P = 0.98 and 0.90, respectively). CONCLUSIONS: In doses used to prevent MAC infection, rifabutin but not clarithromycin decreases the risk of developing cryptosporidiosis in persons with advanced HIV infection who are not receiving potent combination antiretroviral therapy.

Use of passive surveillance data to study temporal and spatial variation in the incidence of giardiasis and cryptosporidiosis.

OBJECTIVE: The goal of this study was to evaluate temporal and spatial variations in the reporting of cases of giardiasis and cryptosporidiosis to a passive surveillance system, and to assess the relationship of those variations to source of drinking water, adjusting for socioeconomic variables. METHODS: The authors analyzed temporal and spatial patterns for 4,058 cases of giardiasis and 230 cases of cryptosporidiosis reported to the Massachusetts Department of Public Health for 1993-1996. They linked each reported case to a database containing information on source of residential water supply and socioeconomic characteristics and evaluated the association between these factors and reporting rates using regression techniques. RESULTS: Reports of giardiasis and cryptosporidiosis were highest for the mixed unfiltered drinking water supply category. Reports of giardiasis were associated with income levels. Increases in reporting for both giardiasis and cryptosporidiosis were seen in summer to early fall. During a suspected outbreak of cryptosporidiosis n the city of Worcester in 1995, a significant increase in reported cases was also observed in the Boston metropolitan area. Following the suspected outbreak, weekly giardiasis rates increased slightly in Worcester and the Boston metropolitan area, while reporting of cryptosporidiosis increased dramatically. CONCLUSIONS: Consistently collected passive surveillance data have the potential to provide valuable information on the temporal variation of disease incidence as well as geographic factors. However, passive surveillance data, particularly in the initial period of surveillance, may be highly sensitive to patterns of diagnosis and reporting and should be interpreted with caution.

The beneficial effects of weekly low-dose vitamin A supplementation on acute lower respiratory infections and diarrhea in Ecuadorian children.

BACKGROUND: Previous studies of large-dose vitamin A supplementation on respiratory morbidity have produced conflicting results in a variety of populations. The influence of malnutrition has not been examined in the majority of these trials. We hypothesized that weekly low-dose vitamin A supplementation would prevent respiratory and diarrheal disease morbidity and that malnutrition might influence the efficacy of vitamin A supplementation. METHODS: In a randomized, double-blind, placebo-controlled field trial of 400 children, 6 to 36 months of age in a high Andean urban slum, half of the children received 10 000 IU of vitamin A weekly and half received placebo for 40 weeks. Children were visited weekly at home by physicians and assessed for acute diarrheal disease and acute respiratory infections. RESULTS: Acute diarrheal disease and acute respiratory infection did not differ globally or by severity between supplement-treated and placebo groups. However, the incidence of acute lower respiratory infection (ALRI) was significantly lower in underweight (weight-for-age z score [WAZ] <-2 SD) supplement-treated children than in underweight children on placebo (8.5 vs 22.3 per 10(3) child-weeks; rate ratio: 0.38 [95% CI: 0.17-0.85]). ALRI incidence was significantly higher in normal-weight (WAZ >-2 SD) supplement-treated children than in normal-weight children on placebo (9.8 vs 4.4 per 10(3) child-weeks; rate ratio: 2.21 [95% CI: 1.24-3.93]). By logistic regression analysis the risk of ALRI was lower in underweight supplement-treated children than in underweight children on placebo (point estimate 0.148 [95% CI: 0.034-0.634]). In contrast, risk of ALRI was higher in normal-weight supplement-treated children (WAZ >-1 SD to mean) than in normal-weight children on placebo in the same WAZ stratum (point estimate: 2.51 [95% CI: 1.24-5.05]). The risk of severe diarrhea was lower in supplement-treated children 18 to 23 months of age than in children on placebo in this age group (point estimate: 0.26 [95% CI: 0.06-1.00]). CONCLUSIONS: Weekly low-dose (10 000 IU) vitamin A supplementation in a region of subclinical deficiency protected underweight children from ALRI and paradoxically increased ALRI in normal children with body weight over -1 SD. Protection from severe diarrhea was consistent with previous trials. Additional research is warranted to delineate potential beneficial and detrimental interactions between nutritional status and vitamin A supplementation regarding ALRI.

Central nervous system manifestations of childhood shigellosis: prevalence, risk factors, and outcome.

BACKGROUND AND OBJECTIVE: Alterations in consciousness, including seizures, delirium, and coma, are known to occur during Shigella infection. Previous reports have suggested that febrile convulsions and altered consciousness are more common during shigellosis than with other childhood infections. Those reports, however, have been from locations where S dysenteriae type 1 was not common, thus making it difficult to assess the specific contribution that S dysenteriae type 1 infection, and Shiga toxin, might make to the pathogenesis of altered consciousness in children with shigellosis. In this study we seek to determine the prevalence, risk factors, and outcome of altered consciousness in children with shigellosis in Bangladesh, a country where infection with all four species of Shigella is common. We particularly focus on the importance of metabolic abnormalities, which we have previously shown to be a common feature of shigellosis in this population. METHODS: This study was conducted at the Diarrhea Treatment Centre of the International Centre for Diarrhoeal Disease Research, Bangladesh in Dhaka, Bangladesh, which provides care free of charge to persons with diarrhea. During 1 year, a study physician identified all inpatients infected with Shigella by checking the logs of the Clinical Microbiology Laboratory daily. Study physicians obtained demographic and historical information by reviewing the patient charts and by interviewing patients, or their parents or guardians, to confirm or complete the history of illness obtained on admission. Patients were categorized as being conscious or unconscious based on a clinical scale; having a seizure documented in the hospital; or having a seizure by history during the current illness that was not witnessed by medical personnel. Patient outcome was classified as discharged improved, discharged against medical advice, transferred to another health facility, or died in the Treatment Centre. Laboratory examinations were ordered at the discretion of the attending physician; all such information was recorded on the study form. Clinical management was by the attending physician. Factors independently predictive of a documented seizure, or of unconsciousness, were determined using a multiple logistic regression analysis. For this analysis variables associated with unconsciousness or a documented seizure in the analysis of variance or chi2 analyses were entered into the regression equation and eliminated in a backward stepwise fashion if the probability associated with the likelihood ratio statistic exceeded .10. RESULTS: During this 1-year study, 83 402 persons with diarrhea came to the Treatment Centre for care, and 6290 patients were admitted to the inpatient unit. Shigella was isolated from a stool or rectal swab sample of 863 (13.7%) of the inpatients. Seventy-one (8%) of the inpatients with shigellosis were >/=15 years old; 61 (86%) were conscious; 10 (14%) were unconscious; none had either a documented seizure or a seizure by history during this illness. Seven hundred ninety-two patients were <15 years old (92%); 654 (83%) were conscious; 73 (9%) were unconscious; 41 (5%) had a documented seizure (compared with >/=15-year age group); 24 (3%) had a seizure by history during this illness. Of the 41 patients with documented seizures, 19 (46.3%) had a seizure at the time of admission, and 22 (53.7%) had a seizure after admission. Twenty-five (61.0%) of the 41 patients with documented seizures were reported to have a seizure during this illness before coming to the Treatment Centre. Clinical features that are known to cause altered consciousness-fever, severe dehydration, hypoglycemia, hyponatremia, or meningitis-were present in 38 (92.7%) of the 41 patients in whom a seizure was witnessed and in 67 (91.8%) of the 73 patients who were unconscious. Nineteen (46. 3%) of the patients who had a seizure documented had two of these five features, 4 (9.8%) had three, and 1 (2. (ABSTRACT TRUNCATED)

Genotypic and phenotypic characterization of Cryptosporidium parvum isolates from people with AIDS.

Genotypic analysis of Cryptosporidium parvum has demonstrated the presence of two subgroups within the species, whereas biochemical and antigenic characterization have shown more heterogeneity. The clinical relevance of these observations is unknown. C. parvum isolates from people with AIDS were studied with respect to parasite genotypes and virulence in cell monolayers and laboratory animals. Ten of 13 oocyst samples had a characteristic human-associated (H) genotype; 3 had a genotype typical of calf-excreted oocysts (C). Virulence in cell culture was mildly or markedly lower in the 5 isolates tested (4 H and 1 C) compared with the GCH1 reference isolate. H isolates did not infect newborn ICR mice, whereas 1 of the 2 C isolates tested did. These findings reinforce the concept of C. parvum genetic subgroupings that correlate to some extent with infectivity and suggest that additional heterogeneity is present within the subgroups.

The gamma interferon gene knockout mouse: a highly sensitive model for evaluation of therapeutic agents against Cryptosporidium parvum.

Cryptosporidiosis is a serious disease in malnourished children and in people with malignancies or AIDS. Current rodent models for evaluating drug therapy against cryptosporidiosis have many limitations, including the need for a high inoculum, the absence of symptoms resembling those seen in humans, and the need to maintain exogenous immunosuppression. We have developed a gamma interferon knockout (GKO) mouse model with which to evaluate therapies against C. parvum and have used paromomycin for evaluation of this model. The GKO model offers considerable improvements over other systems, since it requires no additional immunosuppression and adult mice can be infected with as few as 10 oocysts (compared with 10(7) for SCID mice). Infected mice develop profound gastrointestinal dysfunction due to extensive infection and severe mucosal damage involving the entire small intestine. Clinical symptoms, which include depression, anorexia, weight loss, and wasting, result in death within 2 to 4 weeks. The time of death depends on the oocyst challenge dose. Paromomycin modulated parasitological and clinical parameters in highly predictable and significant ways, including prevention of death. In addition, examination of the extensively infected gut provided an important insight into the dynamics between a specific drug treatment, its impact on the extent and the site of parasite distribution, and clinical outcome. These uniform symptoms of weight loss, wasting, and death are powerful new parameters which bring this model closer to the actual disease seen in humans and other susceptible mammalian species.

Efficacy of nitazoxanide against Cryptosporidium parvum in cell culture and in animal models.

Nitazoxanide (NTZ), a drug currently being tested in human clinical trials for efficacy against chronic cryptosporidiosis, was assessed in cell culture and in two animal models. The inhibitory activity of NTZ was compared with that of paromomycin (PRM), a drug that is partially effective against Cryptosporidium parvum. A concentration of 10 microg of NTZ/ml (32 microM) consistently reduced parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity, in contrast to an 80% reduction produced by PRM at 2,000 microg/ml (3.2 mM). In contrast to its efficacy in vitro, NTZ at either 100 or 200 mg/kg of body weight/day for 10 days was ineffective at reducing the parasite burden in C. parvum-infected, anti-gamma-interferon-conditioned SCID mice. Combined treatment with NTZ and PRM was no more effective than treatment with PRM alone. Finally, NTZ was partially effective at reducing the parasite burden in a gnotobiotic piglet diarrhea model when given orally for 11 days at 250 mg/kg/day but not at 125 mg/kg/day. However, the higher dose of NTZ induced a drug-related diarrhea in piglets that might have influenced its therapeutic efficacy. As we have previously reported, PRM was effective at markedly reducing the parasite burden in piglets at a dosage of 500 mg/kg/day. Our results indicate that of all of the models tested, the piglet diarrhea model most closely mimics the partial response to NTZ treatment reported to occur in patients with chronic cryptosporidiosis.

Paromomycin and geneticin inhibit intracellular Cryptosporidium parvum without trafficking through the host cell cytoplasm: implications for drug delivery.

Cryptosporidium parvum, which causes intractable diarrhea and lethal wasting in people with AIDS, occupies an unusual intracellular but extracytoplasmic niche. No reliable therapy for cryptosporidiosis exists, though the aminoglycoside paromomycin is somewhat effective. We report that paromomycin and the related compound geneticin manifest their major in vitro anti-C. parvum activity against intracellular parasites via a mechanism that does not require drug trafficking through the host cell cytoplasm. We used both normal and transformed aminoglycoside-resistant Caco-2 or MDBK cells in these studies. Timed-exposure experiments demonstrated that these drugs inhibit intracellular but not extracellular parasites. Apical but not basolateral exposure of infected cells to these drugs led to very significant parasite inhibition, indicating an apical topological restriction of action. We estimated intracytoplasmic concentrations of paromomycin, using an intracellular bacterial killing assay, and found that C. parvum infection did not lead to increased paromomycin concentrations compared to those in uninfected cells. Global [3H]paromomycin uptake by Caco-2 cells was approximately 200-fold higher than the estimated intracytoplasmic paromomycin concentration, suggestive of host cell vesicular uptake and concentration (as has been reported with other cell lines). However, preinfection exposure of Caco-2 cells to paromomycin did not result in subsequent inhibition of parasite development, indicating that if exogenous paromomycin enters the infected host cell vesicular compartment, it does not effectively communicate with the parasite. Thus, the apical membranes overlying the parasite and parasitophorous vacuole may be the unsuspected major route of entry for paromomycin and may be of importance in the design and discovery of novel drug therapies for the otherwise untreatable C. parvum.

Cryptosporidium parvum infection of human intestinal xenografts in SCID mice induces production of human tumor necrosis factor alpha and interleukin-8.

The protozoan parasite Cryptosporidium parvum invades intestinal epithelial cells and can cause life-threatening diarrhea in immunocompromised individuals. Despite the clinical importance of this organism, much remains to be learned about the pathogenesis of C. parvum-induced diarrhea. To explore the role of the intestinal inflammatory response in C. parvum disease, using C. parvum oocysts we infected human intestinal xenografts in severe combined immunodeficient (SCID) mice. Seven days after infection, we found levels of human tumor necrosis factor alpha and interleukin-8 in C. parvum-infected human intestinal xenografts that were significantly higher than those seen in uninfected control xenografts. These results demonstrate that human intestinal cells produce proinflammatory cytokines in response to C. parvum infection and establish SCID-HU-INT mice as a model system to study the interactions of C. parvum with the human intestine.

Did Milwaukee experience waterborne cryptosporidiosis before the large documented outbreak in 1993?

The patterns of incidence and pathways of spread for cryptosporidiosis are poorly understood. In this study, we explored the possibility that drinking water caused significant waterborne cryptosporidiosis in Milwaukee well before the massive documented outbreak in April 1993. We generated time series of daily counts of emergency room visits and hospital admissions for gastroenteritis in Milwaukee using the billing records of the Medical College of Wisconsin for January 1, 1992, through May 3, 1993. The Milwaukee Water Works provided us with data on drinking water turbidity for the same period. The service area of the South Plant experienced a sharp rise in turbidity just before the outbreak. During the outbreak period, gastroenteritis events were most strongly associated with turbidity at a lag of 7 days in children and 8 days in adults. It is reasonable to conclude that these lag times reflect the incubation period of Cryptosporidium. During the 434 days before the outbreak, gastroenteritis events were most strongly associated with turbidity at a lag of 8 days among children and 9 days among adults in the service area of the North Plant, the plant that experienced the highest effluent turbidity during this period. These findings are consistent with the conclusion that waterborne cryptosporidiosis was occurring in Milwaukee for more than a year before the documented outbreak.

Natural history and biology of Cryptosporidium parvum.

The taxonomy of the genus Cryptosporidium remains ambiguous, because the current criteria for speciation are insufficient to validate the 6-8 named species. Cross-transmission experiments have shown varying and conflicting results, and the limited genetic data available do not necessarily support currently proposed species designations. The reasons for this ambiguity lie with the ubiquitous nature of Cryptosporidium, probably infecting all vertebrates and variety of tissues therein, and the absence of reference strains with defined virulence attributes that can be linked to genetic markers for comparative analysis. The inability to classify oocysts or confidently to identify their origin, implicate oocysts from all sources as hazardous to humans. Another major issue is the unusual degree of resistance that Cryptosporidium has shown to antiprotozoan and antimicrobial agents. The intracellular but extracytoplasmic domain the parasite occupies is in itself a significant barrier to drug entry. In support of this we outline how the intracellular niche of this parasite differs from the related Apicomplexans, Plasmodium and Toxoplasma, and delineate why the feeder organelle membrane, rather than, or in addition to, the parasitophorous membrane, is the major portal of nutrient entry for Cryptosporidium. The broad conclusion is that anticryptosporidial agents will have to enter the parasite via the multiple apical membranes that camouflage the parasite, or via the host cell, possibly transported by vesicles to the feeder organelle membrane. This may have major implications for rational drug discovery and design.

Human cryptosporidiosis: epidemiology, transmission, clinical disease, treatment, and diagnosis.

Cryptosporidiosis is now recognized as one of the most common human enteric infections. In this critical review, relatively unexplored details of transmission, the interaction with malnutrition and the development of chronic diarrhea, and the need for effective treatment are highlighted. Our inability to detect small numbers of foodborne oocysts limits our understanding of this transmission route, and the possibility of respiratory transmission is yet to be rigorously studied. The toll this disease imposes on children, especially the malnourished, has not been fully appreciated. Indeed, the dynamics of the progression from acute cryptosporidiosis to chronic diarrhea and death of malnourished children is still enigmatic. Our knowledge of the intestinal pathophysiology, while limited, is increasing. The lack of effective drug therapy is both remarkable and sobering. Overall, these unknown areas demonstrate how little we truly know about this parasite.