Andrey I. Egorov

Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts.

ABSTRACT Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

The Elderly and Waterborne Cryptosporidium Infection: Gastroenteritis Hospitalizations before and during the 1993 Milwaukee Outbreak

We used the Temporal Exposure Response Surfaces modeling technique to examine the association between gastroenteritis-related emergency room visits and hospitalizations in the elderly and drinking water turbidity before and during the 1993 Milwaukee waterborne Cryptosporidium outbreak. Before the outbreak, the rate of such events increased with age in the elderly (p<0.002), suggesting that the elderly are at an increased risk. During the outbreak, strong associations between turbidity and gastroenteritis-related emergency room visits and hospitalizations occurred at temporal lags of 5–6 days (consistent with the Cryptosporidium incubation period). A pronounced second wave of these illnesses in the elderly peaked at 13 days. This wave represented approximately 40% of all excess cases in the elderly. Our findings suggest that the elderly had an increased risk of severe disease due to Cryptosporidium infection, with a shorter incubation period than has been previously reported in all adults and with a high risk for secondary person-to-person transmission.

Daily variations in effluent water turbidity and diarrhoeal illness in a Russian city

To assess an association between temporal variations in drinking water quality and gastrointestinal (GI) illness, a cohort study involving 100 randomly selected families (367 individuals) was conducted in the city of Cherepovets, Russia from June through November 1999. Participants maintained daily diaries of gastrointestinal symptoms, water consumption and other behavioural exposure variables, while daily effluent water quality data were provided by the water utility. The cumulative incidence rate of self-reported gastrointestinal diseases, 1.7 cases per person-year, was almost two orders of magnitude higher than that of officially reported GI infections in the city. An interquartile range increase in effluent water turbidity of 0.8 Nephelometric Turbidity Units was associated with a relative risk of self-reported GI illness of 1.47 (95% Confidence Interval 1.16, 1.86) at a lag of 2 days after control for daily rate of consumption of non-boiled tap water, behavioural covariates, day of the week and a seasonally-related linear trend. In the analysis by subsets of study participants stratified by non-boiled tap water consumption, no statistically significant associations between turbidity and GI illness were found for the study participants who always boiled their drinking water. For individuals who drank non-boiled tap water, statistically significant associations between turbidity and GI illness were detected at lags 1, 2 and 7 days.

Serological evidence of Cryptosporidium infections in a Russian city and evaluation of risk factors for infections

PURPOSE Assess the relative frequency of Cryptosporidium infections and risk factors for infection in Cherepovets, Russia. METHODS In June 1999, data on demographic, socioeconomic, hygienic characteristics, and recent gastrointestinal illness were collected on 50 community-recruited adults and 50 blood donors. The community group then maintained daily diaries of exposures and gastrointestinal symptoms for 5 months. Sera samples at the inception and conclusion of the study were tested for antibody responses to the 15/17 kDa and 27 kDa Cryptosporidium antigens using mini-immunoblots. RESULTS At the inception, 68% and 88% of study participants had detectable serological responses to 15/17 kDa and 27 kDa antigens. Older age was associated with stronger antibody responses to both antigens. Attendance at swimming pools was associated with stronger responses to both antigens in predominantly male blood donors. Over the follow-up period, drinking non-boiled water from shallow draw-wells was associated with an increase in serological response to the 27 kDa antigen. Self-reported gastrointestinal illness was not associated with an increase in serological response. CONCLUSIONS The prevalence of positive serological responses in Cherepovets was higher than in most prior Cryptosporidium serosurveys in non-outbreak communities. Drinking water is an important pathway for infection.

Development of an urban green space indicator and the public health rationale

Aims: In this study, the aim was to develop and test an urban green space indicator for public health, as proposed by the World Health Organisation (WHO) Regional Office for Europe, in order to support health and environmental policies. Methods: We defined the indicator of green space accessibility as a proportion of an urban population living within a certain distance from a green space boundary. We developed a Geographic Information System (GIS)-based method and tested it in three case studies in Malmö, Sweden; Kaunas, Lithuania; and Utrecht, The Netherlands. Land use data in GIS from the Urban Atlas were combined with population data. Various population data formats, maximum distances to green spaces, minimum sizes of green spaces, and different definitions of green spaces were studied or discussed. Results: Our results demonstrated that with increasing size of green space and decreased distance to green space, the indicator value decreased. As compared to Malmö and Utrecht, a relatively bigger proportion of the Kaunas population had access to large green spaces, at both shorter and longer distances. Our results also showed that applying the method of spatially aggregated population data was an acceptable alternative to using individual data. Conclusions: Based on reviewing the literature and the case studies, a 300 m maximum linear distance to the boundary of urban green spaces of a minimum size of 1 hectare are recommended as the default options for the indicator. The indicator can serve as a proxy measure for assessing public accessibility to urban green spaces, to provide comparable data across Europe and stimulate policy actions that recognise the importance of green spaces for sustainable public health.

Seasonality of Cryptosporidium oocyst detection in surface waters of Meru, Kenya as determined by two isolation methods followed by PCR

Meru, Kenya has watersheds which are shared by wildlife, humans and domesticated animals. These surface waters can be contaminated by the waterborne pathogen Cryptosporidium. To quantify the seasonality and prevalence of Cryptosporidium in Meru regional surface waters, we used a calcium carbonate flocculation (CCF) and sucrose floatation method, and a filtration and immunomagnetic bead separation method, each of which used PCR for Cryptosporidium detection and genotyping. Monthly water samples were collected from January through June in 2003 and 2004, bracketing two April-May rainy seasons. We detected significant seasonality with 8 of 9 positive samples from May and June (p<0.0014), which followed peak rainy season precipitation and includes some of the subsequent dry season. Six of 9 positive samples revealed C. parvum, and 3 contained C. andersoni. None contained C. hominis. Our results indicate that Meru surface waters are Cryptosporidium-contaminated at the end of rainy seasons, consistent with the timing of human infections reported by others from East Africa and contrasting with the onset of rainy season peak incidence reported from West Africa.

The effect of Helicobacter pylori infection on growth velocity in young children from poor urban communities in Ecuador

OBJECTIVE To characterize the potential effects of Helicobacter infections on growth velocity in low socioeconomic status young children in a developing country. METHODS Children were recruited in poor suburbs of Quito, Ecuador. Normally nourished, mildly and substantially malnourished children (defined using weight-for-age Z-scores at recruitment) formed equal strata. Six height and weight measurements were collected during one year. Enrollment and exit serum samples were analyzed for anti-Helicobacter IgG and exit non-diarrheal feces tested for Helicobacter antigen. RESULTS Among 124 participants (enrollment age 19 ± 9 months), 76 (61%) excreted fecal antigen at exit (were infected). Of these, 44 were seropositive at least once (chronic infections) and 32 tested seronegative both times (new or acute phase infections). The adjusted linear growth velocity during follow-up in children with new infections was reduced by 9.7 (3.8, 15.6) mm/year compared to uninfected controls and 6.4 (0.0, 12.9) mm/year compared to children with chronic infections. The effects of Helicobacter infections on ponderal growth were not significant. CONCLUSION These results suggest that linear growth velocity is reduced in young children during the initial phase of Helicobacter infection.

The SEEDs of two gastrointestinal diseases: socioeconomic, environmental, and demographic factors related to cryptosporidiosis and giardiasis in Massachusetts.

OBJECTIVES We assessed associations between community-level socioeconomic, demographic, and environmental characteristics, and the presence of two potentially waterborne infectious diseases, cryptosporidiosis and giardiasis, as reported to the Massachusetts Department of Public Health. METHODS We created a series of maps showing the spatial distribution of cryptosporidiosis and giardiasis in Massachusetts (1993-2002) overall and by age, using logistic regression to analyze associations between community-level characteristics and the presence of at least one reported case of each disease. This analysis was repeated for communities with predominantly private water supplies. RESULT After adjusting for population size, higher population density and larger than average household sizes were associated with increased odds of reported cases of cryptosporidiosis. Giardiasis was also associated with high population density, but was not associated with household size. In the elderly, income was positively associated with the presence of giardiasis. DISCUSSION These findings suggest that greater population density and larger household sizes may increase the likelihood of protozoan gastrointestinal infection. The results emphasize the necessity to account for distal factors, such as demographic characteristics, that may ultimately play a role in the transmission or reporting of disease.

Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.

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.