Analysis of Serological Responses to Cryptosporidium Antigen among NHANES III Participants FLOYD J. FROST, PHD, TIMOTHY B. MULLER, BS, REBECCA L. CALDERON, PHD, AND GUNTHER F. CRAUN, MPH, PE
PURPOSE: Sera from 1356 National Health and Nutrition Examination Survey (NHANES) III participants from seven primary sampling units were tested for serological responses to two Cryptosporidium antigen groups. Intensity of responses was compared by geographic area, age, sex, race/ethnicity, income, and hepatitis A seropositivity. METHODS: Cryptosporidium seropositivity for the 15/17-kDa and the 27-kDa antigen groups were defined by the intensity of the responses. Conditional and unconditional logistic regression was used to identify significant risk factors. RESULTS: Hispanics, blacks, and females had a higher seropositivity. Younger participants and those with higher income had a lower seropositivity. Being hepatitis A seropositive was strongly related to a weak serological response to the 27-kDa antigen group. Family size was unrelated to Cryptosporidium seropositivity. Significantly higher Cryptosporidium seropositivity was observed for three of the seven primary sampling units. CONCLUSIONS: This study found significant geographical differences in the occurrence and the intensity of serological response. Strong serological responses to the 15/17-kDa antigen occurred more commonly in blacks and Hispanics, individuals not having high incomes, and in older age groups. Ann Epidemiol 2004;14:473–478. Ó 2004 Elsevier Inc. All rights reserved. KEY WORDS: Cryptosporidium, Seroepidemiologic Studies, Water Microbiology, Waterborne Disease, Infectious Disease, NHANES III.
INTRODUCTION Cryptosporidium parvum is recognized as a cause of infectious gastroenteritis. Cryptosporidium oocysts have been found in source and treated drinking water in the United States and elsewhere (1–2). Epidemic cryptosporidiosis has been linked to contaminated food and drinking water, day care attendance, and recreational exposure to water (3–6). The severity and persistence of symptoms are related to the immunocompetence of the host (3, 4) and may be altered by immune responses from prior infections (7–9). Despite the relatively common occurrence of oocysts in source and treated drinking water, Cryptosporidium oocysts are detected in fewer than 2% of stools submitted for ova and parasite analyses (10). However, several studies suggest that Cryptosporidium infections occur more commonly than is suggested by laboratory-based illness surveillance systems From the Lovelace Respiratory Research Institute, Albuquerque, New Mexico (F.J.F., T.B.M.); National Health and Environmental Effects Laboratory, US Environmental Protection Agency, Research Triangle Park, North Carolina (R.L.C.); and Gunther F. Craun and Associates, Staunton, Virginia (G.F.C.). Address correspondence to: Floyd J. Frost, Ph.D., Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive, S.E. Albuquerque, New Mexico 87108. Tel.: (505) 348-8776; Fax: (505) 348-4802. E-mail:
[email protected] This study was funded by the US Environmental Protection Agency Cooperative Agreement CR 8224167-01. Received June 17, 2002; accepted June 6, 2003. Ó 2004 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010
(11–13). Even among persons intentionally exposed to Cryptosporidium oocysts, laboratory-confirmed infections are difficult to detect (9). Furthermore, many infections may not result in overt illness or clinically significant illness (14–15). Serological surveillance has been used to assess occurrences of prior infections. Using the occurrence of serological responses to Cryptosporidium antigens, some studies have estimated the prevalence of prior Cryptosporidium infection in selected populations (11–13, 16–20) while others tracked people who were intentionally or unintentionally exposed to Cryptosporidium oocysts (7, 21, 22). Serological responses to a variety of Cryptosporidium antigens have been studied, with recent interest focusing on responses to 15/17-kDa and 27-kDa antigen groups (7, 11–13, 15–22). Infection usually elicits a serological response to these antigen groups (7) that peaks 4 to 6 weeks after infection (23, 24). The 15/17- kDa marker declines to background levels 4 to 6 months after infection while the 27-kDa marker remains elevated for at least 6 to 12 months (23, 24). Studies have not previously examined serological responses to Cryptosporidium antigens in multiple locations of the United States population. This pilot study was conducted to examine serological responses to two Cryptosporidium antigen groups in a convenience sample of National Health and Nutrition Examination Survey (NHANES) III participants and relates serological responses to other information collected by NHANES III. 1047-2797/04/$–see front matter doi:10.1016/j.annepidem.2003.06.002
474
Frost et al. SEROSURVEY FOR CRYPTOSPORIDIUM ANTIBODY
The study used sera collected in Phase I of NHANES III (1988–1990). Because of the selection of subjects and the inclusion of only seven geographic areas from NHANES III, the results cannot be used to estimate the national prevalence of seropositivity to Cryptosporidium antigens. METHODS
AEP Vol. 14, No. 7 August 2004: 473–478
survey. Personal variables include: race/ethnicity is white, Hispanic, black or other race, female sex, and general health reported as excellent or very good. Laboratory findings include hepatitis A seropositivity. Age was coded as shown in Table 1, but fewer age groups were used for the logistic regression analysis (! 29 years, 30–39 years, 40–49 years, 50–59 years, and 60 years and older).
Subjects NHANES III was a national probability survey of over 20,000 Americans conducted between 1988 and 1994 and employed a stratified, cluster sampling design (25). Participants underwent interviews, completed study questionnaires, and were clinically examined by study personnel (25). For this study, we obtained samples of surplus sera from participants residing in one of seven geographic areas or primary sampling units (PSU) of NHANES III. Since this study was approved after other studies had selected sera for analysis, the sera we used must be considered a convenience sample. In many cases we used the only samples remaining from the PSUs. These PSUs include: two cities with largely Hispanic populations that primarily use chlorinated groundwater, two Midwest cities that primarily use chlorinated and filtered river water from a major river system, one Midwest city that primarily uses filtered and chlorinated surface water supplemented with ground water, one city that primarily uses chlorinated unfiltered surface from a well-protected watershed, and one city that uses chlorinated and filtered river water with minimal human and animal sewage contamination. Measurements Variables collected from sera donors and used in these analyses are shown in Table 1. For purposes of the analyses, we created new binary variables (yes or no). These variables include household characteristics such as: the household income is in the highest quartile of all participants in this sample, the household has more than three members, and more than one household member participated in the TABLE 1. NHANES variables used Variable HSAGEIR HSSEX HSFSIZER DMARETHN HAB1 DMPPIR AHP DMPFSEQ
Description Age (0–10 years, 11–20 years, 31–30 years, 31–50 years, 51–70 years, 71 years and older) Sex (female sex) Family size (greater than three) Race-ethnicity (black, hispanic, other) Would you say your health in general is excellent, very good, good, fair or poor? (Excellent, very good) Poverty/income ratio (highest 25% of the sample) Hepatitis A serologically positive Family sequence number
Protein Gel Blot Procedures Sera were collected and analyzed by immunoblot to measure IgG serological response to the 15/17- and 27-kDa antigen groups. This method, which uses the miniblot format, has been described elsewhere (11–13). The intensity of the serological responses to the 15/17- and 27-kDa antigen groups on the immunoblots were digitally analyzed by an IS-2000 Digital Imaging System (Alpha Innotech, San Leandro, CA). Serological response to the two antigen groups was based on the measured area under the curve of response intensity for each lane at the expected location of the response for the antigen group. The IgG results for each specimen were standardized by calculating the ratio of the response intensity for the unknown sample to the response intensity for a positive control serum contained on each blot. The IgG positive control sera were obtained from individuals with a strong serological response to both antigen groups, approximating the intensity of responses observed from several individuals with laboratory-confirmed cryptosporidiosis. The same positive control sera were used on all blots. The methods employed for serological analysis for hepatitis A responses have been described elsewhere (25).
Statistical Analysis Multivariate analysis of the observed intensity of serological response to the Cryptosporidium antigens was conducted using conditional logistic regression using SAS Proc PHREG. Because of the multistage survey design, the analyses were stratified first by the PSUs and then by family within the PSU. The family unit was not related to the serological response to Cryptosporidium antigens and was, therefore, not considered in the remaining analyses. Four definitions of a positive response were considered: any detectable response and cumulative responses greater than 10%, greater than 20%, or greater than 30% of the positive control. Since the duration of a positive response and factors predictive of the duration of the response are not known, caution should be used in interpreting the prevalence odds ratios. In particular, the prevalence odds ratio calculated using the seropositivity to these antigens is unlikely to accurately estimate the relative risk of prior infections.
AEP Vol. 14, No. 7 August 2004: 473–478
Frost et al. SEROSURVEY FOR CRYPTOSPORIDIUM ANTIBODY
475
TABLE 2. Means serological responses (as a percent of the positive control) by site (PSU) and race City
All races (n)
White (n)
Black (n)
Hispanic (n)
Groundwater #1 (GW1) 15/17-kDa Groundwater #1 (GW1)27-kDa Surface filtered #1 (SW1) 15/17-kDa Surface filtered #1 (SW1) 27-kDa Surface filtered #2 (SW2) 15/17-kDa Surface filtered #2 (SW2) 27-kDa Surface filtered #3* (SW3) 15/17-kDa Surface filtered #3* (SW3) 27-kDa Groundwater #2 (GW2) 15/17-kDa Groundwater #2 (GW2) 27-kDa Surface unfiltered* (SU) 15/17-kDa Surface unfiltered* (SU) 27-kDa Surface filtered #4 (SF4) 15/17-kDa Surface filtered #4 (SF4) 27-kDa
26.0% (158) 25.5% (158) 13.9% (206) 22.3% (206) 28.1% (125) 30.2% (125) 13.4% (128) 23.9% (128) 21.7% (187) 27.1% (87) 13.3% (305) 18.2% (305) 22.7% (247) 26.3% (247)
12.8% (10) 16.9% (10) 13.9% (72) 22.9% (72) 26.5% (59) 23.8% (59) 11.3% (91) 19.6% (16) 18.0% (71) 25.5% (71) 12.7% (230) 18.2% (230) 21.1% (139) 22.7% (139)
–(1) – 14.4% (129) 22.3% (129) 30.0% (65) 36.1% (65) 9.3% (16) 31.2% (16) –(1) –(1) 8.9% (23) 25.1% (23) 24.6% (105) 31.3% (105)
27.0% (147) 26.2% (147) –(3) –(3) –(1) –(1) –(1) 23.6% (114) 28.1% (114) 19.4% (32) 17.8% (32) –(3) –(3)
)Protected or limited access watershed.
RESULTS Serological responses were ascertained for 1356 NHANES III participants. We obtained sera from 158 and 187 participants, respectively, from the two groundwater cities. A high fraction (63%) of these participants was Hispanic. Of the 711 participants from the other five cities, only 12% were Hispanic. From the two cities that obtain all drinking water from surface sources, there were 372 participants, of whom 46% were black. From the city using a mix of ground and surface water for drinking water, there were 206 participants, of whom 63% were black. Of the 305 participants from the city using unfiltered surface water, 10% were Hispanic, 7% black, and 7% other races. Of the 128 participants from the city using filtered surface water derived from a moderately well-protected watershed, 13% were black, none were Hispanic and 16% were other races. The mean serological responses for the 15/17-kDa and 27-kDa antigen groups by city and race are presented in Table 2. Almost one-half of the participants had a serological response to the antigen groups (48% [95% CI, 45%, 50%] for the 15/17-kDa marker and 62% [95% CI, 59%, 64%] for the 27-kDa marker). The percent positive for either marker differed by PSU (e.g., area of residence; p ! 0.001). A higher fraction of participants also had a response intensity exceeding 30% for the 27-kDa than for the 15/17-kDa marker (24.5% [95% CI, 22%, 27%] for the 15/17-kDa marker and 31.6% [95% CI, 29%, 34%] for the 27-kDa marker). The percent of participants with these more intense responses also differed by city of residence (p ! 0.001). The fraction of participants with a detectable response and the mean intensity of response increased with increasing age up to age 50 for both the 15/17-kDa and the 27-kDa markers (p ! 0.01) (Table 3). The conditional logistic regression analyses, stratified by PSU, are presented in Table 4 for the 15/17-kDa antigen
group and Table 5 for the 27-kDa antigen group. Hispanic ethnicity, black race, other race, and increasing age were related to a higher seropositivity to both markers for most definitions of a positive response (p ! 0.01). Female sex was weakly related to an increased occurrence of serological response to both markers. Family size and reporting of health to be good or excellent were not related to serological responses to either antigen group. Higher household income was related to a reduced occurrence of a response to both markers, but was strongly related only to responses to the 15/17-kDa antigen group (p ! 0.001). Being hepatitis A seropositive was related to an increased occurrence of a serological response to both markers but the relationship was statistically significant only for weak responses to the 27-kDa antigen group.
TABLE 3. Serological responses by age: 15/17-kDa and 27-kDa markers Age (years)
N
Mean % of positive control (95% CI)
Percent with detectable response (95% CI)
15/17-kDa marker ! 10 164 4.9% (2.1%, 7.8%) 11–20 191 5.6% (3.5%, 7.7%) 21–30 178 15.8% (12.2%, 19.4%) 31–50 349 20.6% (17.5%, 23.7%) 51–70 249 29.8% (25.9%, 33.8%) 71C 225 29.4% (25.0%, 33.8%) Increasing prevalence by age p ! 0.01
13.4% (8.1%, 18.6%) 21.5% (15.6%, 27.2%) 46.1% (38.7%, 53.4%) 52.1% (46.9%, 57.4%) 67.9% (62.1%, 73.7%) 67.6% (61.4%, 73.7%)
27-kDa Marker ! 10 164 4.7% (2.8%, 6.7%) 11–20 191 10.3% (7.6%, 12.9%) 21–30 178 23.5% (19.3%, 27.7%) 31–50 349 28.9% (25.6%, 32.1%) 51–70 249 35.7% (31.7%, 39.8%) 71C 225 29.8% (25.7%, 33.8%) Increasing prevalence by age p ! 0.01
21.3% (15.1%, 27.6%) 37.7% (30.8%, 44.6%) 65.7% (58.8%, 72.7%) 69.9% (65.1%, 74.7%) 81.5% (76.7%, 86.3%) 74.7% (69.0%, 80.3%)
476
Frost et al. SEROSURVEY FOR CRYPTOSPORIDIUM ANTIBODY
AEP Vol. 14, No. 7 August 2004: 473–478
TABLE 4. Prevalence odds Ratio of 15/19kDa response given different definitions of a positive response Factor
TABLE 6. Unstratified logistic regression analysis
Any response O 10% PCy O 20% PCy O 30% PCy
Female Black race Hispanic ethnicity Other race Age ! 30 years Age 30–39 years Age 40–49 years Age 50–59 years Family size O 3 High income Reported excellent or good health Hepatitis A seropositive
1.3 1.5* 2.0* 2.5* 0.1** 0.4** 0.5** 1.0 1.0 0.7** 1.2
1.2 1.5* 2.0** 3.1** 0.1** 0.4** 0.5** 1.1 0.9 0.6** 1.1
1.3 1.7** 3.2** 2.9** 0.1** 0.4** 0.5** 0.9 1.1 0.7** 1.1
1.3* 1.7* 3.3** 3.6** 0.1** 0.4** 0.4** 0.9 1.1 0.6** 1.1
1.9
1.4
1.3
1.3
)p !0.05. y PC Z Positive Control. ))p ! 0.01.
Variable Female sex Black race Hispanic ethnicity Other race Hepatitis A seropositive High income Age ! 29 years Age 30–39 years Age 40–49 years Age 50–59 years GW1* SF1* SF2* SF3* GW2* SF4*
15/17-kDa response prevalence odds ratio (p-value)
27-kDa response prevalence odds ratio (p-value)
1.3 (0.07) 1.5 (0.02) 1.9 (0.007) 2.5 (0.02) 1.9 (0.11) 0.7 (0.003) 0.1 (0.0001) 0.3 (0.0001) 0.5 (0.002) 1.0 (0.90) 2.2 (0.01) 1.4 (0.14) 3.0 (0.0001) 0.8 (0.35) 2.8 (0.0001) 2.4 (0.0001)
1.2 (0.13) 2.1 (0.0001) 1.7 (0.03) 2.5 (0.02) 4.5 (0.0003) 0.8 (0.12) 0.2 (0.0001) 0.5 (0.0001) 0.6 (0.03) 1.2 (0.5) 1.7 (0.1) 1.1 (0.6) 2.1 (0.004) 0.9 (0.8) 3.4 (0.0001) 1.8 (0.006)
)See Table 2 for description of PSU, comparison is with the SU site.
Serological response to either antigen group occurred more often in participants from three PSUs. These results were statistically adjusted for several factors, identified in Tables 4 and 5, and are presented in Table 6. Differences in serological responses did not correspond to whether the drinking water was obtained from a surface vs. an underground source. However, one city with low levels of serological responses used unfiltered surface-derived drinking water from a well-protected watershed while another used filtered surface water from a moderately well-protected watershed. DISCUSSION An increasing seropositivity to both the 15/17-kDa and the 27-kDa antigen groups were observed with increasing age. TABLE 5. Prevalence odds ratio of 27kDa response given different definitions of a positive response Factor
Any response O 10% PCy O 20% PCy O 30% PCy
Female Black race Hispanic ethnicity Other race Age 0–29 years Age 30–39 years Age 40–49 years Age 50–59 years Large family High income Reported excellent or good health Hepatitis A seropositive )p ! 0.05. y PC Z Positive Control. ))p ! 0.01.
1.2 2.1** 1.8* 2.5* 0.1** 0.4** 0.6* 1.2 1.0 0.8 1.3
1.4** 2.3** 2.2** 2.4* 0.2** 0.5** 0.6* 0.9 0.9 0.7** 1.2
1.5** 2.0** 1.8* 1.6 0.2** 0.6* 0.7 1.2 0.9 0.8 1.2
1.2 2.0** 1.7* 1.7 0.2** 0.5** 0.7 1.1 1.0 0.8 1.2
4.3**
4.4**
1.4
1.3
This suggests that children respond serologically to these two antigen groups, but that a more intense response develops with increasing age. This may occur because repeated infections boost the immune response or because other infectious agents are responsible for the serological response. However, use of the protein gel blot assay rather than an ELISA assay detects serological response to selected Cryptosporidium antigens. This probably reduces the chance that cross-reactions from other infectious agents are responsible for the relationships observed. By age 70, approximately 70% of participants had serologic evidence of prior infection. The high frequency of serological responses suggests that Cryptosporidium infections occur more commonly than is suggested by public health surveillance systems. Many, if not most, of these infections may be either asymptomatic or result in only minor illness. The prevalence of a response to the 27-kDa antigen group was higher for each of the six age groups (p ! 0.05). This finding agrees with prior research (23, 24) that suggests the response to the 27-kDa antigen group is longer-lived than is response to the 15/17-kDa antigen. The longer-lived 27-kDa response may explain why weak responses to the 27-kDa antigen group are also related to hepatitis A seropositivity, since a common set of factors may have contributed to both infections in the distant past. Significant geographic differences were observed in the seropositivity as well as the mean intensity of response to the two antigen groups. These differences may be due to drinking water sources. Low intensities of responses were observed for residents of cities that use surface water from protected sources. This is of interest since watershed
AEP Vol. 14, No. 7 August 2004: 473–478
protection has received less attention in recent years as new technological solutions to water treatment have been developed. This finding, however, suggests that source protection remains an important and effective strategy for minimizing the risk of waterborne pathogen transmission. We found a higher prevalence of serological responses in females than males, possibly suggestive of person-to-person transmission. We found a similar increase in seropositivity in members of smaller families. We found no statistically significant relationships between family size and seropositivity when we considered the number of family members rather than the dichotomized variable. The relationships between Hispanic ethnicity, black race, other race, and the occurrence of serological responses are probably related to socio-economic characteristics that were not accurately measured rather than to race per se. For example, differences in the intensity of serological response by race were not observed in a prior serological study that had a significant fraction of non-white blood donors (13). This suggests that in our study, factors other than race may have been responsible for the observed racial differences in serological responses. High family income (e.g., the highest quartile) was associated with a less frequent 15/17-kDa antigen response and was weakly associated with a less frequent response to the 27-kDa antigen group. Relationships between family income and risk of a parasite infection have not been commonly reported in the United States in recent decades, perhaps because few studies have looked for this relationship. This subject merits further investigation. These results suggest that Cryptosporidium infections occur commonly in the United States. The serological response rates are comparable to those detected in Australia (16), Italy (18), and Canada (17), but substantially lower than those found in a recently published study in Russia (26). Each of these studies used the same positive control to facilitate comparison of findings. This study suggests that demographic and socioeconomic characteristics of individuals do not account for all of the geographic differences in seropositivity observed in this study. Regional differences in the microbial quality of drinking and recreational water should be further investigated. Geographical differences in the risk of endemic Cryptosporidium infections may reduce the power of crosssectional studies such as NHANES to detect drinking water effects, as has been previously observed (13). It may be necessary to pair cities geographically for valid comparisons to be made. In this study, the ground water cities were geographically separated from the surface water cities. Longitudinal surveys using NHANES or data from other random household surveys may be useful for assessing the efficacy of new water treatment technologies to remove or inactivate Cryptosporidium oocysts.
Frost et al. SEROSURVEY FOR CRYPTOSPORIDIUM ANTIBODY
477
REFERENCES 1. Lisle JT, Rose JB. Cryptosporidium contamination of water in the USA and UK, a mini-review. J Water SRT-Aqua. 1995;44:103–117. 2. Rose JBN, Gerba CP, Jakubowski W. Survey of potable water supplies for Cryptosporidium and Giardia. Environ Sci Technol. 1991;25:1393–1400. 3. Meinhardt PL, Casemore DP, Miller KB. Epidemiological aspects of human cryptosporidiosis and the role of waterborne transmission. Epidemiol Rev. 1996;18:118–136. 4. Casemore DP. Human cryptosporidiosis. In: Reeves, D, Geddes, A, eds. Recent Advances in Infection. No. 3. Edinburgh, Scotland: Churchill Livingstone; 1988:209–236. 5. Centers for Disease Control and Prevention (CDC). Outbreak of cryptosporidiosis associated with a water sprinkler fountain – Minnesota. Morb Mortal Wkly Rep. 1997;47:856–860. 6. Craun GF, Hubbs SA, Frost F, Calderon RL, Via S. Waterborne outbreaks of cryptosporidiosis. J Am Water Works Assoc. 1998;90(9):81–91. 7. Moss DM, Chappel CL, Okhuysen PC, DuPont HL, Arrowood MJ, Hightower AW, et al. The antibody response to 27-, 17-, and 15-kDa Cryptosporidium antigens following experimental infection in humans. J Infect Dis. 1998;178:827–833. 8. Frost FJ, Craun GL, Calderon CL, Hubbs SA. So many oocysts, so few outbreaks. J Am Water Works Assoc. 1997;89(12):8–9. 9. Okhuysen PC, Chappell CL, Sterling CR, Jakubowski W, DuPont HL. Susceptibility and serological response of health adults to re-infection with Cryptosporidium parvum. Infect Immun. 1998;66:441–443. 10. Skeels MR, Sokolow R, Hubbard CV, Andrus JK, Baisch J. Cryptosporidium infection in Oregon public health clinic patients 1985–1988; the value of statewide laboratory surveillance. Am J Public Health. 1990;80: 305–308. 11. Frost FJ, de la Cruz AA, Moss DM, Curry M, Calderon RL. Comparisons of ELISA and Western blot assays for detection of Cryptosporidium antibody. Epidemiol Infect. 1998;121:205–211. 12. Frost FJ, Calderon RL, Muller TM, Curry M, Rodman JS, Moss DM, et al. A two-year follow-up of antibody to Cryptosporidium in Jackson County, Oregon following an outbreak of waterborne disease. Epidemiol Infect. 1998;121:213–217. 13. Frost FJ, Muller T, Craun GF, Lockkwood WB, Calderon RL. Serological evidence of endemic waterborne Cryptosporidium infections. Ann Epidemiol. 2002;12(4):222–227. 14. Okhuysen PC, Chappell CL, Sterling CR, Jakubowski W, DuPont HL. Susceptibility and serological response of health adults to re-infection with Cryptosporidium parvum. Infect Immun. 1998;66:441–443. 15. Frost FJ, Craun GF. Serological response to human Cryptosporidium infection (letter). Infect Immun. 1998;66(8):4008. 16. Frost FJ, Muller TB, Fairley CK, Hurley JS, Gunther GF, Calderon RL. Serological evaluation of Cryptosporidium oocyst findings in the water supply for Sydney, Australia. Int J Environ Health Res. 2000;10:35–40. 17. Frost FJ, Muller T, Craun GF, Fraser D, Thompson D, Notenboom R, et al. Serological analysis of a cryptosporidiosis epidemic. Int J Epidemiol. 2000; 29:376–379. 18. Frost FJ, Fea E, Gilli G, Biorci F, Muller TM, Craun GF, et al. Serological evidence of Cryptosporidium infection in southern Europe. Eur J Epidemiol. 2000;16(4):385–390. 19. Frost FJ, Muller T, Craun GF, Calderon RL, Roefer PA. Paired city Cryptosporidium serosurvey. Epidemiol Infect. 2000;126:301–307. 20. Frost FJ, Muller T, Craun GF, Calderon RL. A serological survey of college students for antibody to Cryptosporidium. Epidemiol Infect. 2000;125: 87–92. 21. Moss DM, Bennett SN, Arrowood MJ, Wahlquist SP, Lammie PJ. Enzymelinked immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter. Am J Trop Med Hyg. 1998;51(1): 110–118.
478
Frost et al. SEROSURVEY FOR CRYPTOSPORIDIUM ANTIBODY
22. Moss DM, Bennett SN, Arrowood MJ, Hurd MR, Lammie PJ, Wahlquist SP. Kinetic and isotypic analysis of specific immunoglobulins for crew members with cryptosporidiosis on a US Coast Guard cutter. J Eukaryot Microbiol. 1994;41:52S–55S. 23. Priest JW, Li A, Khan M, Arrowood MJ, Lammie PJ, Ong CS, et al. Enzyme immunoassay detection of antigen-specific immunoglobulin G antibodies in longitudinal serum samples from human cryptosporidiosis patients. Clin Diagn Lab Immunol. 2001;8(2):415–423.
AEP Vol. 14, No. 7 August 2004: 473–478
24. Muller TB, Frost FJ, Craun GF, Calderon RL. Serological responses to Cryptosporidium infection. Infect Immun. 2001;69(3):1974–1975. 25. National Center for Health Statistics. Plan and operation of the Third National Health and Nutrition Examination Survey 1988–1994. Vital Health Stat. 1994;1(32). 26. Egorov A, Frost F, Muller T, Naumova E, Tereschenko A, Ford T. Serological evidence of Cryptosporidium infections in a Russian city and evaluation of risk factors for infection. Ann Epidemiol (in press).
