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Lovelace Respiratory Research Institute, Department of Family and Community Medicine, University of New Mexico
Lovelace Clinic Foundation, Albuquerque
Gunther F. Craun & Associates, Staunton, Virginia
Department of Epidemiology and Preventive Medicine, Monash University, The Alfred Hospital, Victoria, Australia
This study assessed whether serological responses to Cryptosporidium antigens are associated with a reduced risk of diarrheal illness in cases of infection with human immunodeficiency virus (HIV). The association between serological responses to the Cryptosporidium 15/17-kDa and 27-kDa antigen groups and reported diarrheal illness was examined by use of data from a previously published study of cases of HIV infection. In immunosuppressed individuals, a strong serological response to the 27-kDa antigen group was associated with a reduced risk of diarrhea without weight loss. This finding suggests that acquired protective immunity to cryptosporidiosis may be important in controlling the burden of cryptosporidiosis in immunosuppressed individuals.
Cryptosporidium parvum is a major cause of epidemic diarrhea. It has also been reported as being both a cause of severe diarrhea in medically immunosuppressed individuals and, possibly, a life-threatening infection associated with HIV/AIDS infections [1]. Before the introduction of antiretroviral therapy, oocysts were detected in up to 4% of US patients with AIDS [2]. The severity of illness ranged from asymptomatic, or self-limiting, to "fulminate cholera-like" illness [2].
In a previous report, we had postulated that immunocompetent individuals develop protective immunity that could explain the infrequent occurrence of symptomatic illness, the absence of outbreaks in exposed populations, and a disproportionately high fraction of all outbreaks occurring among visitors to communities served by marginally treated surface-derived drinking-water supplies [3]. Because of their lower levels of protective immunity, residents of towns and cities supplied by groundwater that is periodically contaminated by either surface water or sewage may also be at high risk of illness [3].
Cryptosporidium oocysts are commonly found in both raw and treated surface-derived drinking water [4]. A recent study by Aboytes et al. found that low doses of waterborne oocysts occurred even in filtered drinking water that had low levels of turbidity (i.e., <0.5 nephelometric turbidity units [5]. Serological surveys of Cryptosporidium-specific antibodies in humans suggest that a large fraction of the population has been infected [6]. Compared with users of clean groundwater, users of surface-derived drinking water are more likely to have serological responsesand seroconversionsto the 15/17-kDa and 27-kDa Cryptosporidium antigen groups [79]. Despite widespread human exposure to oocysts in surface-derived drinking water, cryptosporidiosis outbreaks are relatively uncommon, even among individuals with HIV/AIDS.
In a recent study in the United States, volunteers from several areas with either groundwater- or surface-derived drinking water were tested for serological responses to the 15/17-kDa and 27-kDa Cryptosporidium-antigen groups; for a 4-month period, these volunteers maintained daily diaries of illness [10]. The study found that strong serological responses to the antigen groups (i.e., 20% of the response in positive control individuals) was associated with a significantly reduced risk of both gastrointestinal and diarrheal illness. These findings also suggest that Cryptosporidium infections may be a common cause of diarrheal and gastrointestinal illness in immunocompetent individuals and that a strong serological response is associated with a lower risk of illness. However, because of their impaired immune responses, it is unclear whether immunosuppressed individuals benefit from protective immunity. To address this question, we examined, in HIV-infected individuals, the association between the occurrence of a strong serological response and diarrheal-illness data reported by these individuals when they provided serum samples.
Subjects and methods.
The present study was reviewed and approved by the institutional review board of the Lovelace Respiratory Research Institute. We used data collected from a previously published cross-sectional serological study of 294 HIV-infected individuals in Melbourne, Australia [11]. Study participants were recruited between March and July 1997, from 1 hospital and 2 community health clinics. Serum samples were analyzed by Western blot, to determine serological responses to Cryptosporidium-specific antigen groups (15/17-kDa and 27-kDa) [6]. The data-collection and serological-analysis methods have been described elsewhere [11]. The earlier study was restricted to 236 men who had sex with men (MSM), to identify risk factors for prior Cryptosporidium infections in this group; the present study includes those 236 men plus 58 non-MSM individuals. The purpose was to determine whether those with a strong serological response had a lower risk of diarrheal illness during the 2 years prior to the time when the serum samples were taken.
In a manner similar to that used in our US study [10], we defined a strong serological response as being an intensity 20% of that in positive control individuals. To facilitate comparisons with the results of our other studies, we used standardized positive control sera for all studies and maintained stringent, standardized quality-control standards for all laboratory analyses [6]. The serological responses were based on the measured intensity of response on a digitized blot, adjusted as a percentage of the positive control value [8]. CD4 cell counts for each individual were obtained during enrollment in the study and were categorized as follows: <75 cells/L, 75199 cells/L, 200374 cells/L, and 375 cells/L.
All study participants completed a short questionnaire on history of illnesses and potential exposures. The questionnaire included 1 question regarding prior occurrence of diarrhea: "Have you had any diarrheal infection (watery stools) in the past two years" Another question asked whether the study participants had lost weight because of that illness. In the present study, the analysis examined (1) whether participants with a strong serological response to either the 15/17-kDa group or the 27-kDa antigen group were less likely to report diarrheal illness and (2) whether the CD4 cell count affected the relationship between serological response and diarrheal illness. Because 35% of the participants reported weight loss, those who reported diarrhea with weight loss were considered separately from those who reported diarrhea without weight loss; for neither group was there any available information on HIV load.
Multivariate analyses were conducted by use of logistic-regression software (Proc Logistic; SAS Institute). Three separate models using different outcome categories(1) any diarrhea, (2) diarrhea with weight loss, and (3) diarrhea without weight losswere examined; the comparison group comprised participants with no reported diarrhea. Independent variables included serological response to the either 15/17-kDa antigen group or the 27-kDa antigen group (no/weak response vs. strong response), CD4 cell count (<75 cells/L vs. 75 cells/L), and age group (<35 years old vs. 35 years old).
Results.
Of the 294 individuals followed, 63 reported having had no diarrhea during the preceding 2 years, whereas 231 reported having had diarrhea (103 with weight loss and 128 without weight loss) during that period (table 1). Approximately 44% of the participants were <35 years old, and 25% had a CD4 cell count of <75 cells/L. Overall, 106 (36.1%) of the participants had a strong serological response the 15/17-kDa antigen group, and 175 (59.5%) had a strong serological response to the 27-kDa antigen group.
A higher percentage of participants who reported either any diarrhea (P = .041) or diarrhea with weight loss (P = .010) were <35 years old, compared with those who reported no diarrhea (table 1). A higher percentage of participants who reported diarrhea with weight loss had CD4 cell counts <75 cells/L, compared with those without diarrhea, but this difference was not significant (P = .061). Between the other groups, there were no significant differences in CD4 cell counts. The percentage of participants with a very strong serological response to the 15/17-kDa antigen group did not differ between any of the groups. Although the difference was not statistically significant, a smaller percentage of participants who reported diarrhea without weight loss showed a strong serological response to the 27-kDa antigen group, compared with those who reported no diarrhea (51.6% vs. 66.7%; P = .068).
Adjusted odds ratios (ORs) for the association between reported diarrheal illness and serological response to both antigen groups are shown in table 2. After the results were adjusted for age group and CD4 cell count, a strong serological response to the 15/17-kDa antigen group was not significantly associated with any of the 3 outcome categories; in contrast, a strong serological response to the 27-kDa antigen group was associated with a lower risk of diarrhea without weight loss (OR, 0.6 [95% confidence interval {CI}, 0.30.9]), whereas no significant association was seen for either the outcome category of any diarrhea or the outcome category of diarrhea with weight loss.
Discussion.
One argument used to justify the regulatory goal of having no Cryptosporidium oocysts in drinking water is the potential high risk of infectious enteric illness in immunosuppressed individualsa group that includes those with HIV/AIDSas well as in those who are either immunosuppressed because of other medical conditions or who are, as in the case of organ-transplant patients, medically immunosuppressed to prevent organ rejection. However, the results of the present study suggest that immunosuppressed individuals with a strong serological response to the 27-kDa antigen group may be at reduced risk of diarrhea without weight loss; if this relationship is causal, then Cryptosporidium may be a significant cause of diarrhea in this population. It is not clear why a similar protective association was not seen for diarrhea with weight loss, although it is possible (1) that more-virulent strains of Cryptosporidium are less affected by immune responses or (2) that much of the weight loss is caused by other factors, including the HIV infection itself. Unfortunately, the intensity of HIV infection could not be assessed, because HIV-load data were not available.
Other studies have found that, compared with those who drink groundwater, individuals who drink surface-derived water are more likely to have a strong serological response to the 15/17-kDa and 27-kDa antigen groups [79], perhaps because of repeated exposure to low doses of Cryptosporidium oocysts. Our earlier studies have found evidence that Cryptosporidium infections are very common and that the exposure may come both from drinking-water sources and from nondrinking-water sources. Cryptosporidium is also likely to be a common infection in immunosuppressed individuals, especially in those lacking strong serological responses. However, it may be an uncommon cause of illness in populations in which the prevalence of a strong serological response to either the 15/17-kDa group or the 27-kDa antigen group is high.
A number of studies have identified risk factors for cryptosporidiosis among individuals with HIV/AIDS. However, less attention has been given to why, even before the introduction of successful AIDS therapies, cryptosporidiosis was a relatively uncommon illness in individual with HIV/AIDS. The results of our earlier studies suggest that the infrequent occurrence of illness is probably not the result of uncommon exposures or infections, because Cryptosporidium infections are common. It is important to better understand why, despite widespread exposure, some individuals become ill and others do not; such understanding may provide guidance as to feasible methods for reducing the risk of Cryptosporidium-related illness, both in the general population and in high-risk, immunosuppressed individuals. It is possible that individuals who are medically immunosuppressed may benefit from having a strong serological response to Cryptosporidium antigens.
The present study's failure to relate diarrhea reports and strong serological responses to the 15/17-kDa antigen group may be the result of the 2-year period covered by the illness questionnaire: after infection, the intensity of serological response to the 15/17-kDa antigen group appears to decline more rapidly, compared with the intensity of serological response to the 27-kDa antigen group [12]. However, in an earlier study of immunocompetent individuals, a study that asked the same question about previous diarrheal illnesses and that covered the same time period, we found a relation between serological responses and illness [13]; in that study, the 15/17-kDa antigen group, but not the 27-kDa antigen group, was found to be related to protection against illness, a finding suggesting that the antigens related to strong serological responses in individuals with HIV-AIDS may be different than those in immunocompetent individuals.
In summary, although additional work is necessary to confirm or refute them, both the results of the present study and those of our earlier studies indicate that Cryptosporidium infections are likely to be a common cause of infection and illness in both immunocompetent and immunosuppressed individuals. Strong serological responses appear to be associated with a reduced risk of diarrheal illness. Previous repeated exposure to low doses of Cryptosporidium oocysts, possibly via drinking water, is a likely reason why users of surface-derived drinking water more commonly have strong serological responses. Another study has reported, for example, that older HIV-infected individuals are at lower risk of cryptosporidiosis than are younger HIV-infected individuals [14], a finding that may be related to the higher levels of serological responses in older individuals [6]. Additional research is necessary to confirm (1) that immunosuppressed individuals with strong serological responses have lower risks of diarrhea and (2) that these responses result from exposure to low doses of Cryptosporidium oocysts, such as that occurring via drinking water; if this is the case, then it is possible that, because of the resulting reduced protective immunity, efforts to reduce or eliminate these exposures to low doses of Cryptosporidium oocysts will increase, rather than reduce, the risk of illness. However, the effort to eliminate periodic exposures to high doses of Cryptosporidium oocysts remains an important public-health activity, which can best be pursued by improving the reliability of drinking-water treatment.
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