Prospective audit of cross-sectional imaging and radiation exposure in general surgical patients.

INTRODUCTION: Most patients admitted with an acute surgical problem undergo some imaging during their in-patient stay. As computed tomography (CT) scanning becomes more readily available, it is becoming the mainstay of assessment. Recent studies have commented on the potential adverse effects of early cross-sectional imaging. This audit aimed to determine the use of early cross-sectional imaging and associated radiation dose in an acute surgical cohort. PATIENTS AND METHODS: Data from 500 acute surgical patients were prospectively collected over a 3-month period. CT scans were undertaken, the effect on subsequent diagnostic and management decisions and the potential associated risks were evaluated. RESULTS: Almost 40% of subjects underwent CT scanning. Of these, 20% did not alter management and could be deemed unnecessary. The radiation exposure equated to an age- and gender-specific risk of fatal cancer induction between 1 in 1675 and 1 in 7130. CONCLUSIONS: Early-cross sectional imaging provides a valuable diagnostic adjunct. Decisions to expose patients to potentially hazardous radiation need to acknowledge the anticipated benefits versus risks.

Characterization of a cytosolic aminopeptidase from Encephalitozoon intestinalis.

Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70.8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4.8. The enzyme was both heat (> 37 degrees C) and cold (< 0 degrees C) labile with an optimal activity at pH 7.2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.

Impact of breast-feeding on Giardia lamblia infections in Bilbeis, Egypt.

A total of 152 infants were followed from birth to 1 year of age in a rural community of Egypt to document Giardia lamblia infection and to determine the effect of breast-feeding on enteric infections by this protozoan. Asymptomatic Giardia infections persisted as long as 4 months, with a mean duration of excretion of 7.18 weeks. The incidence of asymptomatic infection was 4.5 episodes per child-year. Exclusively breast-fed infants had lower risk for asymptomatic (odds ratio [OR] = 0.66, 95% confidence interval [CI] = 0.45-0.96, P < 0.05) and symptomatic infections (relative risk [RR] = 0.50, 95% CI = 0.27-0.90, P < 0.05). Furthermore, breast-fed infants had fewer clinical manifestations, including mucus in stool (23.8% versus 76.2%, P = 0.08), loss of appetite (17.6% versus 82.3%, P < 0.05), and abdominal tenderness (17% versus 82.9%, P < 0.05) compared with infants who were not exclusively breast-fed. Breast-feeding should be considered as an effective means to prevent Giardia infections and should be encouraged in regions where G. lambia is highly endemic.

Expression of IL-15 and IL-4 in IFN-gamma-independent control of experimental human Cryptosporidium parvum infection.

We have previously demonstrated interferon gamma (IFN-gamma) in intestinal mucosa after experimental human Cryptosporidium parvum infection, but expression was limited to sensitized volunteers. To characterize IFN-gamma-independent mechanisms in control of infection, jejunal biopsies from immunocompetent volunteers experimentally challenged with C. parvum were examined by in situ hybridization for interleukin (IL-)15 and IL-4 mRNA with confirmation by immunohistochemistry. Cytokine expression was correlated with prechallenge anti- C. parvum IgG, symptoms, oocyst shedding, and prior IFN-gamma expression data. IL-15 expression was noted only in those without prior sensitization, who did not express IFN-gamma. By contrast, expression of IL-4 was associated with prior sensitization. IL-15 was only detected in those with symptoms (6/14 symptomatic vs 0/3 asymptomatic, P<0.05). Among 14 volunteers who did not express IFN-gamma, oocyst shedding was lower in those expressing IL-15. Overall, 14/15 volunteers who did not shed oocysts expressed either IFN-gamma or IL-15. There was no correlation between expression of IL-4 and symptoms or oocyst shedding. In conclusion, IL-15 expression was associated with control of oocyst shedding in those not expressing IFN-gamma. These data suggest that IL-15 is involved in IFN-gamma independent mechanisms of control of human cryptosporidiosis, perhaps via activation of the innate immune response.

The role of hepatitis C in hepatocellular carcinoma: a case control study among Egyptian patients.

BACKGROUND: Egypt has one of the highest prevalence rates of hepatitis C virus (HCV) infection in the world; however, the risk and attribution related to HCV in Egyptian patients with hepatocellular carcinoma (HCC) remains unknown. GOALS: The current study was undertaken to estimate the risk of HCC in relation to HCV in Egypt. STUDY: Thirty-three patients with HCC and 35 healthy controls who had a similar socioeconomic status were prospectively enrolled at the University of Cairo National Cancer Institute. RESULTS: Anti-HCV antibodies were present in 75.8% of the patients and in 42.9% of the controls (p = 0.01); hepatitis B surface antigen (HBsAg) was present in 15.2% of the patients and in 2.9% of the controls (p = 0.03). In addition, the sex-and age-adjusted odds ratio (OR) for anti-HCV antibodies was 5.1 (95% CI = 1.5-17.4) and for HBsAg was 13.2 (95% CI = 1.2-148.2). Concurrent Schistosoma mansoni and anti-HCV was associated with an OR of 10.3 (95% CI = 1.3-79.8), which was higher than that for anti-HCV (6.5; 95% CI = 1.6-26.6) and S. mansoni infection (0.2; 95% CI = 0.1-6.2) alone. Finally, we estimated the attributable fraction of HCC to HCV to be 64% in this study population and 48% in the general Egyptian population. CONCLUSIONS: Both HCV and hepatitis B virus infection increase the risk of HCC in Egyptian patients, whereas isolated Schistosoma infection does not. Because of the very high prevalence rate of HCV in the general Egyptian population, it accounts for most HCC cases in Egypt.

Standardized method of measuring acanthamoeba antibodies in sera from healthy human subjects.

Acanthamoeba species can cause serious, debilitating, and sometimes life-threatening infections. Three groups have been identified using morphological and immunological comparisons. Previous serological studies have utilized a variety of antigen preparations and assay methods and reported disparate (3 to 100%) results. This study was designed to (i) optimize an enzyme-linked immunosorbent assay for detecting serum antibodies to each of the Acanthamoeba serogroups and (ii) test 55 healthy individuals for specific immunoglobulin G reactivity. The highest signal-to-background ratio was found when 3,000 fixed, intact trophozoites per well were used with a 1:10 serum dilution. Sera yielding optical densities of <0.25 against all three Acanthamoeba serogroups were used to define the cutoff for positive results. The highest background reactivity with these sera was seen with Acanthamoeba polyphaga (serogroup 2), followed by Acanthamoeba culbertsoni (serogroup 3) and Acanthamoeba astronyxis (serogroup 1). Of 55 subjects tested, the highest number of positive results was seen with A. polyphaga (81.8%), followed by A. astronyxis (52.8%) and A. culbertsoni (40%). Seven serum samples (12.7%) were negative for all three Acanthamoeba serogroups, 16 (29.1%) were positive for one serogroup only, 16 were positive for two serogroups, and 16 reacted to all three serogroups. Further analysis showed no significant associations between serogroup reactivity and age or gender. However, some ethnic differences were noted, especially with A. polyphaga antigens. In that case, serum samples from Hispanic subjects were 14.5 times less likely to be positive (P = 0.0025) and had lower mean absorbance values (P = 0.047) than those from Caucasian subjects. Overall, these data suggest that Acanthamoeba colonization or infection is more common than previously thought. Mild or asymptomatic infections may contribute to the observed serum reactivities.

Expression of tumor necrosis factor alpha and interleukin 1 beta in jejuna of volunteers after experimental challenge with Cryptosporidium parvum correlates with exposure but not with symptoms.

Jejunal biopsies from volunteers challenged with Cryptosporidium parvum were examined for tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1 beta mRNA. Postchallenge biopsies from 15 of 28 (54%) volunteers expressed TNF-alpha; 14% expressed IL-1 beta. Cytokine expression did not correlate with enteric symptoms, suggesting that TNF-alpha and IL-1 beta are not key mediators of diarrhea in human cryptosporidiosis.

Risk assessment for Cryptosporidium: a hierarchical Bayesian analysis of human dose response data.

Three dose-response studies were conducted with healthy volunteers using different Cryptosporidium parvum isolates (IOWA, TAMU, and UCP). The study data were previously analyzed for median infectious dose (ID50) using a simple cumulative percent endpoint method (Reed and Muench, 1938). ID50s were derived using two definitions of infection: one as subjects having oocysts detected in stool by direct fluorescence assay, and the other by a clinical finding of diarrhea with or without detected oocysts (Chappell et al., 1998; Okhuysen et al., 1999). In the present study, the data were analyzed using the broader definition of infection (i.e., presence of oocysts in stool and/or diarrheal illness characteristic of cryptosporidiosis). Maximum likelihood dose-response parameter estimates for UCP, IOWA, and TAMU were 2980, 190, and 17.5, respectively. Based on these estimates, the ID50s of the three respective isolates were 2066, 132, and 12.1. The three oocyst isolates were considered representative of a larger population of human-infecting strains and analyzed as combined data using a hierarchical Bayesian model. Hyperparameters defined the distribution of dose-response parameters for the population of strains. Output from Markov Chain Monte Carlo analysis described posterior distributions for the hyperparameters and for the parameters of the IOWA, TAMU, and UCP strains. Point estimates of dose-response parameters produced by this analysis were similar to the maximum likelihood estimates. Finally, the utility of these results for probabilistic risk assessment was evaluated. The risk of infection from single oocyst doses was derived for a mixture of the three isolates (where IOWA, TAMU, or UCP are equally likely), and for an oocyst selected at random from the larger population of strains. These estimated risks of infection were 0.018 and 0.028, respectively.

Fecal antibodies to Cryptosporidium parvum in healthy volunteers.

This study examined the intestinal antibody response in 26 healthy volunteers challenged with Cryptosporidium parvum oocysts. Fecal extracts were assayed for total secretory immunoglobulin A (IgA) and C. parvum-specific IgA reactivity. Specific IgA reactivity was standardized to IgA concentration and expressed as a reactivity index (RI). Anti-C. parvum fecal IgA (fIgA) increased significantly in 17 of 26 (65.4%) following oocyst ingestion. Of those with detectable responses, 59, 76.5, and 94.1% were positive by days 7, 14, and 30, respectively. Volunteers receiving high challenge doses (>1,000 and 300 to 500 oocysts) had higher RIs (RI = 5.57 [P = 0. 027] and RI = 1.68 [P = 0.039], respectively) than those ingesting low doses (30 to 100 oocysts; RI = 0.146). Subjects shedding oocysts and experiencing a diarrheal illness had the highest fIgA reactivity. When evaluated separately, oocyst excretion was associated with an increased fIgA response compared to nonshedders (RI = 1.679 versus 0. 024, respectively; P = 0.003). However, in subjects experiencing diarrhea with or without oocyst shedding, a trend toward a higher RI (P = 0.065) was seen. Extracts positive for fecal IgA were further examined for IgA subclass. The majority of stools contained both IgA1 and IgA2, and the relative proportions did not change following challenge. Also, no C. parvum-specific IgM or IgG was detected in fecal extracts. Thus, fecal IgA to C. parvum antigens was highly associated with infection in subjects who had no evidence of previous exposure and may provide a useful tool in detecting recent infections.

Transforming growth factor beta1 is expressed in the jejunum after experimental Cryptosporidium parvum infection in humans.

Biopsies from volunteers challenged with Cryptosporidium parvum were examined for transforming growth factor beta1 (TGF-beta1). None of the prechallenge biopsies exhibited TGF-beta. Seven of 12 volunteers with oocyst shedding expressed TGF-beta versus 2 of 13 volunteers without detected oocysts. The association of TGF-beta expression with oocyst excretion and the timing of symptoms suggests that TGF-beta mediates intestinal healing.

Interferon-gamma expression in jejunal biopsies in experimental human cryptosporidiosis correlates with prior sensitization and control of oocyst excretion.

To investigate the role of interferon (IFN)-gamma in human cryptosporidiosis, jejunal biopsies from experimentally infected volunteers and chronically infected AIDS patients were examined for IFN-gamma expression by in situ hybridization. IFN-gamma expression was compared with oocyst excretion, baseline serum anti-Cryptosporidium antibody, and symptoms. IFN-gamma mRNA was detected in biopsies from 13 of 26 volunteers after experimental infection but not in biopsies taken before C. parvum exposure or in biopsies from patients with AIDS-associated cryptosporidiosis. After challenge, 9 of 10 volunteers with baseline C. parvum antibody produced IFN-gamma, compared with 4 of 16 volunteers without baseline antibody (P<.01). Furthermore, IFN-gamma mRNA was detected in 9 of 13 volunteers who did not excrete oocysts, compared with 4 of 13 with organisms (P<.05). Thus, expression of IFN-gamma in the jejunum was associated with prior sensitization and absence of oocyst shedding. IFN-gamma production may explain the resistance to infection noted in sensitized persons but may not be involved in control of human primary 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.

Infectivity of Cryptosporidium parvum in healthy adults with pre-existing anti-C. parvum serum immunoglobulin G.

A 50% infectious dose (ID50) of 132 Cryptosporidium parvum oocysts was previously determined in serologically negative individuals (ELISA). In this study, 17 healthy adults with pre-existing anti-C. parvum serum IgG were challenged with 500-50,000 oocysts. Infection and diarrhea were associated with the higher challenge doses. The ID50 was 1,880 oocysts, > 20-fold higher than in seronegative volunteers. Fecal oocysts were detected in only seven (53.8%) of 13 individuals with clinical cryptosporidiosis, indicating that the host response may effectively decrease the number of oocysts produced. Subjects with the highest absorbances prior to challenge had little to no increase in IgG following challenge, whereas volunteers with lower reactivities showed significant postchallenge increases. This suggests that an upper limit of serum IgG was present in some subjects, while others were further stimulated by an additional exposure. These data indicate that prior exposure to C. parvum provides protection from infection and illness at low oocyst doses.

Sequence polymorphism in the beta-tubulin gene reveals heterogeneous and variable population structures in Cryptosporidium parvum.

Restriction fragment length polymorphism (RFLP) analysis of isolates of Cryptosporidium parvum has revealed two subgroups, termed H and C. The limited resolution of the RFLP method precludes an in-depth study of the genetic structure of C. parvum populations. Published C. parvum restriction polymorphisms lie within protein-coding regions known to be more homogeneous than noncoding sequences. To better assess the degrees of heterogeneity between and within C. parvum isolates, sequence polymorphism in the beta-tubulin intron, the only C. parvum intron described to date, was investigated. In contrast to the two genotypes distinguished by multilocus RFLP, several alleles were detected by sequence and RFLP analysis of the beta-tubulin intron and adjacent exon 2. Isolates carrying different beta-tubulin alleles were found. Significantly, one of the beta-tubulin alleles present in two geographically unrelated isolates combined features of C- and H-type isolates, suggesting that it might have arisen from a recombination event. A comparison of multiple samples of a calf-propagated laboratory isolate showed that the ratio of different beta-tubulin alleles fluctuated during serial passage.

The antibody response to 27-, 17-, and 15-kDa Cryptosporidium antigens following experimental infection in humans.

Previous studies have suggested that persons infected with Cryptosporidium parvum develop antibody responses to 27-, 17-, and 15-kDa C. parvum antigens. Studies of volunteers infected with Cryptosporidium species provided an opportunity to evaluate the relationship between antibody reactivity to these antigens and infection outcome. As monitored by immunoblot, increases in specific antibody reactivity were more prevalent among volunteers who developed signs and symptoms of cryptosporidiosis (n = 11) than among asymptomatic infected (n = 7; P = .05) or oocyst-negative volunteers (n = 11; P = .02). Volunteers with preexisting IgG antibody to the 27-kDa antigen excreted fewer oocysts than volunteers without this antibody (P = .003). IgG reactivity to the 17-kDa antigens and IgM reactivity to the 27-kDa antigens were higher at day 0 for asymptomatic infected persons than for those who developed symptoms (P = .03 and P = .04, respectively). These results suggest that characteristic antibody responses develop following C. parvum infection and that persons with preexisting antibodies may be less likely to develop illness.

Combination drug therapy for cryptosporidiosis in AIDS.

Aside from effective antiretroviral therapy, there is no consistently effective antiparasitic therapy for cryptosporidiosis in AIDS. The purpose of this study was to assess safety, efficacy, and durability of combination therapy with paromomycin and azithromycin for chronic cryptosporidiosis. Patients with AIDS, chronic cryptosporidiosis, and < 100 CD4 cells/microL were treated with open-label paromomycin (1.0 g twice a day) plus azithromycin (600 mg once a day) for 4 weeks, followed by paromomycin alone for 8 weeks. In 11 patients, median stool frequency decreased from 6.5/day (baseline) to 4.9/day (week 4) and 3.0/day (week 12). Median reductions in 24-h oocyst excretion were 84%, 95%, and >99% at 2, 4, and 12 weeks, respectively. None of the responses were attributable to antiretrovirals. Of 5 survivors at 12-30 months of follow-up, 3 remain asymptomatic off medications, and 2 have chronic, mild diarrhea. Treatment of cryptosporidiosis with azithromycin and paromomycin was associated with significant reduction in oocyst excretion and some clinical improvement.

Prophylactic effect of bovine anti-Cryptosporidium hyperimmune colostrum immunoglobulin in healthy volunteers challenged with Cryptosporidium parvum.

Bovine hyperimmune anti-Cryptosporidium colostrum immunoglobulin (BACI) decreases the intensity of Cryptosporidium parvum infection in vitro. We investigated the prophylactic effect of BACI in healthy adults challenged with C. parvum. After we established an oocyst dose that resulted in 100% infection in four volunteers (baseline group), 16 volunteers were randomized to receive (1) BACI prior to C. parvum challenge (BACI group) and a nonfat milk placebo 30 minutes later, (2) BACI prior to and 30 minutes after challenge (reinforced BACI group), or (3) nonfat milk placebo prior to and 30 minutes after challenge. Subjects received BACI (10 g) or nonfat milk placebo three times a day for a total of 5 days and were followed for clinical symptoms and oocyst excretion for 30 days. A trend toward less diarrhea (P = .08) was observed for subjects receiving BACI in comparison with occurrences in placebo recipients. Subjects receiving BACI or nonfat milk placebo had a 100-fold reduction in oocyst excretion as compared with excretion in the baseline group.

Susceptibility and serologic response of healthy adults to reinfection with Cryptosporidium parvum.

Healthy adults are susceptible to infection with small numbers of Cryptosporidium parvum oocysts, resulting in self-limited infection. We investigated if infection of humans with C. parvum is protective 1 year after primary exposure. At 1 year after a primary challenge with 30 to 10(6) oocysts, 19 healthy immunocompetent adults were rechallenged with 500 oocysts and monitored for the development of infection and/or illness. Oocyst excretion was quantitated by direct immunofluorescence with a C. parvum-specific monoclonal antibody, and anti-C. parvum antibodies in serum were detected by an enzyme-linked immunosorbent assay. Fewer subjects shed oocysts after the second exposure (3 of 19; 16%) than after the first exposure (12 of 19; 63%) (P < 0.005). Although the rates of diarrhea were comparable after each of the two exposures, the clinical severity as determined by the mean number of unformed stools passed was lower after reexposure (11.25 versus 8.62; P < 0.05). The number of anti-Cryptosporidium immunoglobulin G and A seroconversions increased after secondary exposure. However, the C. parvum serum antibody response did not correlate with the presence or absence of infection.