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ABSTRACT: 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.
Parasitology 02/2002; 124(Pt 1):1-7. · 2.96 Impact Factor
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ABSTRACT: 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.
The current study was undertaken to estimate the risk of HCC in relation to HCV in Egypt.
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.
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.
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.
Journal of Clinical Gastroenterology 09/2001; 33(2):123-6. · 3.16 Impact Factor
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ABSTRACT: 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.
The American journal of tropical medicine and hygiene 09/2001; 65(3):257-60. · 2.59 Impact Factor
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ABSTRACT: 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.
Clinical and Diagnostic Laboratory Immunology 07/2001; 8(4):724-30. · 2.51 Impact Factor
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Clinical Infectious Diseases 06/2001; 32(10):1516-7. · 9.15 Impact Factor
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AIDS 05/2001; 15(6):802-4. · 6.24 Impact Factor
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ABSTRACT: 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.
Infection and Immunity 03/2001; 69(2):1172-4. · 4.16 Impact Factor
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ABSTRACT: 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.
Infection and Immunity 10/2000; 68(9):5405-7. · 4.16 Impact Factor
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ABSTRACT: 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.
Infection and Immunity 09/2000; 68(9):5068-74. · 4.16 Impact Factor
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ABSTRACT: 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.
The Journal of Infectious Diseases 02/2000; 181(2):701-9. · 6.41 Impact Factor
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ABSTRACT: 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.
The Journal of Infectious Diseases 11/1999; 180(4):1275-81. · 6.41 Impact Factor
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ABSTRACT: 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.
The American journal of tropical medicine and hygiene 01/1999; 60(1):157-64. · 2.59 Impact Factor
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ABSTRACT: 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.
The Journal of Infectious Diseases 10/1998; 178(3):827-33. · 6.41 Impact Factor
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ABSTRACT: 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.
The Journal of Infectious Diseases 09/1998; 178(3):900-3. · 6.41 Impact Factor
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ABSTRACT: 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.
Clinical Infectious Diseases 07/1998; 26(6):1324-9. · 9.15 Impact Factor
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ABSTRACT: 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.
Infection and Immunity 02/1998; 66(2):441-3. · 4.16 Impact Factor
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ABSTRACT: Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection.
Journal of Clinical Microbiology 09/1997; 35(8):2013-7. · 4.15 Impact Factor
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ABSTRACT: Cryptosporidiosis is an important cause of diarrhea. We identified 95 patients with cryptosporidiosis over a 6-year period in our county hospital system, including 9 children and 86 adults infected with the human immunodeficiency virus (HIV). Risk factors included male-to-male sexual practices and Hispanic race. Diarrhea, weight loss, and gastrointestinal complaints were the most common symptoms at presentation. Among the HIV-infected adults, 20 (23%) developed biliary tract disease. Biliary involvement was associated with low CD4 counts. Treatment with paromomycin and antimotility agents was effective in reducing diarrheal symptoms in 54 of 70 (77%) patients with the acquired immunodeficiency syndrome (AIDS), although there was a high rate of relapse. Paromomycin did not prevent the development of biliary disease. Biliary disease responded to cholecystectomy or sphincterotomy with stent placement. Though often a cause of morbidity, cryptosporidiosis was only rarely the cause of death, even among patients with HIV. Cryptosporidiosis continues to be an important medical problem even in developed-countries. Current methods of prevention and treatment are suboptimal.
Medicine 04/1997; 76(2):118-39. · 4.35 Impact Factor
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ABSTRACT: Cryptosporidium parvum arginine aminopeptidase (RAP) was studied during in vitro excystation. Specific RAP inhibitors were identified by using C. parvum extracts. Amastatin, a series of alpha-aminoboronic acids, and the chelating agents EDTA and 1,10-phenanthrolene, but not endoproteinase inhibitors, blocked enzymatic activity. RAP inhibitors found to be effective in soluble enzymatic assays were then studied for their effect on in vitro excystation. 1,10-Phenanthrolene, amastatin, and H-boronorleucine (pinacol) inhibited excystation by 84, 57, and 61%, respectively, compared with solvent-treated control oocysts. Sporozoites remained viable within the oocyst as determined by propidium iodide and fluorescein diacetate dye uptake, suggesting that alpha-aminoboronic acids were not directly lethal to the parasite.
Antimicrobial Agents and Chemotherapy 01/1997; 40(12):2781-4. · 4.84 Impact Factor
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ABSTRACT: Data about human Cryptosporidium parvum infection have originated from travelers, community and day care center outbreaks, and persons infected with the human immunodeficiency virus. In addition, experimental infection in 29 antibody-negative, healthy, adult volunteers generated information on the dose-infection response of C. parvum (Iowa strain). In that report, low inocula were sufficient to cause infection in 18 and illness in 7 persons. To further define the duration and intensity of infection in this population, oocyst shedding patterns were investigated in the 18 subjects infected with C. parvum. Oocyst quantitation revealed that volunteers with diarrheal illness (n = 7) excreted more oocysts over the course of the infection than did volunteers without diarrhea (n = 11; P < .05). Symptomatic subjects were more likely to shed oocysts on consecutive days. Further, a statistical nonsignificant inverse trend (r2 = .330, P = .136) was seen between challenge dose and total excreted oocysts. This paradox may relate to receptor saturation or a toxic effect on cells, parasites, or both afforded by a high inoculum.
The Journal of Infectious Diseases 01/1996; 173(1):232-6. · 6.41 Impact Factor
