Method detection limits of PCR and immunofluorescence assay for Cryptosporidium parvum in soil

We determined and compared the method detection limits (MDLalpha) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDLalpha and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.     

Cryptosporidium parvum in patients with chronic diarrhea and AIDS: diagnosis by means of indirect immunofluorescence with monoclonal antibodies

The diagnosis of cryptosporidiosis is difficult when oocyst elimination is poor as occurs in AIDS patients. Aiming to improve the diagnosis, 144 fecal samples coming from AIDS patients with diarrhea, were studied using indirect immunofluorescence with anti-Cryptosporidium monoclonal antibodies. The results were compared with Ziehl Neelsen and safranine stainings. Twenty three samples (15.9%) were positive for Cryptosporidium with at least one of the three methods. Sensitivities were 78.3% for immunofluorescence, 86.9% for Ziehl Neelsen and 91.3 for safranine stainings. The specificity of the three methods was 100%. It is concluded that immunofluorescence does not improve the diagnostic accuracy of cryptosporidiosis and its high cost precludes its use in routine laboratories.     

Simple and rapid measurement of Cryptosporidium excystation using flow cytometry

In vitro excystation is commonly used to determine the viability of samples of purified Cryptosporidium parvum oocysts. Following exposure to conditions that stimulate excystation, samples are examined microscopically to determine the number of excysted oocysts. The microscopy procedure is tedious and time consuming, and difficult to apply to most oocyst samples without a purification step. A simple flow cytometric method was developed for determining the numbers of oocysts that had excysted following the in vitro excystation procedure. Differences in light-scatter properties were used to differentiate intact, partially empty and empty oocysts. By staining samples with a monoclonal antibody specific to the oocyst wall it was possible to apply the technique to unpurified oocysts from faeces. Correlation of the flow cytometric and microscopic method was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. The flow cytometry method is faster and more sensitive than the microscopy procedure, and enables analysis of large numbers of samples and of many thousands of oocysts in each sample.     

Enzyme-linked immunosorbent assay for the detection of Cryptosporidium parvum IgG in the serum of cats

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium parvum IgG in the serum of cats. The ELISA was an indirect ELISA using soluble C. parvum oocyst antigens and a peroxidase-labeled anti-feline IgG secondary antibody. Sera from cats with Toxocara felis, Giardia spp., Aelurostrongylus abstrusus, Isospora felis, Isospora rivolta, Toxoplasma gondii, or Taenia spp. infections were assayed in specificity studies. Following optimization, the ELISA and fecal examination for oocysts were performed on samples from 170 client-owned or humane society source cats and 1 cat inoculated orally with C. parvum oocysts. Cryptosporidium parvum oocysts were detected in feces (4/170; 2.4%), and C. parvum IgG was detected in serum (26/170; 15.3%) from naturally exposed cats. The seroprevalence data suggest that some cats in the geographical area studied were exposed to C. parvum, but persistent oocyst shedding was less common. The ELISA is not useful for predicting oocyst shedding in individual cats.     

Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum

We have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.     

Analyte and label binding assay read by flow cytometry

A new immunometric two-site sandwich assay is introduced, in which a label-scavenging binding partner is added to the sample in addition to the analyte-binding partner. The scavenger binding partner binds excess label antibody, giving a signal proportional to the amount of excess label antibody in the sample solution. A set of two calibration curves is obtained from the two binding partners simultaneously, and a combination of the two signals gives an unambiguous determination of the analyte concentration, even for high analyte concentrations where the hook effect may occur. Two-particle immunofluorometric assays developed for placental alkaline phosphatase and human chorionic gonadotropin on the basis of this principle and yielding signals measured by flow cytometry gave rapid results (2 h) and had working ranges in excess of 5 and 6 orders of magnitude for the respective analytes.     

Detection of intracellular cytokines by flow cytometry

During the last years it has become increasingly clear that production of most cytokines is not confined to one cell type. Thus, a method to detect cytokines at the single cell level would be a helpful tool to study the contribution of different cells to cytokine production in heterogeneous cell populations. Recently, Sander et al. (1991) demonstrated that it is possible to detect intracellular cytokines by fixation with paraformaldehyde, permeabilization with saponin and subsequent indirect immunofluorescent staining using fluorescence microscopy. Here, we describe a modified method to increase the specific intracellular staining which enables us to detect IFN-gamma, IL-2 and IL-4 producing cells by single laser flow cytometry. The carboxylic ionophore monensin was used to interrupt intracellular transport processes leading to an accumulation of the cytokine in the Golgi complex. This resulting increase of the signal/noise ratio permitted us to detect weakly fluorescent cells such as IL-4 producing cells. While IL-4 was detected in approximately 1-3% of peripheral mononuclear cells from healthy donors, up to 30% of the cells produced IFN-gamma and nearly 50% IL-2 after phorbol ester and ionomycin stimulation. Microscopic and flow cytometric analysis showed a highly significant correlation. Using three-color flow cytometry it was possible to measure intracellular cytokines and cell surface markers simultaneously. Subpopulations of human T cells (e.g., CD4+ CD45R0-) producing a restricted cytokine pattern could be identified by cell surface staining and were characterized by their cytokine production. Consequently, there was no further need for cell sorting to determine cytokine producing subsets in heterogeneous cell populations. We have tested human T cell clones for intracellular cytokine production and found a high concordance to ELISA analysis of the supernatants. We conclude that detection of intracellular cytokines by flow cytometry is a rapid, easy and semiquantitative assay which may be used to study individual cells in heterogeneous populations as well as to screen homogeneous cells for their cytokine pattern. This method is particularly relevant in view of the accumulating evidence of the functional role that subsets of (T) cells may play in various diseases depending on the pattern of cytokines they produce.     

Detection of myeloperoxidase by flow cytometry in acute leukemia

BACKGROUND: To evaluate long-term results of intraocular pressure after trabeculectomy for congenital glaucoma. METHODS: Data concerning 55 eyes (30 patients) who underwent trabeculectomy for congenital glaucoma were recorded. Mean age at diagnosis was 3.4 months (range: 2 days to 10 months). Mean follow up was 56.8 months. Associated anterior segment abnormalities, need for one or more new trabeculectomy procedures during follow up, and intraocular pressure at the last examination were noted. RESULTS: Of the 55 eyes, 48 met the success criteria (87.3%). A second and sometimes third or fourth trabeculectomy were necessary during follow up in 17 eyes (31%). Of the seven failures at final examination, six (85%) had been diagnosed and operated on before the age of 1 month, whereas 15 of the 48 eyes with good results (31.2%) were in this group (p < 0.02). Of the seven failures at final examination, six (85%) were operated on two to four times, whereas 10 of the 48 eyes with good results (20.1%) were in this group (p < 0.01). An associated anterior segment abnormality was present in 13 eyes (23%), and did not seem to influence the final outcome. CONCLUSION: Trabeculectomy is an effective procedure for long-term control of intraocular pressure in congenital glaucoma. The early diagnosis and surgical treatment correspond to a poor long-term prognosis, probably related to initially severe cases. In these cases, intraocular pressure is difficult to control despite repeated surgical procedures.     

Hydrophobic and electrostatic cell surface properties of Cryptosporidium parvum

The oocyst wall of Eimeria spp. consists of a 10-nm-thick outer lipid layer and a 90-mm-thick inner layer of glycoprotein which has been described previously to be composed of a single major protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and (125)I labelling of a oocyst wall fragments and of delipidated intact oocysts revealed a molecule of approximately 12 kDa as the major protein component of the oocyst wall of Eimeria tenella. An immunoglobulin M monoclonal antibody (c11B9F3) was produced against this 12-kDa oocyst wall protein sliced from a preparative SDS-polyacrylamide gel. Its reactivity by immunofluorescence against oocyst wall fragments and sporozoites or by immunoperoxidase assays of infected tissue sections was stage restricted to gametocytes and oocysts but pan-specific against all face of the oocyst wall. In chicks passively immunized with C11B9F3, oocyst output was significantly (P<0.01) reduced by 42 to 54% after homologous E. tenella infection and by 35% after heterologous Eimeria maxima infection compared with that of control groups. The results demonstrate the presence of a highly conserved, low-molecular-weight antigen on the oocyst wall and the gametocytes of Eimeria spp. which is a candidate for inclusion in a pan-specific, transmission-blocking vaccine against avian coccidiosis.     

Clinical features of diarrhoea in children caused by Cryptosporidium parvum

A time-series study of suicide and homicide rates in Australia and New Zealand from 1950-85 provided limited support for Durkheim's theory of suicide and no support for Henry and Short's theory of the relationship between suicide and homicide. The need for rival theories is discussed.     

Effects of Cryptosporidium parvum infection on lymphocyte phenotype and reactivity in calves

Cryptosporidium parvum is a protozoan parasite now recognized as a significant cause of neonatal diarrhea in calves, and infection is also widespread in both immunocompetent and immunocompromised humans. No effective treatment or preventive measures against C. parvum infection are available, owing largely to the lack of understanding of immunologic mechanisms of resistance to and recovery from this parasite. In the present study, we compared phenotypes of lymphocytes from peripheral blood, spleen, mesenteric, and prescapular lymph nodes of calves infected or not infected with C. parvum. We also compared reactivity of these lymphocytes to mitogens and C. parvum antigen in vitro. There were more non-T, non-B (null) lymphocytes in all tissues of infected compared with control calves. The percent of CD8+ lymphocytes was significantly increased in spleens of infected compared with control calves, and there were markedly less CD4+ than CD8+ cells in spleens of both groups (i.e. low CD4/CD8 ratios). Splenic lymphocytes showed significantly decreased in vitro proliferation to pokeweed mitogen and C. parvum antigen stimulation compared with lymphocytes from other tissues. These findings suggest that null lymphocytes and CD8+ lymphocytes may be important in the expression and regulation of bovine immune responses to C. parvum in vivo.     

Cryptosporidium parvum: an attempt at experimental infection in rainbow trout Oncorhynchus mykiss

Segmental mediolytic arteriopathy, a rare, noninflammatory arterial disease, is fundamentally a variant of fibromuscular dysplasia. The characteristic angiographic findings of segmental mediolytic arteriopathy include the "string of beads" and microaneurysms which are indistinguishable from those of vasculitis, and the correct diagnosis can be made only after histopathologic evaluation of the arterial lesions. Thrombosis, arterial wall hemorrhage, and dissection are among the complications of segmental mediolytic arteriopathy. We describe herein a patient with segmental mediolytic arteriopathy who presented with hemoperitoneum. The patient underwent urgent surgical repair of a ruptured hepatic artery aneurysm. The postoperative visceral arteriography findings led to a clinical diagnosis of polyarteritis nodosa, and immunosuppressive therapy was initiated. This treatment was stopped as soon as the correct biopsy diagnosis of segmental mediolytic arteriopathy was obtained through outside consultation. The patient recovered without drug treatment and was spared the potentially life-threatening complications of immunosuppression.     

Effects of Cryptosporidium parvum infection in Peruvian children: growth faltering and subsequent catch-up growth

The authors conducted a 2-year (1989-1991) community-based longitudinal study in a shantytown in Lima, Peru, to examine the effect of Cryptosporidium parvum infection on child growth during the year following the onset of infection. A cohort of children, aged 0-3 months at recruitment, was followed monthly for anthropometrics, weekly for stool samples, and daily for diarrheal status. Data from 185 children in the cohort permitted a comparison of growth in C. parvum-infected and noninfected children. The analyses fitted smooth, flexible curves with a linear random-effects model to estimate growth differences between C. parvum-infected and noninfected children. Children infected with C. parvum experienced growth faltering, both in weight and in height, for several months after the onset of infection, followed by a period of catch-up growth. Younger children took longer to catch up in weight than did older children. Catch-up growth, however, did not occur in children infected between ages 0 and 5 months. These children did not catch up in height, and one year after infection they exhibited an average deficit of 0.95 cm (95% confidence interval (CI) 0.38-1.53) relative to noninfected children of similar age. Stunted children who became infected also did not catch up in either weight or height, and one year after infection they exhibited a height deficit of 1.05 cm (95% CI 0.46-1.66) relative to noninfected, stunted children of similar age. These results indicate that Cryptosporidium parvum has a lasting adverse effect on linear (height) growth, especially when acquired during infancy and when children are stunted before they become infected.     

Infectivity of Cryptosporidium parvum oocysts stored in water at environmental temperatures

We showed that the efficacy of the new 2'-deoxycytidine (2'-dCyd) analogue antimetabolite 2'-deoxy-2'-methylidenecytidine (DMDC) correlates well with tumor levels of cytidine (Cyd) deaminase in human cancer xenograft models. DMDC was highly effective in tumors with higher levels of Cyd deaminase, whereas lower levels yielded only slight activity. In contrast, gemcitabine (2',2'-difluorodeoxycytidine), which has action mechanisms similar to those of DMDC, is only slightly active in tumors with higher levels of the enzyme. In the present study, we investigated the roles of Cyd deaminase in the antitumor activity of the two 2'-dCyd antimetabolites in 13 human cancer cell lines. Tetrahydrouridine, an inhibitor of Cyd deaminase, reduced the antiproliferative activity of DMDC (P = 0.0015). Furthermore, tumor cells transfected with the gene of human Cyd deaminase become more susceptible to DMDC both in vitro and in vivo. These results indicate that Cyd deaminase is indeed essential for the activity of DMDC. In contrast, the antiproliferative activity of gemcitabine was increased to some extent by tetrahydrouridine (P = 0.0277), particularly in tumor cell lines with higher levels of Cyd deaminase. This suggests that higher levels of Cyd deaminase may inactivate gemcitabine. Among nucleosides and deoxynucleosides tested, only dCyd, a natural substrate of both Cyd deaminase and dCyd kinase, suppressed the antiproliferative activity of DMDC by up to 150-fold. Because the Vmax/Km of DMDC for dCyd kinase was 8-fold lower than that for dCyd, the activation of DMDC to DMDC monophosphate (DMDCMP) by dCyd kinase might be competitively inhibited by dCyd. In addition, the dCyd concentrations in human cancer xenografts were inversely correlated with levels of Cyd deaminase activity. It is therefore suggested that higher levels of Cyd deaminase reduce the intrinsic cellular concentrations of dCyd in tumors, resulting in efficient activation of DMDC to DMDCMP by dCyd kinase. These results indicate that the efficacy of DMDC may be predicted by measuring the activity of Cyd deaminase in tumor tissues before treatment starts and that DMDC may be exploited in a new treatment modality: tumor enzyme-driven cancer chemotherapy.     

Effect of pasteurization on infectivity of Cryptosporidium parvum oocysts in water and milk

Cryptosporidium parvum is a major cause of diarrheal disease in humans and has been identified in 78 other species of mammals. The oocyst stage, excreted in feces of infected humans and animals, has been responsible for recent waterborne outbreaks of human cryptosporidiosis. High temperature and long exposure time have been shown to render oocysts (suspended in water) noninfectious, but for practical purposes, it is important to know if high-temperature--short-time conditions (71.7 degrees C for 15 s) used in commercial pasteurization are sufficient to destroy infectivity of oocysts. In this study, oocysts were suspended in either water or whole milk and heated to 71.7 degrees C for 15, 10, or 5 s in a laboratory-scale pasteurizer. Pasteurized and nonpasteurized (control) oocysts were then tested for the ability to infect infant mice. No mice (0 of 177) given 10(5) oocysts pasteurized for 15, 10, or 5 s in either water or milk were found to be infected with C. parvum on the basis of histologic examination of the terminal ileum. In contrast, all (80 of 80) control mice given nonpasteurized oocysts were heavily infected. These data indicate that high-temperature--short-time pasteurization is sufficient to destroy the infectivity of C. parvum oocysts in water and milk.     

Detection of intracellular antigens by flow cytometry: comparison of two chemical methods and microwave heating

Detection of intracellular antigens by flow cytometry requires effective fixation and permeabilization of the cell membrane. This study compares three fixation/permeabilization techniques: two commercial chemical reagents, the ORTHOPermeaFix (OPF) and the FIX&PERM Cell Permeabilization Kit (F&P), and a novel method based on microwave heating (MWH). They have been applied to the detection of two nuclear (p53 and rb/p105) and two cytoplasmic (bcl-2 and mdr-1/gp-170) antigens, using positive- and negative-control cell lines and peripheral blood mononuclear cells. Western blotting was performed as a control of protein expression. For the four antigens assessed, cellular morphology, discrimination between intact cells and debris, percentage of positive cells, and mean fluorescence intensity were examined. For this last parameter, the assessment of the MWH technique was performed using SD and a graphical approach inspired by the concepts described by Bland and Altman (Lancet 1986;346: 1085-7) as well as Petersen et al. (Clin Chem 1997;43: 2039-46). The statistical analysis shows that MWH is comparable to the commercial methods and that its reproducibility is also equivalent to OPF and F&P. As assessed for some of the most clinically relevant intracytoplasmic and intranuclear antigens, the MWH method appears to be a valuable and inexpensive alternative. It is worth noting that, unlike commercial reagents, MWH altered surface antigens. Interestingly, this feature, which would prevent cell selection on the basis of combined membrane and intracellular epitopes, is associated with a decrease of nonspecific background fluorescence.     

Genotypic and phenotypic characterization of Cryptosporidium parvum isolates from people with AIDS

Genotypic analysis of Cryptosporidium parvum has demonstrated the presence of two subgroups within the species, whereas biochemical and antigenic characterization have shown more heterogeneity. The clinical relevance of these observations is unknown. C. parvum isolates from people with AIDS were studied with respect to parasite genotypes and virulence in cell monolayers and laboratory animals. Ten of 13 oocyst samples had a characteristic human-associated (H) genotype; 3 had a genotype typical of calf-excreted oocysts (C). Virulence in cell culture was mildly or markedly lower in the 5 isolates tested (4 H and 1 C) compared with the GCH1 reference isolate. H isolates did not infect newborn ICR mice, whereas 1 of the 2 C isolates tested did. These findings reinforce the concept of C. parvum genetic subgroupings that correlate to some extent with infectivity and suggest that additional heterogeneity is present within the subgroups.