Optimization of the Urea Linker of Triazolopyridazine MMV665917 Results in a New Anticryptosporidial Lead with Improved Potency and Predicted hERG Safety Margin.

Cryptosporidiosis is caused by infection of the small intestine by Cryptosporidium parasites, resulting in severe diarrhea, dehydration, malabsorption, and potentially death. The only FDA-approved therapeutic is only partially effective in young children and ineffective for immunocompromised patients. Triazolopyridazine MMV665917 is a previously reported anti-Cryptosporidium screening hit with in vivo efficacy but suffers from modest inhibition of the hERG ion channel, which could portend cardiotoxicity. Herein, we describe our initial development of structure-activity relationships of this novel lead series with a particular focus on optimization of the piperazine-urea linker. We have discovered that piperazine-acetamide is a superior linker resulting in identification of SLU-2633, which has an EC50 of 0.17 µM, an improved projected margin versus hERG, prolonged pharmacokinetic exposure in small intestine, and oral efficacy in vivo with minimal systemic exposure. SLU-2633 represents a significant advancement toward the identification of a new effective and safe treatment for cryptosporidiosis.

The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches.

Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human ß-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human ß-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.

Efficiency of chlorine and UV in the inactivation of Cryptosporidium and Giardia in wastewater.

Wastewater from different sources is contaminated by protozoan parasites including Cryptosporidium and Giardia. Many protozoan parasites are becoming resistant to chemical treatment. The challenge of finding alternatives is presented to researchers by exploring other methods of eliminating protozoan parasites from wastewater. The aim of this study was to assess the speciation and the viability of Cryptosporidium and Giardia in environmental samples with the specific objective of evaluating if effluent chlorination and UV affect the viability. Different doses of chlorine with different exposure times were experimented with both distilled water and waste water spiked with (oo)cysts derived from environmental samples. UV irradiation at different doses was also experimented using the same spiked samples. Two methods of quantification and detection, namely, microscopy and flow cytometry, were used in the experiment. Two vital dyes, Syto-9+PI and DAPI+PI, were the used for staining the collected wastewater samples. It was found that the (oo)cysts responded to chlorination and UV treatments with Giardia responding better than Cryptosporidium. Giardia responded very well to UV irradiations with almost 0 percent remaining viable after a low dose of UV. Cryptosporidium was found to be resistant to chlorination even at high doses but responded well to high UV doses. DAPI+PI dye gave a lower mean percentage viability values than Syto-9+PI. Flow cytometry gave higher mean percentage than microscopy from the results. It is concluded that UV is a promising alternative to Chlorine in removing Cryptosporidium and Giardia from waste water. Appropriate treatment method for wastewater is necessary to minimize water resources pollution when wastewater is released into water systems.

A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.

Treatment of cryptosporidiosis in captive green iguanas (Iguana iguana).

There are no standard guidelines for the treatment of cryptosporidiosis in reptiles. The aim of this study was to evaluate the efficacy of two cryptosporidiosis therapies in captive green iguanas. Eight green iguanas aged 2-6 years, including 6 (1 ♂ and 5 ♀) animals with chronic diarrhea, received treatment for cryptosporidiosis. The presence of Cryptosporidium sp. oocysts was determined in 8 iguanas (100%), Isospora sp. oocysts were detected in 3 animals (37.5%), and Oxyuridae eggs were observed in 5 iguanas (62.5%). The animals were divided into two therapeutic groups (A and B). Group A iguanas were administered halofuginone (Halocur, 0,50 mg/ml Intervet Productions S.A., France) at a dose of 110 mg/kg body weight (BW) every 7 days for 5 weeks. Group B animals were administered sulfadiazine and trimethoprim (Norodine Vet Oral Paste sulfadiazine 288,3 mg/g, trimethoprim 58 mg/g, ScanVet Animal Health A/S, Denmark) at 75 mg/kg BW per os every 5 days for 5 weeks and spiramycin and metronidazole (Stomorgyl, spiramycin 1500000 IU, metronidazole 250 mg, Merial, France) at 200 mg/kg BW every 5 days for 5 weeks. Both groups received hyperimmune bovine colostrum and subcutaneous fluids. Before treatment, the average number of Cryptosporidium sp. oocysts in 1 g of feces was determined at 1.71 * 105 (±313,262.44) in group A and 1.56 * 105 (±262,908.53) in group B; the average number of Isospora sp. oocysts was determined at 3.53 * 103 (±1747.38), and the average number of Oxyuridae eggs was determined at 810 (±496.74). Blood tests were performed once before treatment. The results of blood morphology and biochemistry tests before treatment revealed leukocytosis with a significant increase in heterophile and monocyte counts in all animals. Dehydration, elevated hematocrit values and low levels of Na+, Ca2+, PO4- and Cl- ions were observed in 6 iguanas. Two iguanas died during treatment. The gross necropsy revealed acute inflammation of gastric and duodenal mucosa, mucosal ecchymoses in the gastrointestinal tract, hepatomegaly and liver congestion, cholecystitis, enlarged kidneys and renal edema and congestion, cystitis, and an absence of fat bodies. Parasites were not detected in any developmental form after 40 days of therapy and during an monthly 18-month follow-up period. Effective treatment of cryptosporidiosis in reptiles minimizes the adverse consequences of disease, improves the animals' well-being and decreases euthanasia rates.

A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis.

Diarrhoeal disease is responsible for 8.6% of global child mortality. Recent epidemiological studies found the protozoan parasite Cryptosporidium to be a leading cause of paediatric diarrhoea, with particularly grave impact on infants and immunocompromised individuals. There is neither a vaccine nor an effective treatment. Here we establish a drug discovery process built on scalable phenotypic assays and mouse models that take advantage of transgenic parasites. Screening a library of compounds with anti-parasitic activity, we identify pyrazolopyridines as inhibitors of Cryptosporidium parvum and Cryptosporidium hominis. Oral treatment with the pyrazolopyridine KDU731 results in a potent reduction in intestinal infection of immunocompromised mice. Treatment also leads to rapid resolution of diarrhoea and dehydration in neonatal calves, a clinical model of cryptosporidiosis that closely resembles human infection. Our results suggest that the Cryptosporidium lipid kinase PI(4)K (phosphatidylinositol-4-OH kinase) is a target for pyrazolopyridines and that KDU731 warrants further preclinical evaluation as a drug candidate for the treatment of cryptosporidiosis.

Efficacy of nitazoxanide to treat natural Giardia infections in dogs.

BACKGROUND: Giardia parasites cause gastrointestinal disease in humans, dogs, and many other animals worldwide. The treatment of dogs for giardiasis requires further investigation to ascertain levels of drug efficacy and the possibility of adverse side effects. Nitazoxanide (NTZ) has shown good clinical anti-Giardia activity in humans, yet it has not been evaluated for the treatment of giardiasis in dogs. METHODS: Thirty-five dogs, naturally infected with Giardia were divided into five groups (n = 7): dogs in group NTZ1, NTZ2, and NTZ3 were treated with a single oral dose of 37.5 mg/kg, 75 mg/kg, and 150 mg/kg, respectively, of NTZ on days 0 and 14. The fourth group was treated with a commercially available regimen that includes a combination of pyrantel, praziquantel, and febantel (FEB) administered orally for three consecutive days. Additionally, an untreated control group was established. Giardia cysts from the stool of each dog were quantified on days -3, 0, 5, 7, 9, 11, 14, 18, 25, and 28. Biochemical parameters were evaluated in all dogs, before the first treatment and after concluding the experiment. RESULTS: Shedding of Giardia cysts was reduced in all treated groups when compared to untreated controls (P < 0.01). However, NTZ2, NTZ3, and FEB had a lower risk during the study. Furthermore, NTZ was also effective against another protozoan, Cryptosporidium spp. at doses of 75 mg/kg and 150 mg/kg, in contrast to the combination of febantel + pyrantel + praziquantel. Biochemical parameters of treated animals, namely, aspartate transaminase and alanine transaminase enzymes, remained within physiological ranges. CONCLUSIONS: Based on these results, the implementation of NTZ as a treatment for giardiasis in dogs is proposed. The administration of a single dose is an important advantage of NTZ because it reduces workload, particularly in animals placed in shelters and kennels, where handling of large numbers of animals is required, and personnel is frequently scarce.

Glycoproteins and Gal-GalNAc cause Cryptosporidium to switch from an invasive sporozoite to a replicative trophozoite.

The apicomplexan parasite Cryptosporidium causes cryptosporidiosis, a diarrheal disease that can become chronic and life threatening in immunocompromised and malnourished people. There is no effective drug treatment for those most at risk of severe cryptosporidiosis. The disease pathology is due to a repeated cycle of host cell invasion and parasite replication that amplifies parasite numbers and destroys the intestinal epithelium. This study aimed to better understand the Cryptosporidium replication cycle by identifying molecules that trigger the switch from invasive sporozoite to replicative trophozoite. Our approach was to treat sporozoites of Cryptosporidium parvum and Cryptosporidium hominis, the species causing most human cryptosporidiosis, with various media under axenic conditions and examine the parasites for rounding and nuclear division as markers of trophozoite development and replication, respectively. FBS had a concentration-dependent effect on trophozoite development in both species. Trophozoite development in C. parvum, but not C. hominis, was enhanced when RPMI supplemented with 10% FBS (RPMI-FBS) was conditioned by HCT-8 cells for 3h. The effect of non-conditioned and HCT-8 conditioned RPMI-FBS on trophozoite development was abrogated by proteinase K and sodium metaperiodate pretreatment, indicating a glycoprotein trigger. Cryptosporidium parvum and C. hominis trophozoite development also was triggered by Gal-GalNAc in a concentration-dependent manner. Cryptosporidium parvum replication was greatest following treatments with Gal-GalNAc, followed by conditioned RPMI-FBS and non-conditioned RPMI-FBS (P<0.05). Cryptosporidium hominis replication was significantly less than that in C. parvum for all treatments (P<0.05), and was greatest at the highest tested concentration of Gal-GalNAc (1mM).

Cryptosporidium infection after renal transplantation in an endemic area.

BACKGROUND: Cryptosporidium is one of the common causes of infective diarrhea in post-transplant patients in endemic areas. However, data are limited on Cryptosporidium infection in recipients of solid organ transplantation. The aim of this study was to determine the incidence, disease manifestation, management, and outcome of Cryptosporidium infection in living-donor renal transplant recipients (RTR). METHODS: We performed a detailed retrospective review of the data on all RTR who had diarrheal illness requiring evaluation and hospitalization, and Cryptosporidium infection. RESULTS: During the study period, 119/1235 (8.98%) RTR developed diarrhea, and Cryptosporidium was found in 34/119 (28.5%). Nine of 680 (1.3%) patients were on a cyclosporine (CSA)-based regimen, and 25/643 (3.8%) patients were on a tacrolimus (Tac)-based regimen. The relative risk of developing Cryptosporidium infection was lower on the CSA-based regimen, compared with the Tac-based regimen (odds ratio [OR]: 0.35, 95% confidence interval [CI]: 0.17-0.72, P = 0.003). Twelve of the 34 patients had acute graft dysfunction, mainly caused by combined Tac toxicity and dehydration. Mean serum creatinine and trough Tac level were 2.04 ± 0.65 mg/dL and 8.24 ± 1.19 ng/dL, respectively. Nitazoxanide alone was used in 13 patients, and nitazoxanide in combination with fluoroquinolone in 21 patients, with duration of treatment ranging from 16 to 60 days. Tac was changed to CSA in 8/11 patients. The clearance of cysts and response to nitazoxanide alone were significantly lower, compared with combination therapy (61.53% vs. 95.23%, P = 0.01, 38.46 vs. 85.71%, P = 0.004, respectively). The OR for cyst clearance and response was also significantly lower with nitazoxanide alone, in comparison with combination therapy (OR: 0.65, 95% CI: 0.34-0.92, P = 0.01, OR: 0.45, 95% CI: 0.21-0.81, respectively). Four (16%) of 24 patients with response had relapse. CONCLUSION: Patients with Tac and mycophenolate mofetil combination therapy had a significantly high risk of Cryptosporidium infection. Cryptosporidial infection may require prolonged nitazoxanide therapy, either alone or in combination, with or without reduction in immunosuppression.

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer(®) peptides.

Cryptosporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitazoxanide, only partly efficacious in immunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Cryptosporidium parvum (IMPcp) and Cryptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C. parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function.

Identification and characterization of a novel calcium-activated apyrase from Cryptosporidium parasites and its potential role in pathogenesis.

Herein, we report the biochemical and functional characterization of a novel Ca(2+)-activated nucleoside diphosphatase (apyrase), CApy, of the intracellular gut pathogen Cryptosporidium. The purified recombinant CApy protein displayed activity, substrate specificity and calcium dependency strikingly similar to the previously described human apyrase, SCAN-1 (soluble calcium-activated nucleotidase 1). CApy was found to be expressed in both Cryptosporidium parvum oocysts and sporozoites, and displayed a polar localization in the latter, suggesting a possible co-localization with the apical complex of the parasite. In vitro binding experiments revealed that CApy interacts with the host cell in a dose-dependent fashion, implying the presence of an interacting partner on the surface of the host cell. Antibodies directed against CApy block Cryptosporidium parvum sporozoite invasion of HCT-8 cells, suggesting that CApy may play an active role during the early stages of parasite invasion. Sequence analyses revealed that the capy gene shares a high degree of homology with apyrases identified in other organisms, including parasites, insects and humans. Phylogenetic analysis argues that the capy gene is most likely an ancestral feature that has been lost from most apicomplexan genomes except Cryptosporidium, Neospora and Toxoplasma.

Eradication of cryptosporidium from a defunctionalized colon limb by refeeding stoma effluent.

Over the last 40 years, cryptosporidium has increasingly been recognized as a cause of acute self-limiting diarrhea in normal hosts. In the immunocompromised patient, cryptosporidium may cause severe illness with prolonged diarrhea and malabsorption. Pharmacologic therapy of cryptosporidium relies on adequate delivery of drug metabolites to the colon. Here we describe a patient who developed toxic megacolon during induction therapy for leukemia, requiring ileostomy formation to proceed with leukemia treatment. Although the megacolon resolved promptly, the resulting isolation of the colon from the fecal stream prevented luminal delivery of active metabolites of anti-protozoal drugs, resulting in persistent cryptosporidiosis. Refeeding of the ileostomy output into the colon effectively eradicated cryptosporidium from the colon and permitted closure of the ileostomy.

Characterization of subtilase protease in Cryptosporidium parvum and C. hominis.

Cryptosporidium spp., enteropathogens of humans and other animals, are members of the Apicomplexa. In parasites belonging to this phylum, proteases have been shown to play a key role in the invasion of host cells, organelle biogenesis, and intracellular survival. The subtilases constitute a family of serine proteases present in prokaryotes, eukaryotes, and viruses. The C. parvum subtilase gene, CpSUB1, encodes a transcript of 3972 base pairs (bp) and 1324 amino acids. Using homologous polymerase chain reaction primers, a similar gene, ChSUB1, which has 98% (4007 bp/4050 bp) identity to CpSUB1, was found in C. hominis. The alignment of the CpSUB1 and ChSUB1 nucleotide sequences identified primarily silent substitutions, consistent with the absence of diversifying selection. The catalytic domain of CpSUB1 is very similar to that of other Apicomplexa (> 38% amino acid identity and >57% similarity) and to the bacterial subtilisin BPN from B. subtilis (36 and 47%). Transcriptional upregulation during merozoite development was observed in cell culture, and a predicted 76-bp intron located near the 3' end of the open reading frame was confirmed experimentally. Cryptosporidium parvum infection in cell culture was significantly inhibited by subtilisin inhibitor III and other serine protease inhibitors, emphasizing the importance of the parasite's subtilase for intracellular development and the enzyme's potential as a drug target.

Effect of solar disinfection on viability of intestinal protozoa in drinking water.

The effect of solar disinfection on the viability of intestinal protozoa Giardia lamblia, Microsporidia sp., Cryptosporidium parvum, Cyclospora cyatenensis and Entamoeba histolytica in drinking water was studied as compared to chlorine disinfection. The protozoa were collected from stool samples, to infect to the distilled water. Chlorinated water samples were prepared at concentration of 4 ppm, and the parasites were incubated overnight at room temperature with the treated water. Sun treatment was applied for 2 exposures (6 & 24 hrs), in summer and winter. Sun treated water samples were put in tubes and exposed to sun. The 2 disinfection methods were tested in plastic and glass test tubes. Parasites viability was assessed by viability assay using trypan blue stain (0.4%), and bioassay infectivity tests in experimentally laboratory bred mice. Results proved that all parasites' viability was not affected by chlorine, following solar disinfection treatment, parasites became dark blue in colour and deformed by trypan blue stain. High parasites death was recorded for all parasites except Microsporidia sp. Bioassay infectivity test showed a statistically significant reduction in mean number of all parasites in intestinal sections compared to controls. The best results were tubes exposure to sun for 24 hrs in summer, where G. lamblia, C. parvum and C. cyatenensis were inactivated or absence in intestinal sections. No statistically significant difference was between the use of plastic and glass tubes, either in chlorine or sun treated parasites. So, solar disinfection proved a simple, cheap and effective means for improving water for human use, particularly in developing countries.

Disinfection of sludge with high pathogenic content using silver and other compounds.

A physicochemical sludge with high microbial content (10(2)-10(4) FPU/g TS bacteriophages, 10(6)-10(7) MPN/g TS faecal coliforms, 10(4) MPN/g TS Salmonella spp., 10(4) MPN/g TS Shigella spp., 10(3) MPN/g TS Pseudomonas aeruginosa, 10(2) MPN/g TS Vibrio cholerae, 10(2)-10(3) cysts/g TS Giardia sp., 10(2)-10(4) oocyts/g TS Cryptosporidium sp., 168-215 viable helminth ova/g TS) was disinfected using silver, silver-copper, and silver-copper plus a synergistic agent (SA). Twenty milligrams Ag/g TS inactivated 4.8 log of faecal coliforms in 1 h; however, 40 mg Ag/g TS are needed to reduce helminth ova viability from 84 to 38.4% in the same period of time. Combinations of Ag-Cu (60:600 mg Ag-Cu/g TS) and Ag-SA (60:24 mg Ag-SA/g TS) inactivated 7.8 log of faecal coliforms and around 90% of helminth ova in 60 min. To produce USEPA class A biosolids, 10:100:8 and 5:50:13.3 mg Ag-Cu-SA/gTS are needed. Bacterial regrowth was not observed for all conditions producing <1000 MPN/gTS faecal coliforms, suggesting a residual disinfection effect. Recommended doses to produce class A biosolids inactivated 2-4 log of bacteriophages, 4 log of Salmonella spp., 4 log of Shigella spp., 3 log of Pseudomonas aeruginosa, 2 log of Vibrio cholerae, 87-99.9% of Giardia sp., and 75-99.9% of Cryptosporidium sp.

Risk and control of waterborne cryptosporidiosis.

Cryptosporidium remains at the forefront of studies on waterborne disease transmission and abatement. The impact of environmental land use patterns which contribute animal and human waste, climatic precipitation leading to a strong association with outbreaks, and community infrastructure and water treatment are now recognized as contributing factors in the potential for waterborne spread of the protozoan. Advances in detection methodologies, including the ability to genotype various strains of this organism, have shown that human wastes are often the source of the contamination and cell culture techniques have allowed insight into the viability of the oocyst populations. Currently water treatment has focused on UV and ozone disinfection as most promising for the inactivation of this protozoan pathogen.

Rapid increase of mucosal CD4 T cells followed by clearance of intestinal cryptosporidiosis in an AIDS patient receiving highly active antiretroviral therapy.

Highly active antiretroviral therapy (HAART) suppresses the replication of human immunodeficiency virus (HIV) and leads to an increase in circulating CD4 T lymphocytes, but its effects on other immune compartments such as the intestinal mucosa are not well understood. We describe a severely immunodeficient HIV-infected patient with intractable watery diarrhea and weight loss caused by infection with Cryptosporidium parvum in whom we studied virologic and immunologic changes in both peripheral blood and the intestinal mucosa after initiating HAART. Mucosal biopsies were performed by rectoscopy before and at several time points after HAART was begun. Nucleic acids were extracted from rectal biopsy specimens and blood samples, and HIV RNA was measured by reverse-transcription polymerase chain reaction. Lymphocytes were isolated from rectal biopsy specimens after mechanical disaggregation, and circulating and mucosal CD4 T cells were determined by flow cytometry. HAART led to clinical recovery and eradication of cryptosporidiosis. In both blood and mucosa, HIV RNA decreased below the limit of detection and CD4 T cells increased. Mucosal CD4 T cells increased much faster and to much higher levels than circulating CD4 T cells. Our findings show a rapid repopulation of the intestinal mucosa with CD4 T cells after initiation of HAART that can effectively restore mucosal immunity, leading to eradication of opportunistic pathogens.

Semi-quantitative characterization of electroporation-assisted disinfection processes for inactivation of Giardia and Cryptosporidium.

The effect of electroporation (very short duration pulses of high voltage electricity) on the viability of Giardia cysts and Cryptosporidium oocysts, and on the viability of these organisms in the presence of free chlorine, combined chlorine, hydrogen peroxide and potassium permanganate, was examined. While electroporation itself had only a minor effect on survival, the combination of electrical and chemical treatment produced superior inactivation, particularly with combined chlorine, hydrogen peroxide and potassium permanganate. This enhancement may provide a relatively practical way of achieving enhanced inactivation of resistant protozoa by water disinfection processes. Further study of kinetics and optimum treatment combinations is needed.

Avaliaçäo da influência do formol e do hipoclorito de sódio na pesquisa de oocistos de Cryptosporidium nas fezes, através do método de heine / The evaluation of the effect of formol and sodium hypochlorite on the demonstration of Cryptosporidium oocysts in feces by Heine's method

Cryptosporidium oocysts were searched by Heine's method in stools of nine calves with cryptosporidiosis after stool treatment with two disinfectants, 10 per cent paraformaldehyde solution and 14.5 per cent sodium hypochlorite solution. After 30 minutes exposition to sodium hypochlorite solution oocysts became non refractile and acquired a reddish tinge, making their identification difficult. No morphological alterations occurred in oocysts after paraformaldehyde treatment. We recommend paraformaldehyde at 10 per cent concentration as means of human immunodeficiency virus (HIV) inactivation for routine use in stool examinations and therefore making safer those type of procedures for laboratory personnel, when using Heine's method