Cryptosporidium-Biofilm Interactions: a Review.

Biofilms are increasingly implicated as playing a major role in waterborne cryptosporidiosis. This review aims to synthesize all currently available data on interactions between Cryptosporidium oocysts and biofilms. Initially described following a waterborne outbreak, the integration of Cryptosporidium oocysts in biofilm has been well demonstrated. Biofilms appear important in the dissemination/protection of oocysts in the environment. Consequently, it has been suggested that substrate-associated biofilms should be systematically considered in oocyst water quality assessment. The influence of physicochemical parameters has been studied on oocyst biofilm retention. Biofilm surface roughness, ionic concentration (especially Ca2+), laminar/turbulent flow, shear stress, and electrostatic repulsion forces appear important to consider regarding oocyst release from biofilm. However, data analysis carried out during this review also revealed important gaps in biological interactions within biofilms, offering many perspectives for future work.

Survival and infectivity of Toxoplasma gondii and Cryptosporidium parvum oocysts bioaccumulated by Dreissena polymorpha.

AIMS: The study was aimed to understand the depuration process of Cryptosporidium parvum and Toxoplasma gondii oocysts by zebra mussel (Dreissena polymorpha), to consider the use of the zebra mussel as a bioremediation tool. MATERIALS AND METHODS: Two experiments were performed: (i) individual exposure of mussel to investigate oocyst transfers between bivalves and water and (ii) in vivo exposure to assess the ability of the zebra mussel to degrade oocysts. RESULTS: (i) Our results highlighted a transfer of oocysts from the mussels to the water after 3 and 7 days of depuration; however, some oocysts were still bioaccumulated in mussel tissue. (ii) Between 7 days of exposure at 1000 or 10 000 oocysts/mussel/day and 7 days of depuration, the number of bioaccumulated oocysts did not vary but the number of infectious oocysts decreased. CONCLUSION: Results show that D. polymorpha can release oocysts in water via (pseudo)faeces in depuration period. Oocysts remain bioaccumulated and infectious oocyst number decreases during the depuration period in zebra mussel tissues. Results suggest a degradation of bioaccumulated C. parvum and T. gondii oocysts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the potential use of D. polymorpha as a bioremediation tool to mitigate of protozoan contamination in water resources.

Cryptosporidiosis after treatment with fingolimod: a case report and pharmacovigilance review.

BACKGROUND: Cryptosporidium sp. are common intracellular parasites responsible of severe diarrhea in T-cell-immunocompromised patients. We report the first case of a woman who contracted cryptosporidiosis after treatment with fingolimod, a drug labeled for multiple sclerosis and responsible for marked lymphopenia. CASE PRESENTATION: A 60-year-old woman was admitted for abdominal pain diarrhea and fever. The patient suffered from multiple sclerosis and had been treated with fingolimod from august 2017 to september 2018 time of occurrence of the first digestive symptoms. Stool culture was negative but parasitological examination was positive for Cryptosporidium sp. Blood biological examination profound lymphopenia of 240/mm3 [17 CD4/mm3 (7%) and 32 CD8/mm3 (14%)]. Fingolimod was stopped, and the patient was put on nitazoxanide 500 mg bid for 7 days. The diarrhea resolved and no relapse was observed. Six other cases were found in the Pharmacovigilance database. CONCLUSION: Physicians should be aware of this association and screen for Cryptosporidium in cases of diarrhea in patients treated with fingolimod. Patients should be aware of this risk and advise to take appropriate measures to avoid such contamination.

Assessment of microscopic and molecular tools for the diagnosis and follow-up of cryptosporidiosis in patients at risk.

Cryptosporidiosis is an important though underreported public health concern. Molecular tools might be helpful in improving its diagnosis. In this study, ZR Fecal DNA MiniPrep™ Kit (ZR) and NucliSens® easyMAG® (EM) were compared using four Cryptosporidium-seeded feces and 29 Cryptosporidium-positive stools. Thereafter, ZR was selected for prospective evaluation of Cryptosporidium detection by 18S rDNA and LAXER quantitative PCR (qPCR) in 69 stools from 56 patients after Cryptosporidium detection by glycerin, modified Ziehl-Neelsen (ZN) and auramine-phenol (AP) stainings. The combination of any of the two extraction methods with 18S qPCR yielded adequate detection of Cryptosporidium in seeded stools, but the ZR kit showed the best performance. All 29 Cryptosporidium-positive samples were positive with 18S qPCR, after both ZR and EM extraction. However, false-negative results were found with LAXER qPCR or nested PCR. Cryptosporidiosis was diagnosed in 7/56 patients. All the microscopic methods enabled the initial diagnosis, but Cryptosporidium was detected in 12, 13, and 14 samples from these seven patients after glycerin, ZN, and AP staining respectively. Among these samples, 14 and 12 were positive with 18S and LAXER qPCR respectively. In two patients, Cryptosporidium DNA loads were found to be correlated with clinical evolution. Although little known, glycerin is a sensitive method for the initial detection of Cryptosporidium. When combined with 18S qPCR, ZR extraction, which had not been evaluated so far for Cryptosporidium, was an accurate tool for detecting Cryptosporidium and estimating the oocyst shedding in the course of infection.

Ruling out nosocomial transmission of Cryptosporidium in a renal transplantation unit: case report.

BACKGROUND: Cryptosporidium spp. is a ubiquitous parasite affecting humans as well as domestic and wild vertebrates, causing diarrhea in both immunocompetent and immunocompromised hosts worldwide. Its transmission occurs primarily by the fecal-oral route. In humans, C. parvum and C. hominis are the most prevalent species, whereas immunocompetent and immunocompromised individuals can also be infected by other zoonotic species. Renal transplant patients are prone to develop cryptosporidiosis, which can induce severe and life-threatening diarrhea. CASE PRESENTATION: We report here a series of nearly concomitant cases of acute symptomatic cryptosporidiosis in three renal transplant patients attending the Strasbourg University Hospital Nephrology Unit. The clinical presentation was persistent diarrhea and acute renal failure. The diagnosis was confirmed by microscopic stool examination using a modified Ziehl-Neelsen staining method and species identification by molecular tools. All patients were treated with nitazoxanide and recovered from diarrhea after 14 days of therapy. CONCLUSION: Genotypic species identification was not consistent with an epidemic context, thus underlining the need for genotyping to monitor at risk patients.

Evaluation of water treatment plant UV reactor efficiency against Cryptosporidium parvum oocyst infectivity in immunocompetent suckling mice.

AIM: To assess the efficiency of a medium-pressure UV reactor under full-scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model. METHODS AND RESULTS: Six/seven-day-old mice were administered orally 2-10x10(4)Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm(-2) UV irradiation of ingested oocysts resulted 7 days later in a 3.4-4.0 log10 reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay. CONCLUSION: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development. SIGNIFICANCE AND IMPACT OF THE STUDY: Present results suggest that in WTP conditions, a medium-pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro-organisms present in environmental waters.

In vitro efficacy of nitro- and halogeno-thiazolide/thiadiazolide derivatives against Sarcocystis neurona.

Sarcocystis neurona is an obligate intracellular parasite that causes equine protozoal myeloencephalitis (EPM). The aim of this work was to document inhibitory activities of nitazoxanide (NTZ, [2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide]) and new thiazolides/thiadiazolides on S. neurona in vitro development, and investigate their structure-activity relationships. S. neurona was grown in bovine turbinate cell cultures. At concentrations varying from 1.0 to 5.0mg/L, nitazoxanide and 21 of 32 second generation thiazolide/thiadiazolide agents exerted a > or =95% maximum inhibition on S. neurona development. Most active agents were either NO(2) or halogen substituted in position 5 of their thiazole moiety. In contrast, other 5-substitutions such as hydrogen, methyl, SO(2)CH(3), and CH(3) negatively impacted activity. Compared with derivatives with an acetylated benzene moiety, deacetylated compounds which most probably represent primary metabolites exhibited similar inhibitory activities. Present data provide the first evidence of in vitro inhibitory activities of nitazoxanide and new thiazolides/thiadiazolides on S. neurona development. Active halogeno-thiazolide/thiadiazolides may provide a valuable nitro-free alternative to nitazoxanide for EPM treatment depending on further evaluation of their in vivo activities.

Multicentre study to determine the Etest epidemiological cut-off values of antifungal drugs in Candida spp. and Aspergillus fumigatus species complex.

OBJECTIVES: To determine the Etest-based epidemiological cut-off values (ECVs) for antifungal agents against the most frequent yeast and Aspergillus fumigatus species isolated in 12 French hospitals. METHODS: For each antifungal agent, the Etest MICs in yeast and A. fumigatus isolates from 12 French laboratories were retrospectively collected from 2004 to 2018. The ECVs were then calculated using the iterative statistical method with a 97.5% cut-off. RESULTS: Forty-eight Etest ECVs were determined for amphotericin B, caspofungin, micafungin, anidulafungin, fluconazole, voriconazole, posaconazole and itraconazole, after pooling and analysing the MICs of 9654 Candida albicans, 2939 Candida glabrata SC, 1458 Candida parapsilosis SC, 1148 Candida tropicalis, 575 Candida krusei, 518 Candida kefyr, 241 Candida lusitaniae, 131 Candida guilliermondii and 1526 Aspergillus fumigatus species complex isolates. These ECVs were 100% concordant (identical or within one two-fold dilution) with the previously reported Etest-based ECVs (when available), and they were concordant in 76.1% of cases with the Clinical and Laboratory Standards Institute ECVs and in 81.6% of cases with the European Committee on Antimicrobial Susceptibility Testing ECVs. CONCLUSIONS: On the basis of these and other previous results, we recommend the determination of method-dependent ECVs. Etest ECVs should not be used instead of breakpoints, but may be useful to identify non-wild-type isolates with potential resistance to antifungal agents, and to indicate that an isolate may not respond as expected to the standard treatment.

[Troponin dosage in a patient with asymptomatic myocarditis due to trichinellosis]. / Myocardite asymptomatique au cours d''un cas de trichinellose: intérêt du dosage de la troponine.

Human trichinellosis is a potentially severe parasitic disease occurring after ingestion of undercooked meat infected with Trichinella sp. larvae. We report the case of a patient who ate an undercooked bear meat hunted in Canada; he presented with the usual symptoms of trichinellosis (i.e, facial oedema, myalgias and fever) complicated with an asymptomatic myocarditis. Myocarditis is a rare, but potentially lethal complication of trichinellosis. Myocarditis should be screened systematically even when specific symptoms are missing; dosage of troponin serum is a simple and reliable mean for such screening.

[Cutaneous leishmaniasis in polymyositis]. / Leishmaniose cutanée au cours d'une polymyosite.

INTRODUCTION: The prevalence of cutaneous leishmaniasis is increasing, especially in immunocompromized subjects. CASE RECORD: We report a case of particular interest, where a patient with steroid refractory polymyositis developed cutaneous leishmaniasis. Clinical outcome was favorable after institution of intralesional antimony therapy. DISCUSSION: Cutaneous leishmaniasis is still recognized to be an opportunistic infection. Its frequency is indeed higher in immunocompromized subjects, mainly HIV-infected patients. In our patient, both polymyositis and steroid therapy were predisposing factors of cutaneous leishmaniasis onset; prednisone therapy has been postulated to be associated with immune dysfunction leading to: reduced blood T cells' levels (CD4 et CD8) as well as decreased cytokine synthesis (e.g. interferon gamma).

Pilot-Scale Pulsed UV Light Irradiation of Experimentally Infected Raspberries Suppresses Cryptosporidium parvum Infectivity in Immunocompetent Suckling Mice.

Cryptosporidium spp., a significant cause of foodborne infection, have been shown to be resistant to most chemical food disinfectant agents and infective for weeks in irrigation waters and stored fresh vegetal produce. Pulsed UV light (PL) has the potential to inactivate Cryptosporidium spp. on surfaces of raw or minimally processed foods or both. The present study aimed to evaluate the efficacy of PL on viability and in vivo infectivity of Cryptosporidium parvum oocysts present on raspberries, a known source of transmission to humans of oocyst-forming apicomplexan pathogens. The skin of each of 20 raspberries was experimentally inoculated with five 10-µl spots of an oocyst suspension containing 6 × 10(7) oocysts per ml (Nouzilly isolate). Raspberries were irradiated by PL flashes (4 J/cm(2) of total fluence). This dose did not affect colorimetric or organoleptic characteristics of fruits. After immunomagnetic separation from raspberries, oocysts were bleached and administered orally to neonatal suckling mice. Seven days after infection, mice were euthanized, and the number of oocysts in the entire small intestine was individually assessed by immunofluorescence flow cytometry. Three of 12 and 12 of 12 inoculated mice that received 10 and 100 oocysts isolated from nonirradiated raspberries, respectively, were found infected. Four of 12 and 2 of 12 inoculated mice that received 10(3) and 10(4) oocysts from irradiated raspberries, respectively, were found infected. Oocyst counts were lower in animals inoculated with 10(3) and 10(4) oocysts from irradiated raspberries (92 ± 144 and 38 ± 82, respectively) than in animals infected with 100 oocysts from nonirradiated raspberries (35,785 ± 66,221, P = 0.008). PL irradiation achieved oocyst reductions of 2 and 3 log for an inoculum of 10(3) and 10(4) oocysts, respectively. The present pilot-scale evaluation suggests that PL is an effective mode of decontamination for raspberries and prompts further applicability studies in industrial contexts.

Taenia crassiceps invasive cysticercosis: a new human pathogen in acquired immunodeficiency syndrome?

A fluctuant, painful, subcutaneous, and intermuscular tumor developed in a 38-year-old man with severe acquired immunodeficiency syndrome (AIDS) in which immunodeficiency was severe. Surgery revealed lesions that formed a multilocular pouch embedded in deep tissues in the forearm filled with tapiocalike material containing a viscous fluid, granules, and cysticercilike small vesicles. Pathologic and parasitologic evaluation showed cysticerci embedded in a fibrocollagen reaction with inflammatory granulomatous reaction. Each cysticercus contained an invaginated scolex with two rows of small (i.e., 80 microm) and large (i.e., 114 microm) rostellar hooks, identical to larva of Taenia crassiceps. All clinical, parasitologic, and pathologic features of these cysticerci were very different from those of all other larval cestode (i.e., Taenia solium cysticercosis, coenurosis, sparganosis, cysticercosis due to Taenia saginata [Cysticercus bovis], primary and secondary hydatidosis [Echinococcus species]). T crassiceps cysticerci usually develop in subcutis and pleuroperitoneal cavities of rodents, whereas the adult tapeworm is commonly found in the digestive tract of foxes. Biologic properties of T crassiceps cysticerci and epidemiologic characteristics of pandemic human immunodeficiency virus (HIV) could eventually indicate new potential cases of T crassiceps cysticercosis in humans.

Enzyme immunoassay detection of Cryptosporidium parvum inhibition by sinefungin in sporozoite infected HCT-8 enterocytic cells.

Complete parasite development was obtained in differentiated human enterocytic HCT-8 cells infected at confluence with Cryptosporidium parvum sporozoites, and evaluated in a quantitative enzyme immunoassay. Forty-eight hours after infection, a linear correlation was found between optical density values and the number of parasites determined in an immunofluorescent assay. Sinefungin exerted an inhibitory effect when added within 4 h after sporozoite addition to HCT-8 cultures (MIC50 = 38 mumol L-1), while the inhibitory effects of paromomycin and pentamidine dimethanesulfonate were also easily detected (MIC50 = 0.87 mumol L-1 and 0.27 mumol L-1, respectively). Except for high pentamidine dimethanesulfonate concentrations, no alteration in optical microscopy morphology or trypan blue exclusion of HCT-8 cells was observed in the presence of anticryptosporidial agents, which suggests that they were primarily active against developing parasites. Data suggest that EIA detection of C. parvum development in sporozoite-infected HCT-8 cells provides an accurate and convenient model for in vitro evaluation of parasite infectivity, growth and response to anticryptosporidial agents.

Biliary tract cryptosporidiosis immunosuppressed rat model.

Rats immunosuppressed by hydrocortisone acetate and a low protein diet were challenged with Cryptosporidium Parvum oocysts and studied on days 10, 35 and 70 post-infection. The biliary tract was found to be a major site of parasite infection. C. parvum was visible in the biliary papillary area in association with a proliferation of highly convoluted tubular glands. The papillary lumen was narrowed, and an upstream dilation with bacterial proliferation was seen. The liver was initially free of lesions, and subsequently exhibited late lesions of cholestasis. Parasites were not found in the pancreatic duct, although pancreatitis was frequently observed. Oocysts were consistently present in the distal portion of the ileum. Both challenged and unchallenged immunosuppressed rats, exhibited widespread focal hepatic infarcts and pyelonephritis. Other organs appeared free of lesions. In addition to the intestine, data identified the biliary tract as a major site of C. parvum infection and as a potential protected reservoir which may sustain a chronic infection.

Double-blind, randomized, placebo-controlled study of nitazoxanide in the treatment of fascioliasis in adults and children from northern Peru.

BACKGROUND: Human fascioliasis is a significant world-wide health problem, and massive or repeated infections by Fasciola hepatica can lead to considerable morbidity. AIM: : To evaluate the safety and efficacy of nitazoxanide, when compared with placebo, in the treatment of fascioliasis in adults and children from northern Peru. METHODS: A double-blind, placebo-controlled study was carried out in 50 adults and 50 children infected with F. hepatica. The diagnosis of infection was based on the presence of F. hepatica eggs in one stool sample obtained before inclusion in the study. Patients were randomized to receive treatment with either a 7-day course of nitazoxanide (100 mg b.d., age range 2-3 years; 200 mg b.d., age range 4-11 years; 500 mg b.d., age > 12 years) or matching placebo. Three post-treatment stool examinations were carried out between 30 and 90 days after initiation of treatment. RESULTS: The parasite was eliminated in 18 of 30 (60%) adults completing the study who received nitazoxanide vs. one of eight adults in the placebo group (P = 0.042), and similarly in 14 of 35 (40%) children completing the treatment vs. none of eight in the placebo group (P = 0.038). Only mild, transient adverse events were reported. CONCLUSIONS: A 7-day course of nitazoxanide was effective in adults and children in the treatment of F. hepatica, when compared with placebo.

Randomized clinical study of nitazoxanide compared to metronidazole in the treatment of symptomatic giardiasis in children from Northern Peru.

BACKGROUND: Enteric infection by Giardia intestinalis is a common cause of diarrhoea world-wide and a significant cause of morbidity in children. AIM: To compare the efficacy and safety of nitazoxanide and metronidazole in the treatment of diarrhoea caused by G. intestinalis in children. METHODS: A total of 110 children presenting with diarrhoea caused by G. intestinalis were randomized to treatment with either a 3-day course of nitazoxanide (100 mg b.d., age range 2-3 years; 200 mg b.d., age range 4-11 years) or a 5-day course of metronidazole (125 mg b.d., age range 2-5 years; 250 mg b.d., age range 6-11 years). The patients were followed-up for a determination of clinical response 7 days after the initiation of treatment, and two subsequent stool samples were collected for parasitological examination. RESULTS: Diarrhoea had resolved in 47 children out of 55 (85%) in the nitazoxanide treatment group before the day 7 follow-up visit, compared to 44 out of 55 (80%) for metronidazole. Diarrhoea resolved within 4 days in most cases. Only mild, transient adverse events were reported. CONCLUSIONS: A 3-day course of nitazoxanide suspension is as efficacious as a standard 5-day course of metronidazole suspension in treating giardiasis in children.

Hydrogen peroxide gas plasma sterilization is effective against Cryptosporidium parvum oocysts.

The aim of this work was to evaluate in an immunosuppressed rat cryptosporidiosis model a new method that combines vacuum and low-temperature hydrogen peroxide gas plasma for sterilization of endoscopic material contaminated by Cryptosporidium parvum. Rats were challenged with oocysts either air-dried or air-dried and treated with vacuum alone or associated with gas plasma. No rat was found infected after gas plasma exposure of oocysts, whereas vacuum or air-drying alone resulted only in a decreased infectivity.

Determination of anti-Cryptosporidium coproantibodies by time-resolved immunofluorometric assay.

The role of the mucosal immune response against Cryptosporidium has been suggested by studies on the therapeutic effects of hyperimmune colostrum. In order to study the intestinal response to this infection, we have developed a sandwich-type time-resolved immunofluorometric assay for the determination of anti-Cryptosporidium coproantibodies. This assay has the inherent sensitivity of an immunoassay without the problems due to background responses from other biological compounds, and is thus suitable for faecal samples. The intra-assay coefficients of variation (5.1%, 4.6%, and 5.8% for immunoglobulins (Ig) A, M and G respectively), inter-assay coefficients of variation (9.4%; 10.5% and 12.2% for IgA, IgM and IgG, respectively) and specificity (100% for all 3 isotypes) were all satisfactory. Using this assay to study 12 patients with the acquired immune deficiency syndrome (AIDS) who were infected with cryptosporidiosis, we found a marked elevation of anti-Cryptosporidium IgA and IgM coproantibody titres relative to 18 healthy control values, but no correlation with the gravity of the infection in terms of oocyst shedding. These results suggest that a non-protective mucosal immune response develops to Cryptosporidium in AIDS patients.

Sensitivity of Plasmodium falciparum to amodiaquine and chloroquine in central Africa: a comparative study in vivo and in vitro.

A comparative study in vivo of amodiaquine efficacy (35 mg/kg over 3 d) and chloroquine (25 mg/kg over 3 d) was conducted in 1991 and 1992 in Cameroon and Congo in 123 patients with uncomplicated Plasmodium falciparum malaria. Amodiaquine was more effective than chloroquine, with parasite clearance by day 7 in 79.7% of the patients compared with 59.4%. Sixteen of 32 (50%) P. falciparum isolates tested in vitro were resistant to chloroquine and only 3 of 34 (9%) were resistant to amodiaquine. 5.3% of patients treated with amodiaquine complained of pruritus and 18.7% of nausea, compared with 15.7% and 5% respectively of those treated with chloroquine.