Jean Jacques Ballet

Successful Treatment of Metronidazole- and Albendazole-Resistant Giardiasis with Nitazoxanide in a Patient with Acquired Immunodeficiency Syndrome

A case of metronidazole- and albendazole-resistant giardiasis in a patient with the acquired immunodeficiency syndrome was successfully treated with nitazoxanide (1.5 g twice a day for 30 days). Animal studies and in vitro assays showed that the isolate was resistant to both metronidazole and albendazole and susceptible to nitazoxanide.

Neosporosis in bovine dairy herds from the west of France: detection of Neospora caninum DNA in aborted fetuses, seroepidemiology of N. caninum in cattle and dogs.

Neospora caninum is considered one of the major causes of abortion in cattle in most parts of the world. In this study, the role of N. caninum was investigated in groups of aborted cattle and dairy herds from the west of France. Good correlation was found between parasite DNA detection in fetuses and serologic statuses of dams. In groups with documented abortion status and no antibodies to other pathogens, 17-45% of aborted animals were seropositive for N. caninum, and significant relationship between prevalence of Neospora antibodies and frequency of abortions was found. Neospora-associated abortions were observed all the year round, with a peak in summer. Higher ratios of seropositive abortions were found before the 6th month of gestation. In 12 herds studied in the field, serologic prevalence ranged 6-47%. No difference in age was found between seropositive and seronegative cows. Results indicate that N. caninum is an important and stable cause of abortion in cattle in France.

Quantitative Flow Cytometric Evaluation of Maximal Cryptosporidium parvum Oocyst Infectivity in a Neonate Mouse Model

ABSTRACT The importance of waterborne transmission of Cryptosporidium parvum to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting C. parvum oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.

Reactivity against Sarcocystis neurona and Neospora by serum antibodies in healthy French horses from two farms with previous equine protozoal myeloencephalitis-like cases

Sarcocystis neurona is considered a leading cause of equine protozoal myeloencephalitis (EPM), a common infectious neurological disease in horses in the Americas. EPM-like cases associated with S. neurona peptide reactive antibodies in Western blots were recently described in Normandy, France. In this report, antibodies reacting with S. neurona merozoites were detected using an agglutination assay at titers ranging from 50 to 500 in sera from 18/50 healthy horses from two farms with a previous EPM-like case. Higher values were found in older animals. Four out of six horses which traveled or stayed in the US exhibited titers over 50, a higher figure than in the group which did not travel out of France or stayed in an other European country. No correlation was found between anti-S. neurona and anti-Neospora sp. antibody titers. Data prompt further study of significance of anti-S. neurona antibodies in clinically healthy or diseased European horses, and identification of putative immunizing parasite(s) and their host(s).

Transient neonatal Cryptosporidium parvum infection triggers long-term jejunal hypersensitivity to distension in immunocompetent rats.

ABSTRACT In 5-day-old immunocompetent Sprague-Dawley rats infected with either 102 or 105Cryptosporidium parvum oocysts, transient infection resulted 120 days later in increased cardiovascular depressor response to jejunal distension and jejunal myeloperoxidase activity (P < 0.05). Nitazoxanide treatment normalized jejunal sensitivity (P < 0.001) but not myeloperoxidase levels (P > 0.05). Data warrant further evaluation of the role of early cryptosporidiosis in the development of chronic inflammatory gut conditions.

Long-Term Rasamsonia argillacea Complex Species Colonization Revealed by PCR Amplification of Repetitive DNA Sequences in Cystic Fibrosis Patients.

ABSTRACT The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina, while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients.

Time and temperature effects on the viability and infectivity of Cryptosporidium parvum oocysts in chlorinated tap water.

Abstract The authors compared the viability and infectivity of Cryptosporidium parvum oocysts in chlorinated tap water at various storage durations (i.e., 2 wk, 4 wk, 6 wk, or 8 wk) and at 2 cool temperatures (i.e., 10[ddot]C and 4[ddot]C), using in vitro (excystation) and in vivo (suckling mouse) methods. After 8 wk, mean oocyst excystation decreased to 33.4% and 26.7% at 10[ddot]C and 4[ddot]C, respectively. Suckling mice infectivity was higher after storage at 10[ddot]C than after storage at 4[ddot]C. These data suggest that Cryptosporidium parvum oocysts can survive and remain infectious for 8 wk in cool chlorinated tap water.

Eveluation of Viability and Infective of Waterborne Cryptosporidium parvum Oocysts

Oocysts of Ctyprosporidium parvum are widely distributed in waters and waterborne transmission is frequently responsible for outbreaks of human cryptosporidiosis. The most important was reported in the United States in 1993. Surface as well as ground water may be contaminated by C. parvum oocysts which may remain viable in a buffer solution for up to 12 months at 4 C [l]. Oocysts are strongly resistant to most commonly used disinfectants and chlorination of drinking water is not sufficient to prevent C. purvurn infection. The control of C. parvum contamination of water requires identification and counting of oocysts isolated after filtration which is realised with polycarbonate or cellulose acetate filters. Oocysts are identified by microscopic examination using Henricksen or Heine staining. The oocysts counts may be performed using either an hemocytometer or flow cytometry after labeling using an anti-C. parvum fluorescent monoclonal antiboby (Diagnostics Pasteur). Oocyst counts are not sufficient to evaluate the risk of contamination which requires an evaluation of viability and infectivity of oocysts. MATERIALS AND METHOD. The first step for determination of viability of oocysts consists of the evaluation of their ability to excyst sporozoites. C. parvum oocysts used were obtained from experimently infected neonate calves (h4 Naciri, INR.4, Nouzilly, France). Oocysts were extracted and purified using a double gradient sucrose method (d=1.088 and d=1.044) and excysted in 1% taurocholic acid (Sigma) at 37 C for one h. The oocyst counts performed before and after exystation assays were compared. The percentage of excystation was determined in hemocytometer (cells KOVA Slide 10 Boehringer) and was controled punctually with a flow cytometer (EPICS, Profil I1 coulter). RESULTS AND DISCUSSION. In a first experiment the excystation of oocysts stored in stools without potassium dichromate at 20 C during 47 days was studied. Five days after collecting stools the percentage of excystation was 44.8% 2 7.3%. No significant time effect was found after six weeks (42.9% + 4.5% at day 47). The influence of temperature on oocysts excystation after storage during 10 weeks in 2.5% potassium dichromate solution at 4 C compared to 20 C was also studied. I n three series of experiments the percentage of excystation was found significatively decreased at 20 C compared to 4 C ( p<O.O5). After one week storage at 4C excystation was 96.1% 2.8% and 94.7% 4.5% at 20 C. After 10 weeks storage at 4 C excystation was 89.7% 6.8% at 20 C. A significant difference of the percentages of excystation were found between oocysts stored in water and those stored in 2.5% potassium dichromate solution. When oocysts stored in water were treated by a 2% sodium hypochlorite solution during 10 min, washed and stored again in water, the percentage of excystation was increased and not significantly different from oocysts stored in 2.5% potassium dichromate solution (Tab. 1). A parallel study using scanning electron microscopy showed an alteration of oocysts wall when stored either in a 2.5% potassium permanganate or a 2.5% potassium dichromate solution for one week. Alteration were also observed on oocysts treated with a 2% sodium hypochlorite solution for 10 min when compared to controls. These results suggest that a high redox potential of a solution increases the capability of oocysts to excyst, since the redox potentials of potassium permanganate, potassium dichromate or sodium hypochlorite are very close. In addition, we observed that a 2% glutaraldehyde solution strongly inhibited the oocyst excystation whereas a 1.6% chlorexidine solution did not. The 10% of oocysts excysted after glutaraldehyde treatment were alive and able to infect immunosupressed rat. Infectivity of sporozoftes excysted from oocysts treated with sodium hypochlorite was also studied in a Caco-2 cells model to evaluate oocyst viability [2]. SporozoTtes isolated by filtration using 5 pm filters (Millipore) were added to Caco-2 cells. Three days later, culture was fixed b y acetone and the parasite detected by direct immunofluorescence method [3]. The high level of excystation observed with oocysts treated by hypochlorite suggests that chlorination of water could make oocysts more infective for human as well as the other oxidants studied. Conversely, some disinfectants which partially inhibit excystation, may decrease the risk. Evaluation of the risk of waterborne transmission should also be based on viability of excysted sporozoites in cell cultures and/or infectivity in animal model. [Supported by grants from ANRS no 96020 and 2.3% and 33.8%

Common occurrence of Cryptosporidium hominis in asymptomatic and symptomatic calves in France

Background Cryptosporidium spp. infections are the most frequent parasitic cause of diarrhea in humans and cattle. However, asymptomatic cases are less often documented than symptomatic cases or cases with experimentally infected animals. Cryptosporidium (C.) hominis infection accounts for the majority of pediatric cases in several countries, while C. parvum is a major cause of diarrhea in neonatal calves. In cattle Cryptosporidium spp. infection can be caused by C. parvum, C. bovis, C.andersoni and C. ryanae, and recently, reports of cattle cases of C. hominis cryptosporidiosis cases suggest that the presence of C. hominis in calves was previously underestimated. Methodology/Principal findings From February to November 2015, Cryptosporidium spp. infected calves were detected in 29/44 randomly included farms from 5 geographic regions of France. C. hominis and C. parvum were found in 12/44 and 26/44 farms, respectively with higher C. hominis prevalence in the western region. In 9 farms, both C. parvum and C. hominis were detected. Eighty-six of 412 (73/342 asymptomatic and 13/70 symptomatic) one to nine-week-old calves shed C. hominis or C. parvum oocysts (15 and 71 calves, respectively), with no mixed infection detected. The predominant C. hominis IbA9G3 genotype was present in all regions, and more frequent in the western region. An incompletely characterized Ib, and the IbA13G3, IbA9G2 and IbA14G2 genotypes were present only in the western region. For C. parvum, the most frequent genotype was IIaA16G3R1 with no geographic clustering. Most C. hominis infected calves were asymptomatic, with some exceptions of IbA9G2 and IbA9G3 isolates, while C. parvum IIaA16G3R1 was associated with symptoms. Conclusions/Significance Present results indicate for the first time that in several geographic regions of France, C. hominis was present in about one fifth of both asymptomatic and symptomatic infected calves, with isolated genotypes likely associated with human infection. Further investigations are aimed at documenting direct or indirect transmissions between livestock and humans.

Evaluation of voriconazole anti-Acanthamoeba polyphaga in vitro activity, rat cornea penetration and efficacy against experimental rat Acanthamoeba keratitis

Background Acanthamoeba keratitis (AK) is a sight-threatening infectious disease. Its effective and safe medical therapy remains highly debated. Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically to treat human AK cases. Objectives To measure anti-Acanthamoeba polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of eye-drop and oral voriconazole. Methods A. polyphaga was maintained in axenic cultures. In vitro, amoebicidal and cysticidal activities of voriconazole were measured using an XTT assay. AK lesions of Sprague Dawley rats were scored from grade 0 to grade 3. For 21 days, from day 7 post-infection, voriconazole (1% solution) eye drops were instilled or voriconazole was administered by gavage (60 mg/kg/day). After killing, superficial corneal epithelium scrapings were cultured and analysed by PCR, and eye-globe histology was performed. Cornea and plasma concentrations were determined using 2D HPLC separation and tandem MS. Results In vitro, voriconazole inhibited trophozoite proliferation with an IC50 value of 0.02 mg/L and an IC90 value of 2.86 mg/L; no cysticidal effect was found. In AK rats, eye drops reduced clinical worsening from day 7 to day 14 post-infection and oral voriconazole was not effective. Voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations, which resulted in lower plasma concentrations, whilst oral voriconazole resulted in lower cornea concentrations. Conclusions Present data underline the need for high-frequency eye-drop instillation regimens for efficient AK therapy.