Machine learning and explainable artificial intelligence for the prevention of waterborne cryptosporidiosis and giardiosis.

Cryptosporidium and Giardia are important parasitic protozoa due to their zoonotic potential and impact on human health, and have often caused waterborne outbreaks of disease. Detection of (oo)cysts in water matrices is challenging and extremely costly, thus only few countries have legislated for regular monitoring of drinking water for their presence. Several attempts have been made trying to investigate the association between the presence of such (oo)cysts in waters with other biotic or abiotic factors, with inconclusive findings. In this regard, the aim of this study was the development of an holistic approach leveraging Machine Learning (ML) and eXplainable Artificial Intelligence (XAI) techniques, in order to provide empirical evidence related to the presence and prediction of Cryptosporidium oocysts and Giardia cysts in water samples. To meet this objective, we initially modelled the complex relationship between Cryptosporidium and Giardia (oo)cysts and a set of parasitological, microbiological, physicochemical and meteorological parameters via a model-agnostic meta-learner algorithm that provides flexibility regarding the selection of the ML model executing the fitting task. Based on this generic approach, a set of four well-known ML candidates were, empirically, evaluated in terms of their predictive capabilities. Then, the best-performed algorithms, were further examined through XAI techniques for gaining meaningful insights related to the explainability and interpretability of the derived solutions. The findings reveal that the Random Forest achieves the highest prediction performance when the objective is the prediction of both contamination and contamination intensity with Cryptosporidium oocysts in a given water sample, with meteorological/physicochemical and microbiological markers being informative, respectively. For the prediction of contamination with Giardia, the eXtreme Gradient Boosting with physicochemical parameters was the most efficient algorithm, while, the Support Vector Regression that takes into consideration both microbiological and meteorological markers was more efficient for evaluating the contamination intensity with cysts. The results of the study designate that the adoption of ML and XAI approaches can be considered as a valuable tool for unveiling the complicated correlation of the presence and contamination intensity with these zoonotic parasites that could constitute, in turn, a basis for the development of monitoring platforms and early warning systems for the prevention of waterborne disease outbreaks.

Fate of Cryptosporidium and Giardia through conventional and compact drinking water treatment plants.

Over the past three decades, a notable rise in the occurrence of enteric protozoan pathogens, especially Giardia and Cryptosporidium spp., in drinking water sources has been observed. This rise could be attributed not only to an actual increase in water contamination but also to improvements in detection methods. These waterborne pathogens have played a pivotal role in disease outbreaks and the overall escalation of disease rates in both developed and developing nations worldwide. Consequently, the control of waterborne diseases has become a vital component of public health policies and a primary objective of drinking water treatment plants (DWTPs). Limited studies applied real-time PCR (qPCR) and/or immunofluorescence assay (IFA) for monitoring Giardia and Cryptosporidium spp., particularly in developing countries like Egypt. Water samples from two conventional drinking water treatment plants and two compact units (CUs) were analyzed using both IFA and qPCR methods to detect Giardia and Cryptosporidium. Using qPCR and IFA, the conventional DWTPs showed complete removal of Giardia and Cryptosporidium, whereas Mansheyat Alqanater and Niklah CUs achieved only partial removal. Specifically, Cryptosporidium gene copies removal rates were 33.33% and 60% for Mansheyat Alqanater and Niklah CUs, respectively. Niklah CU also removed 50% of Giardia gene copies, but no Giardia gene copies were removed by Mansheyat Alqanater CU. Using IFA, both Mansheyat Alqanater and Niklah CUs showed a similar removal rate of 50% for Giardia cysts. Additionally, Niklah CU achieved a 50% removal of Cryptosporidium oocysts, whereas Mansheyat Alqanater CU did not show any removal of Cryptosporidium oocysts. Conventional DWTPs were more effective than CUs in removing enteric protozoa. The contamination of drinking water by enteric pathogenic protozoa remains a significant issue globally, leading to increased disease rates. Infectious disease surveillance in drinking water is an important epidemiological tool to monitor the health of a population.

Is there any change in the prevalence of intestinal or cardiopulmonary parasite infections in companion animals (dogs and cats) in Germany between 2004-2006 and 2015-2017? An assessment of the impact of the first ESCCAP guidelines.

Main objective of the present nationwide study was to assess the impact of the ESCCAP guideline for the control of worm infections in dogs and cats 8-10 years after its first publication in Germany. A secondary aim was to determine the prevalence of canine and feline cardiopulmonary nematodes and intestinal protozoa. Faecal samples of 53,693 dogs and 26,491 cats in 2004-2006 as well as of 129,578 dogs and 45,709 cats in 2015-2017 routinely submitted by veterinarians to a private veterinary laboratory were examined using appropriate parasitological methods. In dogs, the prevalence of Toxocara and taeniid egg shedding was significantly lower in 2015-2017 (3.8 % and 0.16 %, respectively) than in 2004-2006 (4.6 % and 0.27 %, respectively). The prevalence of hookworm and Capillaria eggs was higher in the second study period (2.3 % and 0.77 %, respectively) than in the first (1.3 % and 0.6 %, respectively). For Toxascaris leonina (0.55-0.6 %) and Trichuris (0.8-0.9 %), the difference was not significant between the study periods. Dogs shed more often Angiostrongylus vasorum larvae in the second study (3.1 %) than in the first (1.0 %), whereas the prevalence of Crenosoma vulpis did not change significantly (2.2-2.6 %). Cystoisospora canis and C. ohioensis-like infections were less detected in the second study period (1.0 % and 2.1 %, respectively) than in the first (1.8 % and 2.7 %, respectively). Neospora-like oocysts and Sarcocystis sporocysts were more prevalent in the second study period (0.19 % and 0.13 %, respectively) than in the first (0.13 % and 0.06 %, respectively). The percentage of Giardia or Cryptosporidium coproantigen-positive samples was lower in the second study period (18.9 % and 6.7 %, respectively) than in the first (22.8 % and 10.0 %, respectively). In cats, the prevalence of egg shedding of T. cati, Capillaria and taeniids was significantly lower in 2015-2017 (3.5 %, 0.25 % and 0.1 %, respectively) than in 2004-2006 (4.8 %, 0.54 % and 0.22 %, respectively). No difference was recorded for hookworms (0.12-0.13 %) and Ts. leonina (0.04-0.05 %). Aelurostrongylus-like larvae were detected more often in the second study period (6.5 %) than in the first (2.6 %). Infections with Cystoisospora felis, C. rivolta, Toxoplasma-like coccids and Sarcocystis were less prevalent in the second study period (1.9 %, 0.7 %, 0.24 % and 0.02 %, respectively) than in the first (2.7 %, 1.1 %, 0.36 % and 0.1 %, respectively). The percentage of Giardia or Cryptosporidium coproantigen-positive samples was significantly lower in the second study period (10.6 % and 4.8 %, respectively) than in the first (15.4 % and 8.3 %, respectively). Although these results indicate a decline of the occurrence of most canine and feline intestinal parasites in Germany over the years, a transmission risk of zoonotic parasites remains. Therefore, the control of helminth infections in domestic dogs and cats continues to be a challenge for veterinarians and pet owners.

Assessment microbial risks for the presence of Cryptosporidium spp. and Giardia spp. based on the surveillance of the water supply systems in Colombia, 2014-2018.

Cryptosporidium spp. and Giardia spp. cause gastrointestinal diseases of zoonotic origin as well transmitted from person to person, being various reported outbreaks associated with water. The infecting (oo)cyst forms of these parasites are highly resistant to water treatments such as chlorine disinfection and fast filtration. The objective of this study was to assess the microbial risk of infection and symptomatic illness by the ingestion of Giardia spp. and Cryptosporidium spp. in water for human consumption in Colombia, based on the results of water quality surveillance. The detection method was according to the USEPA method 1623. Concentration data of the different points of distribution were grouped according to the pathogen and type of treatment (no treatment; chlorine treatment; chlorine treatment + coagulant). Annual microbial risks of infection and symptomatic diseases were estimated using the quantitative microbial risk assessment approach that included parasite concentrations, the dose-response model, the ingestion rates of water by children and adults, and the morbidity rate of the diseases. The mean annual microbial risk of infection for Giardia spp. was 29.8% for treated water and 50.4% for untreated water, while being 6.0% and 17.7%, respectively, for Cryptosporidium spp. Microbial risk of symptomatic illness for Giardia spp, was 8.2% for treated water and 13.9% for untreated water, while being 3.6% and 10.6%, respectively, for Cryptosporidium spp. The estimated annual microbial risks of infection exceeded the acceptable value of 10-4 (0.01%) recommended by USEPA. Results obtained in this study suggest the need to reduce the microbial risk of infection to protozoan parasites by improving the water treatment, by adopting better handling practices for livestock manure and treatment processes of human feces. PRACTITIONER POINTS: The presence of Cryptosporidium spp was identified in 28 (6.2%) samples and Giardia spp in 29 (6.4%) in water for human consumption in Colombia. The mean annual risk of symptomatic illness due to infection by Giardia spp or Cryptosporidium spp ranges from 33.6%, for treated water, to 58.1%, for untreated water. Annual risks ingestion of protozoa studying in water for human exceed of 10-4 (0.01%) recommended by USEPA.

Delivery of SA35 and SA40 peptides in mice enhances humoral and cellular immune responses and confers protection against Cryptosporidium parvum infection.

BACKGROUND: Cryptosporidium parvum is a major cause of diarrhea in children and ruminants at the earliest stages of life. Maternal antibodies represent the main shield of neonate mammals for most of the infections. Two recombinant antigens (SA35 and SA40), portions of two C. parvum proteins, were tested for their ability to induce immune responses in adult mice and for protection on neonate BALB/c mice born from females immunised by mucosal delivery of both peptides. METHODS: Adult BALB/c mice were intraperitoneally immunised with SA35 and SA40, separately or mixed, and their immune response was characterised. Furthermore, BALB/c pregnant mice were immunised by mucosal delivery with an SA35/40 mix, before and during pregnancy. Soon after birth, their offspring were infected with two doses (1 × 105 and 5 × 103) of C. parvum oocysts and the parasitic burden was determined at 5 and 9 days post-infection. RESULTS: Intraperitoneal immunisation with SA35 and SA40 induced specific IgG and IgG1 in serum, specific IgA in the intestinal mucosa, increase of CD3+/CD4+ and CD30+ cells in splenocytes, which produced IFN-γ. Neonates born from immunised mice and infected with 1 × 105 oocysts showed a significant reduction of oocysts and intestinal forms (23 and 42%, respectively). A reduction of all parasitic forms (96%; P < 0.05) was observed when neonates were infected with 5 × 103 oocysts. CONCLUSIONS: SA35 and SA40 peptides induce specific humoral and cell-mediated immune responses to C. parvum in adult mice. Moreover, mucosal administration of the SA35/40 mix in pregnant mice reduces C. parvum burden in their litters.

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.

Protocol standardization for the detection of Giardia cysts and Cryptosporidium oocysts in Mediterranean mussels (Mytilus galloprovincialis).

Marine bivalve shellfish are of public health interest because they can accumulate pollutants in their tissues. As they are usually consumed raw or lightly cooked, they are considered to be a possible source of foodborne infections, including giardiosis and cryptosporidiosis. Although data indicating contamination of shellfish with Giardia cysts and Cryptosporidium oocysts have been published, comparing results from different studies is difficult, as there is no standardized protocol for the detection and quantification of these parasites in mussels, and different researchers have used different analytical approaches. The aim of this study was to identify and characterize the most sensitive protocol for the detection of Giardia cysts and Cryptosporidium oocysts in shellfish. In an effort to test the sensitivity and the detection limits of the protocol, every step of the process was investigated, from initial preparation of the mussel matrix through detection of the parasites. Comparative studies were conducted, including several methods previously applied by other researchers, on commercial mussels Mytilus galloprovincialis spiked with a known number of (oo)cysts of both parasites. As preparation of the mussel matrix plays an important role in the sensitivity of the method, different techniques were tested. These included: (ia) removal of the coarse particles from the matrix with sieving, (ib) extraction of the lipids with diethyl ether, and (ic) artificial digestion of the matrix with pepsin digestion solution, and (ii) the use or not of immunomagnetic separation (IMS) for the concentration of the (oo)cysts. Pre-treatment of the mussel homogenate with pepsin digestion solution, followed by IMS, then detection with a direct immunofluorescence assay, achieved the highest sensitivity: 32.1% (SD: 21.1) of Giardia cysts and 61.4% (SD: 26.2) Cryptosporidium oocysts were recovered, with a detection limit of 10 (oo)cysts per g of mussel homogenate. The outcome of the current study was the standardization of a protocol, with defined detection limits, for the detection of these two protozoan transmission stages in mussels, in order to be used as a reference technique in future studies. Further advantages of this protocol are that it uses the whole mussel as a starting material and does not require difficult handling procedures. The method has potential to be applied in larger surveys and, potentially, to other species of shellfish for the detection of these parasites. However, the composition (lipid to protein ratio) may be of relevance for detection efficiency for some other species of shellfish.

Assessing viability and infectivity of foodborne and waterborne stages (cysts/oocysts) of Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii: a review of methods.

Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii are protozoan parasites that have been highlighted as emerging foodborne pathogens by the Food and Agriculture Organization of the United Nations and the World Health Organization. According to the European Food Safety Authority, 4786 foodborne and waterborne outbreaks were reported in Europe in 2016, of which 0.4% were attributed to parasites including Cryptosporidium, Giardia and Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidium and Toxoplasma (oo)cysts in food. Therefore, no regulation exists regarding these biohazards. Nevertheless, considering their low infective dose, ingestion of foodstuffs contaminated by low quantities of these three parasites can lead to human infection. To evaluate the risk of protozoan parasites in food, efforts must be made towards exposure assessment to estimate the contamination along the food chain, from raw products to consumers. This requires determining: (i) the occurrence of infective protozoan (oo)cysts in foods, and (ii) the efficacy of control measures to eliminate this contamination. In order to conduct such assessments, methods for identification of viable (i.e. live) and infective parasites are required. This review describes the methods currently available to evaluate infectivity and viability of G. duodenalis cysts, Cryptosporidium spp. and T. gondii oocysts, and their potential for application in exposure assessment to determine the presence of the infective protozoa and/or to characterize the efficacy of control measures. Advantages and limits of each method are highlighted and an analytical strategy is proposed to assess exposure to these protozoa.

TITLE: Estimation de la viabilité et infectiosité des stades (kystes et oocystes) de Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii transmis par la nourriture et l'eau : une revue des méthodes. ABSTRACT: Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii sont des parasites protozoaires qui ont été soulignés comme agents pathogènes émergents dans les aliments par l'Organisation des Nations Unies pour l'alimentation et l'agriculture et l'Organisation Mondiale de la Santé. Selon l'Autorité Européenne de Sécurité des Aliments, 4786 épidémies d'origine alimentaire et hydrique ont été enregistrées en Europe en 2016, dont 0.4% ont été attribuées à des parasites, incluant Cryptosporidium, Giardia et Trichinella. Jusqu'en 2016, aucune méthode standardisée n'était disponible pour détecter les kystes de Giardia et les oocystes de Cryptosporidium et Toxoplasma dans les aliments. Aucune réglementation n'est donc proposée concernant ces dangers. Cependant, compte tenu de leur faible dose infectieuse, l'ingestion d'une quantité d'aliments faiblement contaminés peut entraîner une infection de l'homme. Pour évaluer le risque lié aux protozoaires dans les aliments, des efforts doivent être faits dans l'évaluation de l'exposition pour estimer la contamination le long de la chaîne alimentaire, depuis la matière première jusqu'aux consommateurs. Cette évaluation nécessite de déterminer : (i) la prévalence de parasites infectieux dans les aliments, (ii) l'efficacité des mesures de maîtrise pour éliminer cette contamination. Pour mener une telle évaluation, des méthodes capables d'identifier des parasites viables (vivants) et infectieux sont requises. Cette revue décrit les méthodes actuellement disponibles permettant d'évaluer l'infectiosité et la viabilité des kystes de G. duodenalis et des oocystes de Cryptosporidium spp. et T. gondii, et leur potentiel pour être appliquées dans l'évaluation de l'exposition pour déterminer la présence de parasites infectieux et/ou caractériser l'efficacité des mesures de maîtrise. Les avantages et limites de chaque méthode sont présentés et une stratégie d'analyses est proposée pour évaluer l'exposition à ces protozoaires.

Criptosporidiosis y una salud / Cryptosporidiosis and one health

RESUMEN Criptosporidiosis es una enfermedad diarreica (zoonótica) causada por el parásito Cryptosporidium, el cual infecta al intestino. Se producen tanto en el ser humano como en diferentes animales de granja, mascotas y animales silvestres. Se subestima su papel y poco se conoce sobre su epidemiología, debido al escaso interés que suscita y la limitada disponibilidad de métodos de diagnóstico. La salud de las poblaciones tanto humana como animal, están relacionadas con una estrecha interacción de tres factores: el ambiente, los agentes patógenos, y las poblaciones, lo cual determina el estado de salud y enfermedad. El enfoque, conocido como «Una Salud¼, es una estrategia global en crecimiento que se está adoptando por una diversidad de organizaciones y responsables de las políticas en respuesta a la necesidad de enfoques integrados. Este enfoque puede ser relevante para una amplia gama de objetivos mundiales de desarrollo. Cada vez se habla más de salud integral, de salud holística. Parece que somos más conscientes de que la salud no se reduce a algo puramente biológico, sino que afecta a toda la persona. No es sólo ver al enfermo globalmente, es partir de la complejidad del ser humano y del mundo entero atravesado por la vulnerabilidad e interaccionar con la totalidad de los sujetos.(AU)

ABSTRACT Cryptosporidiosis is a diarrheal disease (zoonotic) caused by the parasite Cryptosporidium, which infects the intestine. It occurs in humans and in different pets, farm and wild animals. Its role is underestimated and little is known about its epidemiology, due to the low interest it draws and the limited availability of diagnostic methods. The population health, both human and animal, is related to a close interaction of three factors: the environment, pathogens, and population, which determines the state of health and disease; some of the diseases considered as emerging, that appeared in the last years. The approach, known as "One Health", is a growing global strategy being adopted by a diversity of organizations and policy makers in response to the need for integrated approaches. This approach may be relevant to a wide range of global development goals. There is more and more talk about holistic health. It seems that we are more aware that health is not reduced to something purely biological, but affects the whole person. It is not only seeing the patient globally, it is starting from the complexity of the human being and the whole world crossed by vulnerability and interacting with the totality of the subjects.(AU)

Cryptosporidium spp. em idosos residentes em instituições de longa permanência no sul do Brasil / Cryptosporidium spp. in older adults living in long-term care facilities in southern Brazil

Introdução: A criptosporidiose é uma doença mundial, levando a quadros desde assintomáticos à diarreia grave. O presente estudo teve como objetivo detectar oocistos de Cryptosporidium spp. em idosos residentes em instituições de longa permanência na cidade de Lages, estado de Santa Catarina, Brasil, buscando avaliar a situação da parasitose, relativamente desconhecida na população idosa, foi avaliada por meio de um método diagnóstico fácil e de baixo custo, porém nem sempre realizado na rotina laboratorial. Métodos: Foram coletadas amostras dos exames Parasitológicos de fezes de 93 idosos residentes em instituições de longa permanência no período de setembro a novembro de 2015. Resultados: Os oocistos do protozoário foram detectados pela técnica de Ziehl-Neelsen modificada. A positividade para oocistos de Cryptosporidium spp. foi de 6,45% (6/93). Em relação ao sexo, a positividade foi de 8,9% (4/45) para mulheres e 2,1% (2/48) dos homens. Todos os pacientes tinham idade entre 60 e 70 anos. Em relação à consistência fecal, todas as amostras foram descritas como diarreia. Conclusão: A criptosporidiose é uma infecção parasitária importante e debilitante que afeta os idosos e precisa ser tratada. Os cuidadores devem estar cientes disso, para que tenham cuidados adequados, especialmente de higiene individual e uso de utensílios que podem transmitir a infecção aos demais idosos que residem nessas instituições. (AU)

Introduction: Cryptosporidiosis is a worldwide disease whose characteristics range from absence of symptoms to severe diarrhea. The present study aimed to detect Cryptosporidium spp. oocysts in older adults living in long-term care facilities in the city of Lages, state of Santa Catarina, Brazil. The situation of this parasitosis, which is relatively unknown in the older population, was evaluated using an easy and low-cost diagnostic method, although not always performed in laboratory routine. Methods: Stool samples were obtained from 93 older adults living in long-term care facilities from September to November 2015. Results: Protozoan oocysts were detected by the modified Ziehl-Neelsen technique. Positivity for Cryptosporidium spp. oocysts was 6.45% (6/93). Regarding sex, 8.9% (4/45) of women and 2.1% (2/48) of men were positive. All patients were aged between 60 and 70 years. Regarding fecal consistency, all samples were described as diarrhea. Conclusion: Cryptosporidiosis is an important, debilitating parasitic infection that affects older age and needs to be addressed. Caregivers must be aware of it in order to provide appropriate care, especially in terms of individual hygiene and use of tools that may transmit infection to other older adults living in these facilities. (AU)

Innate immune responses play a key role in controlling infection of the intestinal epithelium by Cryptosporidium.

Cryptosporidium infection leads to acute diarrhea worldwide. The development of cryptosporidiosis is closely related to the immune status of its host, affecting primarily young ruminants, infants, and immunocompromised individuals. In recent years, several studies have improved our knowledge on the immune mechanisms responsible for the control of the acute phase of the infection and have highlighted the importance of innate immunity. The parasite develops in the apical side of intestinal epithelial cells, giving these cells a central role, as they are both the exclusive host cell for replication of the parasite and participate in the protective immune response. Epithelial cells signal the infection by producing chemokines, attracting immune cells to the infected area. They also actively participate in host defense by inducing apoptosis and releasing antimicrobial peptides, free or incorporated into luminal exosomes, with parasiticidal activity. The parasite has developed several escape mechanisms to slow down these protective mechanisms. Recent development of several three-dimensional culture models and the ability to genetically manipulate Cryptosporidium will greatly help to further investigate host-pathogen interactions and identify virulence factors. Intestinal epithelial cells require the help of immune cells to clear the infection. Intestinal dendritic cells, well known for their ability to induce and orchestrate adaptive immunity, play a key role in controlling the very early steps of Cryptosporidium parvum infection by acting as immunological sentinels and active effectors. However, inflammatory monocytes, which are quickly and massively recruited to the infected mucosa, seem to participate in the loss of epithelial integrity. In addition to new promising chemotherapies, we must consider stimulating the innate immunity of neonates to strengthen their ability to control Cryptosporidium development. The microbiota plays a fundamental role in the development of intestinal immunity and may be considered to be a third actor in host-pathogen interactions. There is an urgent need to reduce the incidence of this yet poorly controlled disease in the populations of developing countries, and decrease economic losses due to infected livestock.

Measurement of Elastic Modulus and Vickers Hardness of Surround Bone Implant Using Dynamic Microindentation - Parameters Definition

The clinical performance of dental implants is strongly defined by biomechanical principles. The aim of this study was to quantify the Vicker's hardness (VHN) and elastic modulus (E) surround bone to dental implant in different regions, and to discuss the parameters of dynamic microindantion test. Ten cylindrical implants with morse taper interface (Titamax CM, Neodent; 3.5 mm diameter and 7 mm a height) were inserted in rabbit tibia. The mechanical properties were analyzed using microhardness dynamic indenter with 200 mN load and 15 s penetration time. Seven continuous indentations were made distancing 0.08 mm between each other perpendicularly to the implant-bone interface towards the external surface, at the limit of low (Lp) and high implant profile (Hp). Data were analyzed by Student's t-test (a=0.05) to compare the E and VHN values obtained on both regions. Mean and standard deviation of E (GPa) were: Lp. 16.6 ± 1.7, Hp. 17.0 ± 2.5 and VHN (N/mm2): Lp. 12.6 ± 40.8, Hp. 120.1 ± 43.7. No statistical difference was found between bone mechanical properties of high and low profile of the surround bone to implant, demonstrating that the bone characterization homogeneously is pertinent. Dynamic microindantion method proved to be highly useful in the characterization of the individual peri-implant bone tissue.

O desempenho clínico de implantes dentais é fortemente definido por princípios biomecânicos. Este trabalho objetivou quantificar a Dureza Vickers (VHN) e módulo de elasticidade (E) do osso periimplantar e discutir parâmetros metodológicos de ensaio dinâmico de indentação. Foram utilizados 10 implantes de corpo cilíndrico com interface cone morse, (Titamax CM; Neodent, Curitiba, PR, Brasil), diâmetro de 3.5 mm e altura de 7 mm inseridos em tíbia de coelho recém obtidas após abate dos animais. As propriedades mecânicas foram analisadas usando penetrador dinâmico de microdureza Vickers (CSM Micro-Hardness Tester; CSM Instruments, Peseux, Switzerland) com carga de 200 mN e tempo de penetração de 15s. Foram feitas 7 indentações no osso cortical na base da rosca (Br) e na ponta da rosca (Pr) na direção perpendicular ao implante, com distância entre elas de 0,08 mm perpendicular a interface osso implante em direção a superfície esterna. Os dados foram analisados por meio de teste t-Student (P<0,05). O valores médios e desvio padrão de E (GPa) foram: Br. 16,6 ± 1,7A; Pr. 17,0 ± 2,5A e VHN (N/mm2): Br. 125,6 ± 40,8A; Pr. 120,1 ± 43,7A. Não houve diferença significativa entre as propriedades mecânicas avaliadas no osso na base e na ponta da rosca do implante, demonstrando que a caracterização desta estrutura de forma homogênea em análises computacionais é pertinente. O método de indentação dinâmica mostrou ser altamente útil na caracterização individualizada do tecido ósseo periimplantar.

Elongation factor-1α is a novel protein associated with host cell invasion and a potential protective antigen of Cryptosporidium parvum.

The phylum Apicomplexa comprises obligate intracellular parasites that infect vertebrates. All invasive forms of Apicomplexa possess an apical complex, a unique assembly of organelles localized to the anterior end of the cell and involved in host cell invasion. Previously, we generated a chicken monoclonal antibody (mAb), 6D-12-G10, with specificity for an antigen located in the apical cytoskeleton of Eimeria acervulina sporozoites. This antigen was highly conserved among Apicomplexan parasites, including other Eimeria spp., Toxoplasma, Neospora, and Cryptosporidium. In the present study, we identified the apical cytoskeletal antigen of Cryptosporidium parvum (C. parvum) and further characterized this antigen in C. parvum to assess its potential as a target molecule against cryptosporidiosis. Indirect immunofluorescence demonstrated that the reactivity of 6D-12-G10 with C. parvum sporozoites was similar to those of anti-ß- and anti-γ-tubulins antibodies. Immunoelectron microscopy with the 6D-12-G10 mAb detected the antigen both on the sporozoite surface and underneath the inner membrane at the apical region of zoites. The 6D-12-G10 mAb significantly inhibited in vitro host cell invasion by C. parvum. MALDI-TOF/MS and LC-MS/MS analysis of tryptic peptides revealed that the mAb 6D-12-G10 target antigen was elongation factor-1α (EF-1α). These results indicate that C. parvum EF-1α plays an essential role in mediating host cell entry by the parasite and, as such, could be a candidate vaccine antigen against cryptosporidiosis.

Determination of the recovery efficiency of cryptosporidium oocysts and giardia cysts from seeded bivalve mollusks.

The intestinal parasites Cryptosporidium and Giardia are transmitted by water and food and cause human gastroenteritis. Filter-feeding bivalve mollusks, such as oysters and mussels, filter large volumes of water and thus concentrate such pathogens, which makes these bivalves potential vectors of disease. To assess the risk of infection from consumption of contaminated bivalves, parasite numbers and parasite recovery data are required. A modified immunomagnetic separation (IMS) procedure was used to determine Cryptosporidium oocyst and Giardia cyst numbers in individually homogenized oysters (Crassostrea gigas) and mussels (Mytilus edulis). About 12% of the commercial bivalves were positive, with low (oo)cyst numbers per specimen. The recovery efficiency of the IMS procedure was systematically evaluated. Experiments included seeding of homogenized bivalves and whole animals with 100 to 1,000 (oo)cysts. Both seeding procedures yielded highly variable recovery rates. Median Cryptosporidium recoveries were 7.9 to 21% in oysters and 62% in mussels. Median Giardia recoveries were 10 to 25% in oysters and 110% in mussels. Giardia recovery was significantly higher than Cryptosporidium recovery. (Oo)cysts were less efficiently recovered from seeded whole animals than from seeded homogenates, with median Cryptosporidium recoveries of 5.3% in oysters and 45% in mussels and median Giardia recoveries of 4.0% in oysters and 82% in mussels. Both bivalve homogenate seeding and whole animal seeding yielded higher (oo)cyst recovery in mussels than in oysters, likely because of the presence of less shellfish tissue in IMS when analyzing the smaller mussels compared with the larger oysters, resulting in more efficient (oo)cyst extraction. The data generated in this study may be used in the quantitative assessment of the risk of infection with Cryptosporidium or Giardia associated with the consumption of raw bivalve mollusks. This information may be used for making risk management decisions.

Assessing the infection risk of Giardia and Cryptosporidium in public drinking water delivered by surface water systems in Sao Paulo State, Brazil.

A survey of Giardia and Cryptosporidium was conducted in surface water used as drinking water sources by public water systems in four densely urbanized regions of Sao Paulo State, Brazil. A Quantitative Microbial Risk Assessment, based on protozoa concentrations, was performed to estimate the probability of protozoa infection associated with drinking water ingestion. A total of 206 source water samples were analyzed over a 24 month period using the USEPA Method 1623. The risk of infection was estimated using an exponential dose response model, children and adults exposure and a gamma distribution for (oo)cyst concentrations with three scenarios for treating censored data. Giardia was detected in 102 of the samples, and 19 of them were also positive for Cryptosporidium, with maximum concentrations of 97.0 cysts/L and 6.0 oocysts/L, respectively. Risk distributions were similar for the three scenarios. In the four regions, the estimated risk of Giardia infection per year, for adults and children, ranged from 0.29% to 2.47% and from 0.08% to 0.70%, respectively. Cryptosporidium risk infection varied from 0.15% to 0.29% for adults and from 0.04% to 0.08% for children. In both cases, the calculated risk surpassed the risk of infection of 10(-4) (1:10,000) defined as tolerable by USEPA for a yearly exposure. The probability of Giardia infection was very close to the rates of acute diarrheic disease for adults (1% to 3%) but lower for children (2% to 7%). The daily consumption of drinking water was an important contributing factor for these differences. The Microbiological Risk Assessment carried out in this study provides an indication of infection risks by Giardia and Cryptosporidium in the population served by these source waters. Strategies for source water protection and performance targets for the water treatment should be established to achieve the required level of public health risk.

Direct and indirect QMRA of infectious Cryptosporidium oocysts in reclaimed water.

Water scarcity leads to an increased use of reclaimed water, which in turn calls for an improvement in water reclamation procedures to ensure adequate quality of the final effluent. The presence of infectious Cryptosporidium oocysts (IOO) in reclaimed water is a health hazard for users of this resource. Here, we gathered information on Cryptosporidium (concentrations, infectivity and genotype) in order to perform quantitative microbial risk assessment (QMRA). Moreover, data concerning the spores of sulphite-reducing clostridia (SRC) were used to undertake QMRA at a screening level. Our results show that the probability of infection (PI) by Cryptosporidium depends on the tertiary treatment type. The mean PI using the exponential dose-response model was 3.69 × 10(-6) in tertiary effluents (TE) treated with UV light, whereas it was 3 log(10) units higher, 1.89 × 10(-3), in TE not treated with this disinfection method. With the ß-Poisson model, the mean PI was 1.56 × 10(-4) in UV-treated TE and 2 log(10) units higher, 4.37 × 10(-2), in TE not treated with UV. The use of SRC to perform QMRA of Cryptosporidium showed higher PI than when using directly IOO data. This observation suggests the former technique is a conservative method of QMRA.

Effectiveness of water treatment for the removal of Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia are intestinal parasites of humans and of many other species of animals. Water constitutes an important route of transmission for human infections in both developed and developing countries. In Poland, contamination of water sources with oocysts/cysts is not routinely monitored and scientific research in this field is scarce. Our aim was to compare the contamination of surface and treated water and thus the success of water treatment processes. Water samples (n=94) of between 30 l (surface water) to over 1000 l for tap water, were taken in the period of 2008-2009 using specially constructed equipment with cartridge filtration (Filta-Max; IDEXX, USA). Immunofluorescent assay, and nested polymerase chain reaction were used for the detection of parasites. Cryptosporidium oocysts were found in 85% of surface water and in 59% of raw (intake) water samples. Oocysts were also detected in treated water (16%) but were absent in samples of swimming pool water. The highest mean number of Cryptosporidium oocysts [geometric mean (GM)=61/10 l] was found in samples of rinsing water. Giardia cysts were observed in 61% of surface water samples, in 6% of raw water and in 19% of treated water, with the highest number of cysts noted in rinsing water samples (GM=70 cysts/10 l). Our study highlights the frequent occurrence of parasites in surface waters in Poland and the effectiveness of water treatment for the removal of parasites from drinking water.

Cryptosporidiosis: host immune responses and the prospects for effective immunotherapies.

Cryptosporidium spp. that develop in intestinal epithelial cells are responsible for the diarrhoeal disease cryptosporidiosis, which is common in humans of all ages and in neonatal livestock. Following infection, parasite reproduction increases for a number of days before it is blunted and then impeded by innate and adaptive immune responses. Immunocompromised hosts often cannot establish strong immunity and develop chronic infections that can lead to death. Few drugs consistently inhibit parasite reproduction in the host, and chemotherapy might be ineffective in immunodeficient hosts. Future options for prevention or treatment of cryptosporidiosis might include vaccines or recombinant immunological molecules, but this will probably require a better understanding of both the mucosal immune system and intestinal immune responses to the parasite.

Immunogenicity of orally administrated recombinant Lactobacillus casei Zhang expressing Cryptosporidium parvum surface adhesion protein P23 in mice.

Cryptosporidium parvum, an intestinal apicomplexan parasite, is a significant cause of diarrheal diseases in both humans and animals. What is more, there is no promising strategy for controlling cryptosporidiosis. In this study, the P23 immunodominant surface protein of C. parvum sporozoites was stably expressed in the Lactobacillus casei Zhang strain and its immunogenicity was evaluated in a mouse model. The molecular weight (23 kDa) and immunogenicity of p23 gene expressed by L. casei Zhang were similar to that of the native P23 protein. Oral immunization with control L. casei Zhang and recombinant L. casei Zhang-p23 activated the mucosal immune system to elicit serum immunoglobulin G (IgG) and mucosal IgA in mice. Furthermore, the expression of cytokines such as IL-4, IL-6, and IFN-γ in splenocytes of mice was detected by real-time PCR after oral immunization. P23-specific immunocyte activation was also verified. These findings indicate that the live L. casei Zhang vector may be a new tool for the production of mucosal vaccines against cryptosporidiosis in animals.

[Epidemiological aspects of gastrointestinal infections]. / Epidemiologische Aspekte gastrointestinaler Infektionen.

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate seasonal patterns and age-associated trends of the main bacterial, viral, and parasitic enteric pathogens in Southwest Germany. PATIENTS AND METHODS: From January 2002 through December 2008 a total of 99,057 patients were tested for Norovirus, Rotavirus, bacterial pathogens, Cryptosporidium parvum (C. parvum), and Giardia lamblia (G. lamblia). RESULTS: All these pathogens were detected throughout the whole year. But there were distinctive seasonal patterns of activity of the following pathogens being detected: norovirus was detected mainly from September through April. The highest rotovirus activity was observed from December through June. But bacterial pathogens und C. parvum were found mainly from June to November. The percentage of positive results during the months with the highest activity was 10 - 49% for norovirus, 25% - 41% for rotavirus, 14 - 18% for bacterial infection and 3 - 4 % for C. parvum. G. lamblia and adenovirus were found throughout the year in 7 - 15% and 3 - 10% of samples, respectively. Moreover, the detection rate of different pathogens depended on patient age. In infants younger than one year, rotavirus, norovirus and adenovirus were most frequently isolated pathogenes. Stool samples from kindergarden- and school-age children were positive largely for bacterial pathogens such as Salmonella and Campylobacter particularly in late summer or early autum. In patients older than 60 years, norovirus, rotavirus, and toxin producing Clostridium difficile strains were the most common pathogens. CONCLUSIONS: In view of the age and season related frequency of detection of enteric pathogens, a step-by-step diagnosis of gastrointestinal tract infections is recommended. Considering that most pathogens are detected sporadically over the whole year, the analysis of negative samples should be appropriately expanded. The knowledge of seasonal occurrence can also be applied to improve the application of hygienic measures.