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The study's aim was to analyze the population structure of enterococci causing human invasive infections in a medium-sized Argentinian Hospital coincidental with a 5 year-period of increased recovery of antibiotic resistant enterococci (2010-2014). Species identification (biochemical testing/MALDI-TOF-MS), antimicrobial susceptibility (disk-diffusion) and clonal relatedness (PFGE/MLST/BAPS) were determined according to standard guidelines. β-lactamase production was determined by a nitrocefin test and confirmed by PCR/sequencing. The isolates were identified as Enterococcus faecalis and Enterococcus faecium at a 2:1 ratio. Most of the E. faecalis isolates, grouped in 25 PFGE-types (ST9/ST179/ST236/ST281/ST388/ST604/ST720), were resistant to high-levels (HLR) of gentamicin/streptomycin. A ST9 clone ( bla + /HLR-gentamicin) was detected in patients of different wards during 2014. E. faecium isolates were grouped in 10 PFGE-types (ST25/ST18/ST19/ST52/ST792), with a low rate of ampicillin resistance. Five vancomycin-resistant E. faecium, three vanA (ST792/ST25) and two vanB (ST25) were detected. The ST25 clone carried either vanA or vanB . The recovery of a bla + -ST9- E. faecalis clone similar to that described in the late 1980s in Argentina suggests the possibility of a local hidden reservoir. These results reflect the relevance of local epidemiology in understanding the population structure of enterococci as well as the emergence and spread of antimicrobial resistance in predominant enterococcal clonal lineages.

Cystic echinococcosis caused by Echinococcus granulosus is a major zoonosis of public health significance in the Patagonian region of Argentina. This investigation sought to test the hypothesis that the persistence and dispersion of the parasite eggs can be explained by physical and meteorological parameters along with final host infection and behaviour. This observational study was carried out over a five-year period within an enclosure where two dogs harbouring a worm burden ranging from 100 to 1000 mature adult E. granulosus, as well as two uninfected dogs, had previously been kept for six months. Environmental canine faeces, topsoil, pond water, and sediment samples were examined to control for the presence of eggs and coproantigens of the parasite using microscope-based techniques and copro-ELISA plus copro-Western Blot tests. The parasite eggs were detected up to 41 months later in faeces from infected dogs, soil and sediment, and coproantigen tests remained positive for up to 70 months in faeces. Overall, parasite eggs were found within a maximum distance of 115 m from the contaminated dog faeces deposition site. Our findings indicate that under Patagonian environmental conditions, egg persistence and dispersion seem to be related to the worm burden and habits of the infected dog, to prevailing wind direction and to the existence of low bushes as well as natural bodies of water. The present study is the first to provide direct evidence of interaction between bioclimatic conditions and E. granulosus egg dispersion under Patagonian field conditions.

The degree of antagonism exercised by fungi on geohelminth development varies according to the morphological alterations caused by different fungal species. Saprophytic fungi may exert ovicidal or ovistatic effects. The aim of this study was to apply scanning electron microscopy (SEM) to observe the action of two soil saprophytic species of Chrysosporium (C. indicum and C. keratinophylum) on Toxocara canis eggs. The fungal strains to be tested were incubated for 28 days at 28°C in 2% water agar with a suspension of unembryonated T. canis eggs. A suspension of T. canis eggs in 2% water agar was used as control group. The assay was done in triplicate for each fungus and the control group. SEM observations were performed on the 4th, 7th, 14th, 21st, and 28th day after inoculation. The effect of the fungi on eggs was evaluated in accordance with the alterations observed on the surface and the changes in the normal characteristics of the eggs. Hyphae around the eggs, appresoria penetrating the shell and changes in the typical egg membrane were observed in this assay. Type 3 effect (alterations that occur both in the embryo and the shell, and hyphal penetration of the eggs) was the prevalent effect. SEM allowed us to observe clearly the morphological alterations in T. canis eggs due to the effect of C. indicum and C. keratinophylum. Both saprophytic species of Chrysosporium alter the egg structure and alterations increase as exposure increases., (Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Taenia hydatigena eggs were investigated for morphological and physiological changes under water stress conditions. Fresh eggs were exposed at 31%, 47% and 89% of relative humidity (RH), and survival, size and ultrastructural changes were accounted up to 365 days of exposition. The article shows how each RH environment affects the vitality of the eggs. Results of this study suggest that T. hydatigena eggs have mechanisms to withstand water stress, indicating that the eggs clustering improves protection against desiccation, and that endogenous metabolism using triacylglycerols play an important role in the maintenance of embryo vitality under low, medium and high relative humidity conditions. This contributes to understanding the water stress resistance mechanism in eggs belonging to Taeniidae family. The findings shown herein have provided a basis to better comprehend basic biology and epidemiology of the cysticercosis caused by T. hydatigena., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Blastocystis spp. is the most common protozoan detected in human stool samples. In developing countries, infection rates are higher than 20%. The presence of this parasite in the feces of several host species suggests its zoonotic potential. The clinical relevance and the pathogenic role of Blastocystis spp. in the intestinal tract remain unclear. There are several clinical reports that recognize it as the etiologic agent of several intestinal disorders such as diarrhea, inflammatory bowel disease and ulcerative colitis, although the pathogenicity of this parasite has not been proved yet. This wide range of clinical manifestations could be related to the genetic diversity exhibited by this parasite., (Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Echinococcus granulosus sensu lato (E. granulosus sl) must be considered as a species complex, comprising Echinococcus granulosus sensu stricto (E. granulosus ss, genotypes G1-G3), Echinococcus equinus (G4), Echinococcus ortleppi (G5) and Echinococcus canadensis (G6-G10) although the species status of E. canadensis is still controversial. These genotypes closely match the intermediate hosts associated strains described in earlier times among which E. canadensis G6 corresponds to the camel strain. As there are no studies concerning the development of adult stages of the G6 genotype from non-camel origin, the aims of the present study were: to characterize for the first time the development of E. canadensis G6 in dogs experimentally infected with protoscoleces derived from goats, to describe the resultant adult morphology, to evaluate the growth of their rostellar hooks from larval to adult stages and to determine the prepatent period of the strobilar stage of E. canadensis G6 derived from goats. The development of the strobilar stage of E. canadensis G6 genotype of goat origin was examined by studying the growth (variation of the total worm length) and segmentation in experimentally infected dogs at 14, 25, 35 and 56days post infection. A morphological characterization of 35-day-old worms as well as of larval and adult rostellar hooks was also carried out by conventional optical microscopic observations and/or by scanning electron microscopy. The prepatent period of the strobilar stage was assessed by microscopic examination of faeces from 2 infected dogs. Our results were compared with published data from the camel and other strains. The roles of the host, genotype and species in morphological and developmental features as well as the taxonomic position of E. canadensis G6 were discussed. The prepatent period of E. canadensis G6 genotype of goat origin was determined as at least, 41days. The obtained results contribute to increase the knowledge about the biology and genetics of E. granulosus sl complex and are also of practical usefulness for the design of disease control strategies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cryptosporidium is an opportunistic protozoan parasite of humans and animals worldwide and causes diarrheal disease that is typically self-limiting in immunocompetent hosts but often life threatening to immunocompromised individuals. However, there is a lack of completely efficient therapy available. Probiotics have attracted the attention as potential antiparasite compounds against protozoa involved in intestinal infections. This study investigated the effects of administration of probiotic Enterococcus faecalis CECT 7121 on Cryptosporidium parvum infection in immunosuppressed mice. Effects on C. parvum infection at the intestinal mucosa were studied and scored at each portion of the gut. It was demonstrated that Ef CECT 7121 interfered with C. parvum infection when both probiotic and parasite were present in the same intestinal location suggesting that Ef CECT 7121 supplementation can alleviate the negative effects of C. parvum infection.

The study of scientific production is a good indicator of the progress in research and knowledge generation. Bibliometrics is a scientific discipline that uses a set of indicators to quantitatively express the bibliographic characteristics of scientific publications. The scientific literature on the epidemiology of intestinal parasites in Argentina is scattered in numerous sources, hindering access and visibility to the scientific community. Our purpose was to perform a quantitative, bibliometric study of the scientific literature on intestinal parasites in humans in Argentina published in the period 1985-2014. This bibliometric analysis showed an increase in the number of articles on intestinal parasites in humans in Argentina published over the past 30 years. Those articles showed a collaboration index similar to that of the literature, with a high index of institutionality for national institutions and a very low one for international collaboration. The original articles were published in scientific journals in the American Continent, Europe and Asia. The use of bibliometric indicators can provide a solid tool for the diagnosis and survey of the research on epidemiology of intestinal parasites and contributes to the dissemination and visibility of information on the scientific production developed in Argentina., (Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Cystic echinococcosis caused by Echinococcus granulosus sensu lato is one of the most important helminth zoonoses in the world; it affects both humans and livestock. The disease is endemic in Argentina and highly endemic in the province of Neuquén. Considerable genetic and phenotypic variation has been demonstrated in E. granulosus, and ten different genotypes (G1-G10) have been identified using molecular tools. Echinococcus granulosus sensu lato may be considered a species complex, comprised of E. granulosus sensu stricto (G1-G3), E. equinus (G4), E. ortleppi (G5) and E. canadensis (G6-G10). In endemic areas, the characterization of cystic echinococcosis molecular epidemiology is important in order to apply adequate control strategies. A cut-off value for larval large hook total length to distinguish E. granulosus sensu stricto isolates from those produced by other species of the complex was defined for the first time. Overall, 1780 larval hooks of 36 isolates obtained from sheep (n= 11, G1), goats (n= 10, G6), cattle (n= 5, G6) and pigs (n= 10, G7) were analysed. Validation against molecular genotyping as gold standard was carried out using the receiver operating characteristic (ROC) curve analysis. The optimum cut-off value was defined as 26.5 μm. The proposed method showed high sensitivity (97.8%) and specificity (91.1%). Since in most endemic regions the molecular epidemiology of echinococcosis includes the coexistence of the widely distributed E. granulosus sensu stricto G1 strain and other species of the complex, this technique could be useful as a quick and economical tool for epidemiological and surveillance field studies, when fertile cysts are present.

Cryptosporidium parvum from 73 dairy calves less than two months old from Buenos Aires province (Argentina) were molecularly characterized using sequence analysis of the GP60 gene. Seventy-five sequences were obtained, and seven different subtypes were identified, all belonging to the IIa subtype family. The most common subtypes were IIaA20G1R1 (27/75), IIaA22G1R1 (16/75), and IIaA18G1R1 (13/75). Subtypes IIaA21G1R1, IIaA23G1R1, IIaA16G1R1 and IIaA19G1R1 were found sporadically. Two samples contained mixed infections with IIaA21G1R1 and IIaA22G1R1. A significant association was found between subtypes and geographic location, whereas there was no relation between subtypes and presence of diarrhea. Three of the subtypes found in this study (IIaA16G1R1, IIaA18G1R1, and IIaA19G1R1) were previously identified in humans. These findings suggest that cattle could play an important role in the transmission of cryptosporidiosis to humans in Buenos Aires province., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Fecal specimens were obtained from a total of 70 dairy calves less than two months old on 11 municipalities in Buenos Aires, Argentina. After removal of fecal debris by sieving and sucrose flotation, specimens were subjected to PCR to detect the presence of Enterocytozoon bieneusi. PCR revealed a 14.3% of prevalence for E. bieneusi with 10 positive calves from 7 municipalities. Gene sequence analysis conducted in all samples positives by PCR revealed the presence of six genotypes; four previously reported in cattle as well as humans (D, I, J, and BEB4), one never reported in cattle before but previously reported in humans (EbpC), and one novel genotype (BEB10). These results constitute the first molecular characterization of E. bieneusi in Argentina, and suggest a potential risk of zoonotic transmission in this area., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cryptosporidium parvum subtype IIaA21G1R1 oocysts were used to infect dexamethasone immunosuppressed N: NIH Swiss mice. This is the first Cryptosporidium mouse model in which the relationship between infection and apoptosis has been histologically studied at each portion of the gut in order to observe this dynamic in chronic cryptosporidiosis. Histology showed developmental stages in the duodenum, proximal and distal jejunum, ileum, cecum and colon, with the small intestine remaining infected until day 35 post infection. At proximal jejunum an inverse correlation between infection and apoptosis was observed at days 28 and 35 p.i. Data suggests that jejunum could be an interesting place to carry out further studies on the dynamics of Cryptosporidium infection and apoptosis. Based on these findings, this mouse model was useful to evaluate clinical, parasitological and histological aspects of C. parvum subtype IIaA21G1R1 infection, and it will be an appropriate tool to investigate different aspects of Cryptosporidium infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Human cystic echinococcosis is a highly endemic zoonotic disease in the province of Neuquén, Patagonia Argentina, although a hydatid control programme has been carried out since 1970. Human infection due to Echinococcus canadensis (G6 genotype) is frequent in Neuquén. However, the reservoir for this species remains undetermined in a region where camels are absent. We investigated the fertility, viability and molecular epidemiology of hydatid cysts obtained from local goats, pigs and sheep in order to identify the possible reservoirs of E. canadensis (G6). We also analyzed isolates from infected dogs. A total of 67 isolates were identified by the DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene. Cysts from sheep (n=16), goats (n=23) and pigs (n=18) and adult worms from 10 infected dogs were analyzed. The fertility of the hydatid cysts was 78.6%; 90.4% and 94.4% for sheep, goats and pigs, respectively. We detected E. canadensis (G6) in 21 of 23 goat samples and in 1 dog isolate, E. canadensis (G7) in all the pig isolates, E. granulosus sensu stricto (G3) in 1 sheep and the G1 genotype in 15 sheep, 2 goats and 9 dog samples. The G1 haplotypes included the common sheep strain sequence and 2 microvariants of this sequence. E. granulosus sensu stricto (G3) is described for the first time in South America. We conclude that goats act as reservoir for E. canadensis (G6) in Neuquén, and that control strategies may have to be adapted to local molecular epidemiology to improve the control of parasite transmission., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Hydatidosis is endemic in Neuquén, Patagonia, Argentina, even though sanitary authorities have been performing a control programme since 1970. At present, the programme is in consolidation phase, and dogs have being evaluated by arecoline purgation. The aims of this study were to evaluate diagnostic performance of a coproantigen (CAg) ELISA test developed "in house" and to assess CAg detection in prepatent period. We examined 8 dogs experimentally infected with Echinococcus granulosus and 403 rural dogs in an endemic area in Neuquén using CAg ELISA test and arecoline purgation. Within the experimental dog group, sensitivity and specificity of the test were 93.6% and 88.5% respectively. In rural dogs group, the overall prevalence of canine echinococcosis was 3.7% using arecoline purgation and 12.4% by the CAg test; sensitivity and specificity of the test using arecoline purge as standard were 73.3% and 89.9% respectively. Possible cross reactions in CAg test were evaluated in rural dogs: CAg was undetectable in 96.4% of the dogs infected only with taeniids non-E. granulosus, and in 90.1% of dogs infected with non-taeniid helminths. The CAg test could detect infections within prepatent period and produced negative results after worm expulsion. Our test showed adequate diagnostic performance with experimentally and naturally infected dogs, in the epidemiological situation of Neuquén. Employment of this sensitive and practical method for surveillance in the control programme in Neuquén would improve screening of canine echinococcosis by detecting infected dogs even with low burdens or within prepatent period.

The aim of the present paper was to evaluate the larvicidal effect of Enterococcus faecalis CECT7121 (Ef7121) on the Toxocara canis cycle both in vitro and in vivo. For the in vitro experiments, T. canis larvae were incubated with the supernatants of Ef7121 (EI) and mutant Ef7121 (EIm), in a pre-culture of Ef7121 (EII) and in a fresh culture with Ef7121 (EIII) and the Ef7121 mutant strain (EIIIm). The viability of the larvae was calculated after a 48 h incubation. A significant reduction of the viability of T. canis larvae was observed in EI, EII and EIII. A decrease of this inhibitory effect was observed in EIm and EIIIm (p = 0.008). In the in vivo experiments, mice were orally inoculated with three doses of Ef7121. To study the probiotic persistence in the intestine, the animals were sacrificed every four days and their intestines were dissected. The initial average bacterial levels were 9.7 x 10(4) for Ef7121 (colony forming units/g). At the end of the assay the levels were 1.46 x 10(4). No bacterial translocation was detected in mesenteric lymphatic nodules and spleen. Ef7121 interference with the biological cycle was evaluated in mice challenged with T. canis. The interference was significant when the mice were challenged with probiotic and T. canis simultaneously (p = 0.001), but it was not significant when the challenge was performed 15 days after administration of the bacterial inoculum (p = 0.06). In conclusion, Ef7121 possessed in vitro and in vivo larvicidal activity.

The objective of this study was to evaluate the influencing factors in the occurrence of coliforms in the drinking water in La Plata (Argentina) from June 1999 to June 2001. A total of 180 samples were collected from Rio de La Plata (102 samples) and Puelche Aquifer (78 samples); 45 samples were collected for each of the four seasons. The membrane filter procedure was used for isolating bacteria, and each sample was tested for chlorine and pH. The highest percentage of samples positive for coliforms in the two tested media was obtained in summer while the highest percentage of negative samples was obtained in winter. No Escherichia coli was isolated. The percentage of injured coliforms fluctuated between 70 and 100%. The most frequently isolated bacteria was Enterobacter cloacae in summer, Enterobacter agglomerans in autumn and Klebsiella oxytoca in winter and spring. Significant correlations were observed between coliforms and the distance from the initial treatment point, and with the level of free chlorine. We conclude that drinking water contamination in La Plata occurs in the distribution system due to increased temperatures and reduced disinfectant levels, which result in bacterial regrowth.

The aim of this work was to investigate the lipid content of Taenia hydatigena eggs and to evaluate the role of lipids in the maintenance of embryo viability. The total lipid content of the egg was 4.5% (w/w). Five classes of neutral lipids were identified: esterified cholesterol, free cholesterol, triacylglycerols, diacylglycerols and free fatty acids. Our results suggest that triacylglycerols play a key role in the maintenance of embryo viability. In addition, we found that T. hydatigena eggs remain metabolically active by mobilisation of stored triacylglycerols. This study contributes to the understanding the survival strategies of a member of the Taeniidae family in the environment outside the host.

The presence of parasites was investigated by the examination of 1944 dog faecal samples collected from urban (n=646) and rural (n=1298) areas of the province of Neuquén, Patagonia, Argentina. Parasitic agents (PA) were found in 37.86% of samples. A total of 15 different PA were detected, including Toxocara canis (16.35%), Taenia spp./Echinococcus spp. (12.65%), Trichurisvulpis (6.06%), Giardia spp. (1.29%), Toxascaris leonina (0.56%), Ancylostomacaninum (0.41%), Dipylidium caninum (0.31%), Diphyllobothrium spp. (0.10%), among others. Several of these PA are recognized as zoonotic agents. Therefore, the results of this investigation revealed that local population is exposed to a broad spectrum of zoonotic parasites by means of environmental contamination with dog faeces. Prevalence of PA was slightly higher in rural (40.06%) than in urban (33.44%) locations. Distribution of groups of PA (cestodes, nematodes, and protozoa) showed statistical differences between both habitats. Prevalence of cestodes (18.18%) and protozoa (11.86%) was significantly higher in the rural environment than in urban areas and nematodes (29.10%) were more frequent in urban locations. Infection of dogs with Linguatula serrata and Cryptosporidium sp. was demonstrated for the first time in Neuquén. Rural dogs of the study area are under hydatic disease control program, which includes treatment with praziquantel every 6 weeks; thus, the finding of high level of cestode infection in these areas is of great relevance. The epidemiology of zoonotic parasitic infections in urban and rural dogs showed different patterns and, in consequence, different control measurements should be applied in each location., (Copyright 2009 Elsevier B.V. All rights reserved.)

Objectives: To design, implement, and evaluate a complex plan of actions aimed at reducing parasitoses in a rural town in the province of Buenos Aires, Argentina, with the involvement of the community., Methods: The project took place in General Mansilla, Buenos Aires province, in three stages. First, the initial epidemiological situation of intestinal parasitoses in the community was assessed by analyzing for parasites a series of stool samples and anal swabs taken from a sample of 522 individuals. Second, interventions were implemented: the pharmacological treatment of the infected individuals, and health education for the public involving local promoters. Third, the pharmacological treatment was evaluated with followup stool testing for those treated; the health education effort was evaluated through a survey and stool testing for parasites of those who had participated solely in the education., Results: The frequency of intestinal parasitoses was 58.2%; of the total, 43.9% were protozoa and 35.2%, helminths. The most common pathogens were Enterobius vermicularis, Blastocystis hominis, and Giardia lamblia. The deworming treatment reduced intestinal parasitoses to 15.1% (P < 0.001), and was more effective in helminths than in protozoa. Intestinal parasitoses declined following the health education intervention, in general (from 58.2% to 47.9%; P = 0.019), as well as for helminths (from 35.2% to 20.3%; P < 0.001), and hygiene habits improved significantly., Conclusions: Treatment of parasitosis and the educational intervention provided by local promoters made it possible to reduce parasitic diseases-particularly those caused by helminths-in the community under study. We recommend extending this experience to other rural communities and broadening the interventions to cutoff additional modes of transmission, such as water and food.

The genus Cryptosporidium, responsible for producing cryptosporidiosis, includes several species. Humans and livestock are the main sources of infection. Waterborne cryptosporidiosis outbreaks are associated with drinking water. The infective parasite stage is the oocyst, which is resistant to conventional potabilization treatments. In immunocompetent hosts it produces acute, self-limiting diarrhoea. In immunocompromised people, it could develop severe, life-threatening pattern forms of the infection. People with AIDS are especially susceptible to these clinical forms. Cryptosporidium infections are also considered a major cause of morbimortality in calves, which leads to important economic losses. In the last years, there has been an increase of patients suffering from different causes of immunosuppression, and the need to find an effective therapy against Cryptosporidium has become greater. In spite of the many attempts of the pharmaceutical industry to develop an effective antiparasitic agent to treat cryptosporidiosis, this infection and its clinical consequences still constitute a major public health problem. This article analizes the taxonomy, morphology, biology and life cycle of Cryptosporidium. Clinical, immunological, epidemiological features and diagnosis of cryptosporidiosis are also included. Treatment and prevention of the infection are discussed, and future tendencies are suggested for this emerging parasitic infection.

Dairy calves less than 1 month of age are commonly infected with Cryptosporidium spp. The objective of the present study was to evaluate the prevalence of Cryptosporidium spp. among dairy calves or=810 oocysts/field. This study shows that Cryptosporidium spp. is one of the causes of calf neonatal diarrhoea in a rural area of Buenos Aires, Argentina. The highest intensity of infection reported for the

The purpose of this study was to investigate the genotypes of Giardia lamblia from human and animal feces and their epidemiological and clinical characteristics in Argentina, South America. Seventy isolates, 60 from humans (adults and children), eight from dogs and two from cows were processed by polymerase chain reaction-restriction fragment length polymorphism. Data corresponding to demographic, socio-cultural and environmental variables and presence/absence of signs/symptoms were collected. The triosephosphate isomerase gene was amplified from 43 (71.66%) of the 60 human fecal samples. Among these, 3/43 (6.98%) were genotype AII and 40/43 (93.02%) were genotype B. Assemblage AII was detected in three children who lived together in a shantytown and they were oligosymptomatic and none had diarrhea. This genotype was not found in animals. Genotype B showed a high prevalence in both adults and children. It was also found in polysymptomatic people, many of whom presented diarrhea. It was also found only in one dog. The present study represents the first contribution to the knowledge of G. lamblia genotypes in Argentina.

We evaluate the prevalence of intestinal parasites in 504 people and the degree of association between environmental variables and parasites found in population, soil and water in a rural area of Argentina during 2002-2003. A structured survey was used to evaluate the environmental variables and fecal-human, soil and water samples were analyzed. The prevalence of parasites was 45.4%. Most prevalent protozoa were Blastocystis hominis (27.2%) and Giardia lamblia (6.9%), while the most prevalent helminth was Ascaris lumbricoides (3.8%). The analyzed environmental variables showing association (p < 0.05) with presence of parasites in population were: cardboard-tin or wooden house, dirt floor, home or communal water pump, faucet outside the house or public faucet and cesspool or latrine. Parasite forms were found in 82.3% of the soil samples and in 84.2% of the water samples. In both samples we found parasites that were also found in people. In this study we have found deficient sanitary conditions associated with presence of parasites in population and we have evidenced that contaminated soil and water were the source of these parasites.

The present study is the first comprehensive analysis of human cystic echinococcosis (CE) epidemiological data carried out in the province of Neuquén, Patagonia Argentina, after 34 years of uninterrupted control program. A retrospective study of all the official records of confirmed human CE cases between 1995 and 2004 was carried out. A total of 1107 cases were reported. The overall mean annual incidence (MAI) was 24.4 per 100,000 inhabitants for the total population and 9.7/100,000 in the 0-14 year group. Distribution of cases by age and sex as well as frequency of cyst locations was analysed. Children accounted for 13.3% of total cases, indicating active transmission of the disease. Territorial distribution of cases was highly heterogeneous: MAI per 100,000 ranged from 7.9 in the Metropolitan Sanitary Area to 78.4 in the western rural areas. Higher values were recorded in small communities as Pilo Lil (800/100,000). MAI showed a significant decrease from 1995 to 1999 (43.9-18.8) but remains stable from 2000 to 2004 (15.9-15.5). These results suggest that standard control measures, despite long-term implementation, are not able to produce a sustained improvement of the epidemiological status of the disease. Further studies about local transmission cycles, definite and intermediate hosts present, Echinococcus granulosus strains or cultural behaviours in small communities are required in order to adequate the control actions in Neuquén.

Objective: To evaluate the effectiveness of an intervention based on training and the use of a protocol with an automatic stop of antimicrobial prophylaxis to improve hospital compliance with surgical antibiotic prophylaxis guidelines., Design: An interventional study with a before-after trial was conducted in 3 stages: a 3-year initial stage (January 1999 to December 2001), during which a descriptive-prospective survey was performed to evaluate surgical antimicrobial prophylaxis and surgical site infections; a 6-month second stage (January to June 2002), during which an educational intervention was performed regarding the routine use of a surgical antimicrobial prophylaxis request form that included an automatic stop of prophylaxis (the "automatic-stop prophylaxis form"); and a 3-year final stage (July 2002 to June 2005), during which a descriptive-prospective survey of surgical antimicrobial prophylaxis and surgical site infections was again performed., Setting: An 88-bed teaching hospital in Entre Ríos, Argentina., Patients: A total of 3,496 patients who underwent surgery were included in the first stage of the study and 3,982 were included in the final stage., Results: Comparison of the first stage of the study with the final stage revealed that antimicrobial prophylaxis was given at the appropriate time to 55% and 88% of patients, respectively (relative risk [RR], 0.27 [95% confidence interval {CI}, 0.25-0.30]; P<.01); the antimicrobial regimen was adequate in 74% and 87% of patients, respectively (RR, 0.50 [95% CI, 0.45-0.55]; P<.01); duration of the prophylaxis was adequate in 44% and 55% of patients, respectively (RR, 0.80 [95% CI, 0.77-0.84]; P<.01); and the surgical site infection rates were 3.2% and 1.9%, respectively (RR, 0.59 [95% CI, 0.44-0.79]; P<.01). Antimicrobial expenditure was 10,678.66 US$ per 1,000 patient-days during the first stage and 7,686.05 US$ per 1,000 patient-days during the final stage (RR, 0.87 [95% CI, 0.86-0.89]; P<.01)., Conclusion: The intervention based on training and application of a protocol with an automatic stop of prophylaxis favored compliance with the hospital's current surgical antibiotic prophylaxis guidelines before the intervention, achieving significant reductions of surgical site infection rates and substantial savings for the healthcare system.

We compared three preparation techniques for critical point drying of fungus Paecilomyces lilacinus (Thom) Samson with Toxocara canis (Werner) Johnston and Taenia hydatigena Linneo eggs by scanning electron microscopy. We evaluated filtration (first), centrifugation (second), and phytoplankton network (third) in critical point drying methods. The first and third methods were advantageous for T. canis eggs because they preserved the quantity and quality of samples to obtain better images definition. The best technique for T. hydatigena eggs was the addition of phytoplankton network in critical point drying which preserved these helminth eggs.

We studied the viability and infectiousness of aged Echinococcus granulosus eggs by in vivo evaluation in ovines. Our results demonstrated that after 41 months of ageing of the eggs under environmental conditions of an inferior arid climate (Patagonia, Argentina), they were still able to produce infection in 4/4 ovines challenged with 1200 eggs per ovine. In the ovines experimentally infected with these aged eggs, the occurrence of hepatic and pulmonary cysts was determined by necropsy, histologic and genetic studies. The eggs were found in a semi-senescent stage, thus keeping their capacity to generate an infection in the intermediary ovine host.

Paecilomyces lilacinus (Thom) Samson is a saprophytic hyphomycete from the soil with biological activity on helminth eggs. We evaluated the influence in vitro of P. lilacinus on the viability of the oncospheres from Taenia hydatigena, a parasite cestode of dogs and sheep. The eggs were exposed to the fungus strain in sterile distilled water and observed by light microscopy at days 4, 7 and 14 post-inoculation, and the viability was evaluated. The viability found in the exposed P. lilacinus oncospheres was significantly different in all observations. P. lilacinus exercised a negative biological activity on T. hydatigena eggs in vitro.

Aim: To evaluate the relationships between the personal, sociocultural, and environmental characteristics, and the presence or absence of symptoms with the detection of Enterobius vermicularis (E. vermicularis) in a population sample in our region (General Mansilla, Province of Buenos Aires, Argentina), by individual and familiar analyses., Methods: E. vermicularis was diagnosed in 309 people from 70 family units residing in the urban area and the rural area of the city of General Mansilla. Each of them was surveyed so as to register personal, environmental and sociocultural data. Questions about the presence or absence of anal itch, abdominal pain and sleeping disorder were also asked. Significant associations were determined by square chi tests. Logistic regression models were adjusted by using a backward conditional stepwise method to determine the presence of this parasite in the individuals and in the families., Results: The parasites were found in 29.12% (90/309) of the individuals, with a frequency of 14.28% (20/140) among the heads of the families and of 41.42% (70/169) among the children. The only variables showing a significant association were affiliation, where the risk category was "being the son/daughter of", and the symptoms were abdominal pain, sleeping disorder, and anal itch. Families with a member infected with parasite were considered Positive Families (PF) and they were 40/70 (57.14%), only 5% (2/40) of the PF had 100% of their members infected with the parasite. The logistic regression models applied showed that the risk categories were mainly affiliation (son/daughter) and housing (satisfactory) among others., Conclusion: The presence of E. vermicularis was proved in one third of the studied population. The frequency of families with all their members infected with the parasite was very low. Most of the studied personal, sociocultural, and environmental variables did not turn out to be significantly associated with the presence of the parasite. An association with the category of "son/daughter" and housing classified as "satisfactory" was determined. The latter may be due to the fact that the people living in that category of housing have hygienic practices at home that favour the distribution of the eggs in the environment. The presence of the analysed symptoms was associated with the presence of the parasite, thus strengthening the need of periodical control of the population showing at least one of these symptoms., (Copyright 2004 The WJG Press ISSN)

The aim of this study was to relate personal data, socio-cultural and environmental characteristics, and the presence of symptoms/signs with the frequencies of Giardia spp. and Blastocystis hominis among a rural population in Buenos Aires Province, Argentina. Of the surveyed population (350), 3.7% were infected with only Giardia spp. or 22.9% with B. hominis, and 2.3% were infected with both protozoa. The frequency of infection according to sex; 6.1% of males were infected and 1.6% of females by Giardia spp., 26.7% and 19.5% by B. hominis, and 2.4% and 2.2% by both parasites, respectively. Giardia spp. was detected in only three adults (over 14 years), but B. hominis was more frequent in adults than in children. The prevalences of these protozoa in this community are lower than those reported by other Argentinean studies, which is probably associated with the low density of the studied population (5.95 inhab/km2). Statistical analysis revealed that a male sex, flooding of the home, the use of a latrine, and an abdominal pain were correlated with the presence of these parasites, which indicate the importance of these factors in rural communities.

The purpose of this study was to optimize and evaluate the purification techniques, isolation and breaking of cysts of Giardia spp from fecal samples to isolate DNA. Filtrated fecal samples were tested in 3 purification techniques: Telleman solution, sucrose and Telleman plus sucrose. The sucrose solution let us to isolate the cysts with less detritus. The cleaned cysts were splited in 3 techniques to test the breaking: osmotic shock and heat, chemistry degradation and thermic shock, enzymatic action and mechanic effect. Only the last method was successful and showed bands in agarose gel. The result of this study shows a routine and common method which could be used in the previous steps to the PCR technique for the genotypification of these parasites.

The objectives of the present work were to screen topsoil samples collected from public squares in two cities within the Argentine Patagonia for the presence of infective forms of intestinal parasites and to examine the possible relationship between positive findings and the environmental, socioeconomic, and cultural conditions of that region. For this purpose we studied 13 public squares, their 13 custodians, and 44 family groups within their respective surrounding areas. Of the 226 topsoil samples analyzed, 44.3% proved positive for infective forms of intestinal parasites, with 17.3% of these containing more than one species. The frequency of appearance of positive samples was dependent on the season of the year (p < 0.001), while presence of the parasites was related to the soil pH (p < 0.05) but independent of the soil relative humidity (p > 0.05). Some of the organisms detected are associated with zoonoses. We observed the presence of Capillaria spp. and Spirocerca spp. under cool desert climatic conditions. Within the group of custodians we detected hematologic alterations one positive serology for toxoplasmosis and documented behavior conducive to risk of infection with the parasites found in those squares. Within the family group an acquaintance with parasitic zoonoses and their prevention was an inconsistent finding, with toxocarosis and toxoplasmosis being the diseases associated with the greatest degree of ignorance. Furthermore, we consider the failure to de-parasitize pets and the practice of feeding them with raw meat, as typically found in our family survey, to be factors contributing to a greater likelihood of public square contamination. From the results obtained here, we propose a spatial organization approach for the purpose of detecting zones at risk of contracting zoonotic parasitoses within urban environments.

We investigated the presence of intestinal parasites in canine feces collected from public squares in Comodoro Rivadavia, Chubut, Argentina (45 degrees S, 68 degrees W) and determined the persistence of Echinococcus granulosus eggs in those droppings under natural environmental conditions in that region. In the first experiment, we analyzed 163 fecal samples collected from urban squares during 8 months time and found parasitic elements in 46.6%. The presence of parasites was independent of the condition of the feces (fresh or dried; P>0.05). Parasites potentially pathogenic in man were present, such as Toxocara species (spp.), Taenia spp./Echinococcus spp., Uncinarias spp., and Entamoeba spp. In the second experiment, we analyzed two canine fecal samples contaminated with E. granulosus eggs, deposited for 41 months within the natural environment. These parasitic elements persisted during the entire study as attested by light microscopy and the ELISA coproantigen test. We propose the study of the presence of intestinal parasites in canine feces within the environment as a general strategy for identifying and monitoring areas of risk for canine-related zoonoses since we were able to demonstrate the persistence of E. granulosus eggs in deposited canine feces for over 3 years within the area studied.

In a study, carried out in 2000, of the clinical and parasitological status of a Wichi Aboriginal community living in the suburbs of Tartagal, northern Salta, Argentina, 154 individuals were screened for parasitic infections. Ninety-five faecal samples were also obtained from the same population. Ninety-three percent of the subjects were positive for 1 or more of the parasites investigated by direct test and 70.5% of them had parasitic superinfection. The most frequent helminths were Strongyloides stercoralis (50.5%) and hookworm (47.4%). We found low reinfection rates and a long reinfection period after treatment and provision of safe water and sanitation. Serum reactivity of these patients was analysed by enzyme-linked immunosorbent assay and indirect immunofluorescent assay and 22.1% of them had anti-Toxocara antibodies, 16.2% were positive for a complex antigen of Leishmania braziliensis, 29.9% were positive for a complex Trypanosoma cruzi antigen, and 17.5% were positive for a specific Trypanosoma cruzi antigen, Ag 163B6/cruzipain.

Objective: To determine the presence of eggs, larva, cysts and oocysts of intestinal parasites in the soil of a suburb of Neuquén city during 1 year in order to evaluate their seasonal fluctuations in relation to climatic data and soil characteristics in the studied area., Methods: A total of 107 soil samples were processed for parasite isolation by sedimentation and flotation methods during the four seasons of the year. Meteorological data were registered and physical, chemical and structural characteristics of the soil were analysed., Results: About 28.9% of the soil samples were positive for at least one parasite form. Six protozoa species (cysts of Entamoeba sp., Enteromonas sp., Endolimax sp., Giardia sp., Iodamoeba sp. and coccidia oocysts) were recovered, but neither larvae nor eggs of human or animal helminths parasites were detected. The percentage of contaminated soil samples and the diversity of species showed a marked decrease in the warm and dry months of the summer. The soil was sandy, without vegetable cover, well drained, and with scarce organic matter content., Conclusion: The frequencies of parasite recovery and the number of species show seasonal fluctuations related to the rainfall. The importance of soil as a risk factor for the transmission of intestinal parasites in the studied area is conditioned by its structural characteristics, which prevent retaining the humidity, and by climatic variables. The interrelation of both factors determines unfavourable conditions that could explain the low level of contamination observed in soil as well as the absence of eggs and helminth larvae.

Environmental parameters influencing the distribution of parasite species in three neighbourhoods of differing socioeconomic conditions in La Plata, Argentina were analysed. Coproparasitological screenings were performed in children up to 14 years old from a marginal zone (100), a suburban neighbourhood (101), and an urban area (91) in 1999--2000. The presence of parasite species in environmental samples (water and soil) and the degree of association among parasite communities was documented and evaluated. The prevalence of infection in each population was 73.0%, 54.4% and 35.2%, respectively. The frequencies of helminths and pathogenic protozoa were both higher in the marginal zone, where sanitary and environmental conditions were significantly inferior compared with the other zones. The high prevalence of intestinal parasites in this infantile population was related to parasitic contamination of the soil and water sources in addition to deficient sanitary and sociocultural conditions. Calculation of an equitability index revealed that the specific richness was less equitable once socioeconomic conditions and hygienic practices were improved. This study demonstrates the need to implement management practices for the control of intestinal parasitoses in accordance with the environmental and sociocultural characteristics of a given ecosystem.

Fungi from the soil of public areas in La Plata, Argentina were isolated and evaluated for their biological interaction with Toxocara canis eggs in vitro. We isolated and identified two fungal species: Fusarium pallidoroseum and Mucor hiemalis. Each species was co-cultured with T. canis eggs in sterile distilled water. The samples were observed by light and scanning electron microscopy (SEM) at days 4, 7 and 14 post-inoculation. Under the conditions of our experiments, F. pallidoroseum exhibited a high ovicidal activity on T. canis eggs, whereas M. hiemalis exhibited no such effects.

The frequency of intestinal parasites (IP) in 69 children between 2 and 14 years of age was studied within the city of Neuquén, Patagonia, Argentina in relation to socioeconomic, cultural and environmental conditions. Soil type was determined and contamination with IP was investigated in 75 soil samples. Frequency of IP in children was 50.7%. Blastocystis hominis was the most frequent species (24.6%). Simultaneous infection with 2 to 4 species was found in 47,5% of the infected children. Statistical association was found between parasitic infection and family hygienic level (p < 0.001). Socioeconomic level was defined as lower middle class, however, infrastructure and public services were adequate. The climate is continental semiarid and the soil is sandy with a low organic matter level. Intestinal protozoan organisms were found in 33.3% of soil samples and Sarcocystis sp. was the most frequent species (14.6%). The parasites present in soil are related to the environmental conditions that allow their viability and development. Socioeconomic and cultural factors are important for the appearance and spread of IP in communities where sanitary conditions and infrastructure are adequate.

We studied the antibiotic sensitivity of injured coliforms isolated from drinking water of La Plata, Argentina. The antibiotic sensitivity test by the agar diffusion method were proved in: Klebsiella oxytoca (14 strains), Enterobacter aerogenes (4 strains) and Enterobacter cloacae genomic group 3 (14 strains). We found that while these impaired total coliforms were sensitive to piperacillin-tazobactam (TAZ), netilmicin (NTL), ofloxacin (OFLX), and norfloxacin (NFLX) (100%), they had resistant to aminopenicillin-sulbactam (AMS) and nitrofurantoin (NIT) (100%). The resistance to antibiotics demonstrated in these strains would point to the need to promote a rational and judicious use of antimicrobial agents while at the same time implementing a program of active vigilance aimed at ensuring the highest quality of drinking water throughout the system.

During two years (1997-1999) an investigation of possible infections of chlamydial etiology in outpatients with follicular conjunctivitis was carried out, through the use of specific assays. Fifty-seven selected patients with presumptive inclusion conjunctivitis were diagnosed by means of ophthalmoscopic examination and bilateral tarsal-conjunctiva swabbing for microorganisms. The possible presence of Chlamydia trachomatis was tested by immunofluorescence microscopy and isolation in cell culture of McCoy line. Of the 57 conjunctivitis patients screened, 37 (65%) proved to be positive by cell culture (CC) and 27 (47%) by direct immunofluorescence (IFD). A good agreement between the two assays was observed, where the CC was more sensitive than IFD. Of these 37 patients with chlamydial conjunctivitis, 23 (62%) were women, with over one-third of them ranging in age from 45 to 65 years. Their clinical records revealed an evolution period of 1 to 12 months. Eighteen (78%) of these women reported previous genital pathology, while 4 (29%) of the 14 men had a history of urethritis by Chlamydia trachomatis. A high frequency of follicular conjunctivitis by Chlamydia (65%) in the screened patients was observed, without any evidence of urogenital signs and symptoms at the moment of the study.

We screened the La Plata drinking water distribution network for fecal and total coliform bacterial indicator by purification procedures, cultivating 66 membrane-filtered samples from the two networks on m-T7 agar. Subterranean and river-derived water yielded 13 and 18 confirmed gram-negative bacillus isolates, with 54% and 72% representing total coliforms, respectively. Those from the former source were Klebsiella oxytoca, Enterobacter agglomerans, and Enterobacter aerogenes and from the latter Klebsiella oxytoca, Enterobacter agglomerans, and Enterobacter cloacae, genomic group 3. Since 58% of the samples were positive using m-T7 medium it is suggested that the inclusion in standard quality control protocols should be implemented.

Saprophytic soil fungi can exert ovicidal and ovistatic effects on helminths with differing degrees of efficiency. The representatives of such fungi from temperate regions, Paecilomyces lilacinus (Thom) Samson and P. marquandii (Masse) Hughes, exhibit recognized ovicidal activity on some nematodes. We evaluated the action in vitro of P. lilacinus and P. marquandii on the zoonotic canine roundworm eggs of Toxocara canis. Eggs exposed and unexposed to fungal samples were observed by both light and scanning electron microscopy on days 4, 7 and 14 post-inoculation. Ovicidal activity of P. lilacinus on T. canis eggs was considered to be high and that of P. marquandii to be intermediate.

The anthelmintic effect of tinidazole (100 mg/kg per day for 3 successive days) was tested in male Swiss CF-1 mice infected with second-stage Toxocara canis larvae at challenge doses of 250, 500, 1000, and 1500 embryonated eggs per mouse. The drug was given orally on days 3-5 postinfection (p.i.) to one-half of the animals, and all mice were killed on day 40 p.i. The number of larvae recovered from each mouse's brain and skeletal muscle was then scored in both groups. Tinidazole yielded a highly significant reduction in the total recovery of larvae from the test animals' brains at the second and third inoculum levels but no statistically significant reduction at the highest larval dose as compared with the values obtained in the untreated control animals.

Cryptosporidium sp. causes self-limiting gastroenteritis in immunocompetent individuals and can produce a life-threatening chronic diarrhea in immunodeficient patients. In order to obtain populations of selectively infective oocysts for inoculation in biological experiments, we developed an operational protocol for the enrichment of viable oocysts from crude fecal material. Using either fresh or formaldehyde-fixed feces as sources of viable and nonviable oocysts, respectively, we fractionated the samples on parallel discontinuous sucrose gradients and evaluated oocyst viability at different banding densities by an in vitro excystation assay. The formaldehyde-inactivated fecal samples formed no bands after centrifugation and 91.66% of the oocysts became concentrated in the pellet. Fresh fecal samples formed three bands at densities 1.062, 1.092, and 1.121, in addition to a sediment. Here the viability of the gradient-sedimented oocysts was 92.3% overall, and of those in the second band 100%. Modifications in oocyst permeability thus seems to alter their sedimentation characteristics so that consequent distribution on sucrose gradients can be correlated with oocyst viability. Discontinuous sucrose density gradient sedimentation would thus constitute a simple and rapid mean to obtain viable oocysts for use in biological models both in vivo and in vitro.

The relationship between asthma and covert toxocariasis was studied in 38 patients with asthma and 44 control individuals (without asthma). Inclusion/exclusion criteria were determined. An ELISA test based on the detection of Anti-Toxocara canis (E/S antigen) serum immunoglobulin G (Ig G) and E (Ig E) was determined in both groups. Ordinary allergens and E/S antigen of T. canis injections were used to evaluate cutaneous reactivity. The seroprevalence in patients with asthma was 68.42%, and in the control individuals was 13.63%. This difference was significant. The percentage of asthmatic patients with two antitoxocara antibodies was 26.31% and 4.54% in control individuals. All asthmatic patients with antitoxocara IgE had cutaneous reactivity to Ag E/S. We conclude that the asthmatic patients with IgE and IgG antitoxocara suffer a covert toxocarosis.

To assess the viability of the oocysts from Cryptosporidium sp, an in vitro excystation technique was developed and optimized. To this end, oocysts from Cryptosporidium sp were isolated from a sample of fecal material from a child with symptomatic cryptosporidiosis. The specimen was processed by a discontinuous-sucrose-gradient technique yielding a final suspension of purified and concentrated oocysts. The resulting oocysts were quantified by counting an aliquot in a Neubauer hemocytometer chamber and the remainder were preserved in phosphate-buffered saline at pH 7.2 and 4 degrees C. The two protocols tested through the use of 100-microliter aliquots containing 178,000 oocytes/mm3 were: I. Treatment at 37 degrees C for 24 h with 1% (v/v) bile plus 0.44% (w/v) sodium bicarbonate. Group A: pretreated with acidified trypsin (pH 2.75) at 37 degrees C for 1 h; Group B: not pretreated. II. Treatment at 37 degrees C for 24 h with 1% (v/v) bile at pH 6 and at pH 8 either with or without 0.44% (w/v) sodium bicarbonate in air or an atmosphere containing 10% (v/v) CO2. Group 1: bile, pH 6; Group 2: bile, pH 6 plus CO2; Group 3: bile, pH 6 plus bicarbonate; Group 4: bile, pH 8 plus bicarbonate; Group 5: bile, pH 6 plus bicarbonate plus CO2; Group 6: bile, pH 8 plus bicarbonate plus CO2; Group 7: bile, pH 8; and Group 8: bile, pH 8 plus CO2. We then assessed the degree of excystation in all the groups by counting the number of intact oocysts in the hemocytometer. For the first protocol, the oocyst count after excystation revealed an average of 28,000 oocysts/mm3 in Group A and 22,000 in Group B. These values indicate excystation frequencies of 84.3% and 87.6% for these two groups, respectively. The corresponding excystation percentages for groups 1-8 of protocol II were 49.5, 56.7, 83.2, 88.3, 88.3, 92.7, and 94.1, respectively. Preincubation with trypsin failed to augment oocyst excystation in Cryptosporidium in any of these experimental circumstances. The optimal conditions for oocyst excystation in these 24 h incubations were thus seen to be in the presence of 1% (v/v) bile at pH 8, and 37 degrees C under an atmosphere of 10% (v/v) CO2.

The prevalence of intestinal parasites was studied as a function of socioeconomic conditions within La Plata, Argentina. Age, sex, and environmental factors were considered. Thus, from each of three areas within the city - the first a 'marginal' zone, the second a lower-income suburb, the third a middle-income urban district - 100,101, and 91 children up to 14 years old, respectively, were examined for intestinal parasites. Giardia lamblia was the most frequent species found. The respective prevalences of intestinal parasites overall (73, 54.4, and 35.1%), of polyparasitism (61.6, 27.2, and 12.5%), and of helminthic infection (32, 10.9, and 0.0%) were the highest within the population group having significantly inferior sanitary and environmental conditions. A positive statistical association between the prevalence of intestinal parasitosis and age was observed in all three of the neighborhoods. We also noted a correspondence between the frequency of such infections and school attendance in the two suburban districts. Management practices in accordance with the specific characteristics of an urban environmental and sociocultural ecosystem are thus important for the control of intestinal-parasite infection within municipal populations.