Identification of pathogen bacteria and protozoa in treated urban wastewaters discharged in the Ebro River (Spain): water reuse possibilities.

The aim of this research work is to identify the presence of pathogens, bacteria and protozoa, in different treated urban wastewaters and to relate biological pollution with the processes used in wastewater treatment plants. A study of the possibilities for water reuse is carried out taking into account bacterial and parasite composition. The analysed bacteria and protozoa are: Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Clostridium perfringens (spore), Salmonella spp., Legionella spp., helminths eggs, Giardia, Cryptosporidium spp. and free-living amoebae (FLA). The selected municipal wastewater treatment plants (MWTPs) are located in Navarra (Spain) and the main difference between them is the use of natural lagoons as tertiary treatment in some plants. The results concerning bacteriological identification showed contamination of mainly faecal origin, and the use of natural lagoons as tertiary treatment in some MWTPs produced an important disinfection effect. Moreover, pathogen parasites such as Giardia and Cryptosporidium were not detected in the samples studied although FLA were identified in all cases.

Evaluation of an immunochromatographic dip strip test for simultaneous detection of Cryptosporidium spp, Giardia duodenalis, and Entamoeba histolytica antigens in human faecal samples.

Immunochromatographic (IC) tests may play an important role in the future diagnosis of parasitic diseases because of their speed and simplicity of use. A recently developed test to detect Cryptosporidium spp, Giardia duodenalis and Entamoeba histolytica was evaluated. Microscopy and PCR were the "gold standard" reference techniques and the results of this IC test were compared with those obtained with ELISA and IC single test for the three parasites. One hundred sixty stool samples were assayed. Using microscopy, 22 samples were diagnosed as positive for Cryptosporidium spp., 31 for Giardia duodenalis, 41 for Entamoeba histolytica/dispar, and 68 had a negative diagnosis for the three parasites. Results of IC tests show sensitivities of 70-72% for Cryptosporidium, 90-97% for Giardia and 62.5% for Entamoeba histolytica. Specificities were of 93.6-94.9%, >99% and 96.1%, respectively. In all diagnoses, agreement with microscopy and PCR was over 90%, except in the triple test and microscopy in E. histolytica detection that was 76.3%, due to the inability of microscopy to differentiate E. histolytica from nonpathogenic species such as E. dispar or E. moshkovskii. The triple stool immunoassays provide adequate sensitivities and specificities for use in outbreak situations, for screening proposals and for massive assays in endemic areas where a large number of samples must be analysed or as complementary test for individual diagnosis.

Sustainable laser-based technology for insect pest control.

Aphids damage directly or indirectly cultures by feeding and spreading diseases, leading to huge economical losses. So far, only the use of pesticides can mitigate their impact, causing severe health and environmental issues. Hence, innovative eco-friendly and low-cost solutions must be promoted apart from chemical control. Here, we have investigated the use of laser radiation as a reliable solution. We have analyzed the lethal dose required to kill 90% of a population for two major pest aphid species (Acyrthosiphon pisum and Rhopalosiphum padi). We showed that irradiating insects at an early stage (one-day old nymph) is crucial to lower the lethal dose without affecting plant growth and health. The laser is mostly lethal, but it can also cause insect stunting and a reduction of survivors' fecundity. Nevertheless, we did not notice any significant visible effect on the offspring of the surviving irradiated generation. The estimated energy cost and the harmless effect of laser radiation on host plants show that this physics-based strategy can be a promising alternative to chemical pesticides.

CHARACTERISING THE EOS SLOT-SCANNING SYSTEM WITH THE EFFECTIVE DETECTIVE QUANTUM EFFICIENCY.

As opposed to the standard detective quantum efficiency (DQE), effective DQE (eDQE) is a figure of merit that allows comparing the performances of imaging systems in the presence of scatter rejection devices. The geometry of the EOS™ slot-scanning system is such that the detector is self-collimated and rejects scattered radiation. In this study, the EOS system was characterised using the eDQE in imaging conditions similar to those used in clinical practice: with phantoms of different widths placed in the X-ray beam, for various incident air kerma and tube voltages corresponding to the phantom thickness. Scatter fractions in EOS images were extremely low, around 2 % for all configurations. Maximum eDQE values spanned 9-14.8 % for a large range of air kerma at the detector plane from 0.01 to 1.34 µGy. These figures were obtained with non-optimised EOS setting but still over-performed most of the maximum eDQEs recently assessed for various computed radiology and digital radiology systems with antiscatter grids.

Absorbed dose-to-water protocol applied to synchrotron-generated x-rays at very high dose rates.

Microbeam radiation therapy (MRT) is a new radiation treatment modality in the pre-clinical stage of development at the ID17 Biomedical Beamline of the European synchrotron radiation facility (ESRF) in Grenoble, France. MRT exploits the dose volume effect that is made possible through the spatial fractionation of the high dose rate synchrotron-generated x-ray beam into an array of microbeams. As an important step towards the development of a dosimetry protocol for MRT, we have applied the International Atomic Energy Agency's TRS 398 absorbed dose-to-water protocol to the synchrotron x-ray beam in the case of the broad beam irradiation geometry (i.e. prior to spatial fractionation into microbeams). The very high dose rates observed here mean the ion recombination correction factor, k s , is the most challenging to quantify of all the necessary corrections to apply for ionization chamber based absolute dosimetry. In the course of this study, we have developed a new method, the so called 'current ramping' method, to determine k s for the specific irradiation and filtering conditions typically utilized throughout the development of MRT. Using the new approach we deduced an ion recombination correction factor of 1.047 for the maximum ESRF storage ring current (200 mA) under typical beam spectral filtering conditions in MRT. MRT trials are currently underway with veterinary patients at the ESRF that require additional filtering, and we have estimated a correction factor of 1.025 for these filtration conditions for the same ESRF storage ring current. The protocol described herein provides reference dosimetry data for the associated Treatment Planning System utilized in the current veterinary trials and anticipated future human clinical trials.

A specific primer pair for the diagnosis and identification of Acanthamoeba astronyxis by random amplified polymorphic DNA-polymerase chain reaction.

Random amplified polymorphic DNA (RAPD) is a useful tool for species identification. The obtained band patterns can be used for specific primer pair design that is useful for species identification. In this study, a distinctive 485-bp band in Acanthamoeba astronyxis band patterns was found, using the OPC20 primer (ACTTCGCCAC). The band specificity was confirmed by hybridization, using it as a probe, against all OPC20 amplifications from different Acanthamoeba species. Once the fragment was sequenced, we used it to design a specific primer pair that was useful for the identification of different isolates as A. astronyxis species.

Comparison of 2 centrifugation procedures in the formalin-ethyl acetate stool concentration technique for the detection of Cryptosporidium oocysts.

A comparison between the standard formalin-ethyl acetate (FEA) stool concentration procedure (centrifugation at 400 g for 2 min) and a modification proposed to increase the recovery of Cryptosporidium oocysts (500 g for 10 min) was performed on 73 formalin-preserved stool specimens known to be positive for Cryptosporidium. False-negative results were obtained with both methods, but the increase in centrifugation time and force resulted in the detection of a higher number of oocysts and positive samples. The modified procedure had a sensitivity significantly higher than the standard method (99% compared to 86%, P = 0.0045).

Fuchsin fluorescence and autofluorescence in Cryptosporidium, Isospora and Cyclospora oocysts.

Cryptosporidium parvum and Isospora belli oocysts stained with carbol-fuchsin, as in a modified Ziehl Neelsen technique, fluoresce bright red under green light (546nm). Cryptosporidium oocysts tend to fluoresce more brightly the less intensely stained they appear under transmitted light; this is not the case with Isospora. Fuchsin-stained Cyclospora cayetanensis oocysts fluoresce rather dimly, but those not taking the dye retain their typical autofluorescence. Cryptosporidium and Isospora oocysts are also autofluorescent, appearing violet under u.v. light (365 nm), and green under violet (405 nm) and blue-violet light (436 nm). Their autofluorescence does not survive the staining procedure.

Use of operative transluminal coronary angioplasty as an adjunct to coronary artery bypass.

Operative transluminal coronary angioplasty (OCTA) was used to improve coronary artery bypass graft (CABG) runoff in patients having complex segmental and diffuse coronary artery obstructions. OTCA was performed during CABG through the bypass arteriotomy on 64 arteries in 58 patients. An angioplasty system specifically designed for operative use was employed. In 13 cases, angioplasty was performed both proximal and distal to the coronary arteriotomy, for a total of 77 angioplasty sites. Elective restudy was performed on 36 angioplasty sites in 28 arteries in 24 patients: Sixteen patients were restudied between 18 and 21 days (mean 16) and eight between 4 and 32 months (mean 20.5). Patency rate, as assessed at each angioplasty site, was 86.1% (20/24 studied early and 11/12 studied late). Three coronary perforations (4.7%) occurred and were repaired without perioperative infarction or other sequelae. The one operative death (1.7%) occurred in a patient with preoperative refractory cardiogenic shock. There were seven perioperative infarctions (12.1%), of which three (4.7%) were in the distribution of the coronary artery undergoing OTCA. The favorable short-term and medium-term patency rates indicate that OTCA is a useful adjunct to CABG that permits more complete revascularization of small or diffusely diseased coronary arteries.

Diplogonoporiasis presumably introduced into Spain: first confirmed case of human infection acquired outside the Far East.

A 58-year-old man who was very fond of eating raw fish and had not left Zaragoza Province in Spain in the last 20 years excreted a short chain of gravid proglottids. Treatment with 50 mg/kg of paromomycin sulfate was divided into three doses, all given within the same day, followed by administration of a laxative at night. On days 1 and 2 post-treatment, several chains of degenerated proglottids were evacuated. Only eggs were expelled on days 3 and 4. A long complete strobila including the scolex, and immature, mature, and gravid segments was spontaneously discharged on day 25 after treatment. It was identified as Diplogonoporus balaenopterae, and was the first confirmed case of diplogonoporiasis outside the Far East (all previous cases were reported from Japan, except for one recent case reported from Korea). The origin of this case, outside of its normal geographic location, may be associated with the importation of fish into Spain. The viability of the infective larval stage is evidence of its resistance to export/import conditions. Treatment with paromomycin sulfate did not result in the whole worm being discharged but several short strobilar chains showed drug-induced degeneration.

Short report: one case of nasal human myiasis caused by third stage instar larvae of Oestrus ovis.

This is a report of a case of human nasal myiasis caused by third instar larvae of the sheep nasal bot fly, Oestrus ovis. Female flies rarely deposit first instar larvae in the eye, nostrils, and external auditory canal of humans, where they usually survive only a few days without further development. One human infestation by third instar larvae of O. ovis has been recorded in an patient infected with human immunodeficiency virus (HIV) in the United Kingdom. Our report describes a new case of nasal myiasis caused by the third instar larvae of the sheep bot fly in a patient without HIV or signs of any other disease.

Western blot applied to the diagnosis and post-treatment monitoring of human hydatidosis.

The serologic diagnosis of hydatidosis (caused by Echinococcus granulosus) can be made by different techniques, although the lack of standardization of the antigens affects the sensitivity, specificity and concordance among the different tests. We have applied the Western-Blot (WB) technique, associated with a purified antigen from sheep hydatid fluid, at 60 samples of serum from 14 patients suffering echinococcosis in different bodily locations, monitored for 3 years. The WB test enabled the detection of antibodies in the pre-surgical samples for proteins of 12-14, 16, 20, 24-26, 34, 39 and 42 kDa in molecular weight in 15-96% of the patients. The combination involving 2 of the 3 proteins of 20, 39 and 42 kDa has made it possible to diagnose 100% of the cases. The antibodies specific to proteins 39 and 42 kDa disappeared in less than one year in the patients cured after surgery, while in patients with persistent or recurrent parasitism the bands present before surgery persisted or other new ones appeared. The WB with purified antigens proved to be highly useful in the diagnosis and post-surgical monitoring of hydatidosis patients. The antigen used is proposed as a standard antigen for the diagnosis and follow-up of pre- and postsurgical hydatidosis.

The renin-angiotensin system is not responsible for hypertension following coronary artery bypass grafting.

Systemic hypertension following coronary artery bypass graft (CABG) procedures has been reported to occur in 15% to 80% of cases. Previous reports have implicated the renin-angiotensin system as being responsible, at least in part, for this phenomenon. In this prospective study, 18 previously normotensive subjects were studied before, during, and after CABG. In 4 patients (22%), paroxysmal postoperative hypertension developed (systolic blood pressure greater than 150 mm Hg). There were no differences between the normotensive and hypertensive groups in plasma renin activity, angiotensin II level, or aldosterone level. Despite the trend toward elevation of these variables during cardiopulmonary bypass (CPB), all had returned to control levels within two hours after CPB, whether or not hypertension developed. Serum norepinephrine levels were elevated (.10 greater than p greater than .05) in the hypertensive group at the time hypertension developed. No other relationship or pattern could be defined to distinguish the hypertensive from the normotensive group. The renin-angiotensin system does not appear to be responsible for paroxysmal hypertension following CABG.

Emerging and reemerging pathogens.

From 1973 to 1995, 29 new and reemerging pathogenic microbes were recognized. However, in discussions about emerging infectious diseases, the focus is often on the clinical effects of the host-parasite relationship, rather than the examination of the biology of the pathogen. Many of what we refer to as emerging diseases are characterized better as 'diseases of human progress'. Thus, the aerosolization of water has played an important role in the emergence of Legionella pneumophila infections. New diseases are superimposed on endemic diseases such as diarrhoeal diseases, malaria and tuberculosis. In addition, many pathogens are becoming increasingly resistant to standard antimicrobial drugs, making treatment difficult and in some cases impossible. We summarize our experience on emerging parasitic diseases (primary amoebic meningoencephalitis, respiratory cryptosporidiosis, and diplogonoporiasis), and selected problems of bacterial resistance (MDR tuberculosis caused by Mycobacterium bovis and macrolide-resistance mechanisms of Streptococcus pneumoniae and S. pyogenes).

Detection of Blastocystis sp. in pigs in Aragon (Spain).

Infection by Blastocystis sp. has been detected for the first time in pigs in Spain. Detection was carried out by the ethyl acetate-formalin concentration method and direct microscopy. Vacuolated cells of Blastocystis sp. were found in stool samples from 27 of 360 pigs examined (7.5%). The protozoan was present on nine of 17 pig farms studied (53%). Infected pigs ranged from 1 month old to adults, but the organism was most frequent in 1-2 month old (18.4%) and 2-6 month old pigs (15.4%). No correlation was found between Blastocystis sp. infection and occurrence of diarrhoea in pigs.

Detection of Cryptosporidium oocysts in extra-intestinal tissues of sheep and pigs.

Extra-intestinal infections by Cryptosporidium parvum have been detected in pigs and sheep. Detection was carried out by imprints of the mucosa of different organs and viscera in 55 sheep and 57 pigs slaughtered at three abattoirs in Zaragoza (northeast Spain). Imprints were stained by using a modified Ziehl-Neelsen technique. In addition to intestinal infections, cryptosporidial oocysts were found in the gall-bladders of two pigs which were 2 months old, and in some organs of sheep aged 5 days or more, including the gall-bladder (5), mesenteric lymph nodes (2), trachea (7), lung (3) and the uterus of one lamb.

Prevalence of Cryptosporidium and Giardia infections in cattle in Aragón (northeastern Spain).

Faecal samples from 554 bovines randomly selected at 30 farms in Aragón were examined to investigate the prevalence of Cryptosporidium and Giardia infections. C. parvum oocysts were identified by using the Ziehl-Neelsen modified technique in 109 (19.7%) bovines ranging from 3 days old to adults. Positive animals were found in 19 (63.3%) farms. As much as 44.4% of calves aged 3-4 days were infected, but infection rates peaked at 6-15 days of age (76.7%). Nevertheless, prevalence was also high in weanling calves aged 1.5-4 months (14%), fattening calves and heifers 4-24 months old (7.7%) and adults (17.8%). Diarrhoea was recorded in 78.6% of suckling and 29.4% of weanling calves infected by C. parvum, but it was only found to be statistically associated with infection in suckling calves (P < 0.01). All calves shedding moderate or many oocysts had diarrhoea, whereas asymptomatic infection was always correlated with few oocysts in faeces. Cryptosporidial infections were always asymptomatic in bovines older than 4 months. Giardia cysts were identified in 65 bovines (11.7%) from 16 (53.3%) of the farms surveyed. Infection rates were significantly higher in suckling (14.1%) and weanling calves (38%) than in bovines older than 4 months (2.2%) (P < 0.001). Diarrhoea was recorded in 45.5% of suckling and 10.9% of weanling calves infected by Giardia, but it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic calves.

Comparison of an acid-fast stain and a monoclonal antibody-based immunofluorescence reagent for the detection of Cryptosporidium oocysts in faecal specimens from cattle and pigs.

A commercially available direct immunofluorescence (IF) assay with monoclonal antibodies (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) was compared with the modified Ziehl-Neelsen (MZN) acid-fast technique for the detection of Cryptosporidium oocysts in faecal samples from cattle and pigs. Stool specimens individually collected from 108 bovines and 90 pigs were examined in a blind test. The results of the two procedures corresponded (both positive or negative) in 102 (94.4%) cattle samples and 80 (88.9%) pig faecal samples. However, the remaining six (5.5%) cattle specimens and 10 (11.1%) pig stool samples, all of them harboring few oocysts (0-1 oocysts per 20 x field), were negative by MZN and positive by IF. False-negative results of the acid-fast stain occurred in suckling (17.2% of discrepant results) and weaned calves (2.9%) as well as weaned piglets (43.7%) and fattening pigs (10%). Stool specimens from the remaining age groups were negative by both techniques. The MacNemar's chi-square test showed that differences between both methods were statistically significant (P < 0.05). Compared with immunofluorescence procedure, the sensitivity of MZN technique in samples from cattle and pigs was 79.3% and 67.7% and the negative predictive value was 92.9% and 85.5% respectively. The specificity and positive predictive values of the acid-fast stain were 100% in both animal species. It is concluded that the monoclonal antibody-based immunofluorescence reagent evaluated is more efficient that the MZN technique, especially for detecting a low number of Cryptosporidium oocysts, in faecal specimens from both cattle and pigs.

Prevalence of Cryptosporidium infections in pigs in Aragón (northeastern Spain).

Faecal samples from 620 pigs randomly selected from 27 farms throughout Aragón were examined to determine the prevalence of Cryptosporidium infections. Detection of oocysts was performed using the ethyl-acetate stool concentration method and the modified Ziehl-Neelsen technique. Cryptosporidium parvum oocysts were identified in 136 (21.9%) pigs from 21 (77.8%) farms. Infected animals ranged from 1 to 6 months old and oocysts were not detected in suckling piglets or adults. Infection rates were significantly higher in weaned, 1-2 month old piglets (59.2%) than in fattening, 2-6 month old pigs (34.3%) (P < 0.001). Cryptosporidial infections were asymptomatic in most of the pigs (90.4%) and usually of low intensity, since 92.6% of the infected pigs excreted few oocysts (0-1 oocysts per field at x 200 magnifications). Although 24.1% of weaned and 5.6% of fattening pigs infected by C. parvum had diarrhoea, it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic pigs, in both weaned (64.7% and 46.7%, respectively) and fattening pigs (34.3% and 33.3%).

[Quinolone resistance in Salmonella enterica]. / Resistencia a quinolonas en Salmonella enterica.

Nalidixic acid resistance is a marker of quinolone sensibility in Salmonella spp. In our laboratory we observed an increase of nalidixic acid resistant strains from 0.44% in 1990 to 13.7% in 1996. The purpose of this work was to study the evolution of nalidixic acid resistance in Salmonella spp., as well as the association with certain serotypes and the cross-resistance to others agents. A total of 1,782 isolates of Salmonella enterica were studied from fecal and blood sources over a 7-year period; 45 strains (2.52%) were resistant to nalidixic acid (MIC > 16 microgram/ml). Two isolates required 2 microgram/ml of ciprofloxacin for inhibition, while the remaining 43 strains required 1 microgram/ml. The most frequent serotypes in nalidixic acid resistant strains were the following: enteritidis (33.3%), hadar (31.11%) and virchow (15.5%). In 1996, enteritidis was the predominant serotype (13/31), and hadar serotype was found to have the greatest nalidixic acid resistance at 80% of strains. No differences of sensibility to other agents were detected between the nalidixic acid resistant group and nalidixic acid sensitive strains. This type of resistance appears regularly in uncommon serotypes such as hadar. The increasing incidence of resistant isolates in enteritidis in 1996 is of even greater concern.