The osmotic response capacity of the Antarctic fish Harpagifer antarcticus is insufficient to cope with projected temperature and salinity under climate change.

Over the last decades, climate change has intensified. Temperatures have increased and seawater has become "fresher" in Antarctica, affecting fish such as Harpagifer antarcticus. Thus, this study aimed to evaluate changes in the osmoregulatory response of the Antarctic notothenioid fish Harpagifer antarcticus and evaluate how it will cope with the future climate change and environmental conditions in the Antarctic, and in the hypothetical case that its geographical distribution will be extended to the Magellanes region. The present study was undertaken to determine the interaction between temperature and salinity tolerance (2 °C and 33 psu as the control group, the experimental groups were 5, 8, and 11 °C and 28 and 23 psu) and their effect on the osmoregulatory status of H. antarcticus. We evaluated changes in gill-kidney-intestine NKA activity, gene expression of NKAα, NKCC, CFTR, Aquaporins 1 and 8 in the same tissues, muscle water percentage, and plasma osmolality to evaluate osmoregulatory responses. Plasma osmolality decreased with high temperature, also the gill-kidney-intestine NKA activity, gene expression of NKA α, NKCC, CFTR, Aquaporins 1, and 8 were modified by temperature and salinity. We demonstrated that H. antarcticus can not live in the Magallanes region, due to its incapacity to put up with temperatures over 5 °C and with over 8 °C being catastrophic.

Comparison between urine culture profile and morphology classification using fluorescence parameters of the Sysmex UF-1000i urine flow cytometer.

AIMS: To determine the usefulness of the fluorescence parameters generated by Sysmex UF-1000i flow cytometer for the rapid diagnosis of urinary tract infection by bacilli or cocci. METHODS AND RESULTS: Urine samples (n = 1924) were studied by culture and microbiology and subsequently by cytometry, using BACT-Morph software and considering forward-scattered light (FSC) and fluorescent light scatter fluorescence parameters. BACT-Morph software showed moderate diagnostic accuracy (78·4%) to detect rod-shaped bacteria, with sensitivity of 82·4% and specificity of 62·5%. Forward-scattered (B_FSC) values of the bacterial channel were significant higher for the Gram-positive cocci category (P < 0·001). A cut-off of B_FSC ≥24·2, expressed in arbitrary units (analytical channel, ch), provided higher sensitivity (90·0%) but lower specificity (38·9%), and the diagnostic accuracy for Gram-positive cocci classification reached 62·0%. CONCLUSIONS: Utilization of BACT-Morph software and bacterial channel fluorescence parameters (B_FSC ≥24·2 ch) offered an approximate discrimination of bacilli and cocci but the specificity was low, especially for FSC. SIGNIFICANCE AND IMPACT OF THE STUDY: Further research is needed to establish the usefulness of flow cytometry for aetiological diagnosis.

Fine-mapping butyrophilin family genes revealed several polymorphisms influencing viral genotype selection in hepatitis C infection.

Host-viral genetic interaction has a key role in hepatitis C infection (HCV) and maybe in the viral selection. In a preliminary GWAS analysis, we identified BTN3A2 rs9104 to be associated with HCV genotype 1. Therefore, our aim was to determine the influence of BTN family on the selection of HCV genotype. We performed a fine-mapping analysis of BTN gene region in a cohort of chronic HCV infection (N=841), validating significant results in another independent chronic HCV infection cohort (N=637), according to selection of viral genotype. BTN3A2 rs9104, BTN3A2 rs733528, BTN2A1 rs6929846, BTN2A1 rs7763910 and BTN3A3 rs13220495 were associated with viral genotype selection. Interestingly, BTN3A2 rs9104 GG genotype was closely related to genotype 1 infection (80.7% (394/488) compared with genotype 3 infection (53.5% (23/43); P=0.0001) in patients harboring IL28B-CT/TT genotype, although this effect was not observed in IL28B-CC genotype. Similarly, BTN3A3 rs13220495 CC genotype was linked to genotype 3 infection (100% (32/32)) compared to genotype 1 (87.3% (137/157); P=0.028) in patients harboring IL28B-CC genotype, but did not in IL28B-CT/TT genotype. Genetic variants in the butyrophilin family genes may alter susceptibility to infection, selecting HCV genotype and influencing disease progression. BTN3A2 rs9104 was strongly associated with genotype 1 infection and the haplotype BTN3A3 rs13220495 CC+IL28B genotype CC was universal in patients with hepatitis C genotype 3a.

Hepatotoxicity associated with statin use: analysis of the cases included in the Spanish Hepatotoxicity Registry.

OBJECTIVES: The hepatotoxic potential of statins is controversial. The objectives of this study were to describe the relative frequency of hepatotoxicity caused by statins and the phenotypes found in Spain. PATIENTS AND METHODS: The incidence of hepatotoxicity attributed to statins in the Spanish Hepatotoxicity Registry (REH) were studied and compared with those attributed to other drugs. RESULTS: Between April 1994 and August 2012, the REH included a total of 858 cases of which 47 (5.5 %) were attributed to statins. Of these, 16 were due to atorvastatin (34 %); 13 to simvastatin (27.7 %); 12 to fluvastatin (25.5 %); 4 to lovastatin (8.5 %) and 2 to pravastatin (4.3 %). Statins represented approximately half of the cardiovascular group which occupied 3rd place (10 %), after anti-infectious agents (37 %) and central nervous system drugs (14 %). The hepatocellular pattern was predominant, especially in the simvastatin group (85%), the cholestatic/mixed pattern was more frequent with fluvastatin (66 %) and had a similar distribution to atorvastatin. Patients with statin-induced toxicity were older (62 years versus 53 years, p < 0.001) and more often demonstrated anautoimmune hepatitis phenotype (8.5 % versus 1.4 %, p < 0.003). CONCLUSIONS: Statins are not a common cause of hepatotoxicity in Spain. Atorvastatin is the statin involved in the greatest number of incidents. The liver injury pattern varies among the different statins. The hepatitis phenotype with autoimmune features appears to be a characteristic signature of statin-induced hepatotoxicity.

Ingestion of paralytic shellfish toxins in a carnivorous gastropod (Chorus giganteus): effects on their elemental composition and reproductive traits.

The producer of paralytic shellfish toxin (PST), Alexandrium catenella, is one of the main generators of HABs in the coasts of Chile. Its presence produces ecological and economic damage, directly affecting filter-feeding organisms, and indirectly to other organism through the trophic chain. The objective of this research was to identify the effect of a toxic diet on the energetic and reproductive parameters of the carnivorous snail Chorus giganteus. Two groups of snails were used, one fed with toxic prey (bivalves fed with A. catenella), and the other fed with non-toxic prey. Both treatments were maintained under these conditions for 63 days, then, elemental composition (C, N) and energy content were estimated, and fecundity parameters were analyzed. The results indicate that snails fed with toxic prey had a lower percentage of C and C/N ratio. The energy content was significantly lower in intoxicated snails. Regarding fecundity parameters, a higher number of egg-masses were produced by toxic snails, however, only 62% of these showed embryonic development, with 57% hatching success. A negative relationship was identified between the mean PST concentration, quantified in snails, and the number of egg-masses produced per aquarium. In the aquarium where the snails had highest average PST concentration (1200 ± 820 µg STX.2HCL eq. Kg-1) there was no oviposition, while egg-masses were only produced by snails in aquaria where the average concentration did not exceed 360 ± 160 µg STX.2HCL eq. Kg-1. It is likely that, with low levels of accumulated PST, C. giganteus activates its oviposition process as a response to toxin-induced stress, generating a higher energy expenditure supported by a redirection of its reserves. However, when the intoxication presents higher levels, the reproductive process could be inhibited, similar to what has been identified in other molluscs.

Upwelling as a stressor event during embryonic development: Consequences for encapsulated and early juvenile stages of the marine gastropod Acanthina monodon.

Upwelling phenomena alter the physical and chemical parameters of the sea's subsurface waters, producing low levels of temperature, pH and dissolved oxygen, which can seriously impact the early developmental stages of marine organisms. To understand how upwelling can affect the encapsulated development of the gastropod Acanthina monodon, capsules containing embryos at different stages of development (initial, intermediate and advanced) were exposed to upwelling conditions (pH = 7.6; O2 = 3 mg L-1; T° = 9 °C) for a period of 7 days. Effects of treatment were determined by estimating parameters such as time to hatching, number of hatchlings per capsule, percentage of individuals with incomplete development, and shell parameters such as shell shape and size, shell strength, and the percentage of the organic/inorganic content. We found no significant impacts on hatching time, number of hatchlings per capsule, or percentage of incomplete development in either the presence or absence of upwelling, regardless of developmental stage. On the other hand, latent effects on encapsulated stages of A. monodon were detected in embryos that had been exposed to upwelling stress in the initial embryonic stage. The juveniles from this treatment hatched at smaller sizes and with higher organic content in their shells, resulting in a higher resistance to cracking 30 days after hatching, due to greater elasticity. Geometric morphometric analysis showed that exposure to upwelling condition induced a change in the morphology of shell growth in all post-hatching juveniles (0-30 days), regardless of embryonic developmental stage at the time of exposure. Thus, more elongated shells (siphonal canal and posterior region) and more globular shells were observed in newly hatched juveniles that had been exposed to the upwelling condition. The neutral or even positive upwelling exposure results suggests that exposure to upwelling events during the encapsulated embryonic phase of A. monodon development might not have major impacts on the future juvenile stages. However, this should be taken with caution in consideration of the increased frequency and intensity of upwelling events predicted for the coming decades.

Viral infections of the central nervous system in Spain: a prospective study.

The aim of the study was to determine the incidence of viruses causing aseptic meningitis, meningoencephalitis, and encephalitis in Spain. This was a prospective study, in collaboration with 17 Spanish hospitals, including 581 cases (CSF from all and sera from 280): meningitis (340), meningoencephalitis (91), encephalitis (76), febrile syndrome (7), other neurological disorders (32), and 35 cases without clinical information. CSF were assayed by PCR for enterovirus (EV), herpesvirus (herpes simplex [HSV], varicella-zoster [VZV], cytomegalovirus [CMV], Epstein-Barr [EBV], and human herpes virus-6 [HHV-6]), mumps (MV), Toscana virus (TOSV), adenovirus (HAdV), lymphocytic choriomeningitis virus (LCMV), West Nile virus (WNV), and rabies. Serology was undertaken when methodology was available. Amongst meningitis cases, 57.1% were characterized; EV was the most frequent (76.8%), followed by VZV (10.3%) and HSV (3.1%; HSV-1: 1.6%; HSV-2: 1.0%, HSV non-typed: 0.5%). Cases due to CMV, EBV, HHV-6, MV, TOSV, HAdV, and LCMV were also detected. For meningoencephalitis, 40.7% of cases were diagnosed, HSV-1 (43.2%) and VZV (27.0%) being the most frequent agents, while cases associated with HSV-2, EV, CMV, MV, and LCMV were also detected. For encephalitis, 27.6% of cases were caused by HSV-1 (71.4%), VZV (19.1%), or EV (9.5%). Other positive neurological syndromes included cerebellitis (EV and HAdV), seizures (HSV), demyelinating disease (HSV-1 and HHV-6), myelopathy (VZV), and polyradiculoneuritis (HSV). No rabies or WNV cases were identified. EVs are the most frequent cause of meningitis, as is HSV for meningoencephalitis and encephalitis. A significant number of cases (42.9% meningitis, 59.3% meningoencephalitis, 72.4% encephalitis) still have no etiological diagnosis.

Embryonic encapsulated development of the gastropod Acanthina monodon is impacted by future environmental changes of temperature and pCO2.

Egg capsules of the gastropod Acanthina monodon were maintained during the entire period of encapsulated development at three temperatures (10, 15, 20 °C) and two pCO2 levels (400, 1200 µatm). Embryos per capsule, size at hatching, time to hatching, embryonic metabolic rates, and the resistance of juveniles to shell breakage were quantified. No embryos maintained at 20 °C developed to hatching. The combination of temperature and pCO2 levels had synergistic effects on hatching time and developmental success, antagonistic effects on number of hatchlings per capsule, resistance to juvenile shell cracking and metabolism, and additive effect on hatching size. Juveniles hatched significantly sooner at 15 °C, independent of the pCO2 level that they had been exposed to, while individuals hatched at significantly smaller sizes if they had been held under 15 °C/1200 µatm rather than at 10 °C/low pCO2. Embryos held at the higher pCO2 had a significantly greater percentage of abnormalities. For capsules maintained at low pCO2 and 15 °C, emerging juveniles had less resistance to shell breakage. Embryonic metabolism was significantly higher at 15 °C than at 10 °C, independent of pCO2 level. The lower metabolism occurred in embryos maintained at the higher pCO2 level. Thus, in this study, temperature was the factor that had the greatest effect on the encapsulated development of A. monodon, increasing the metabolism of the embryos and consequently accelerating development, which was expressed in a shorter intracapsular development time, but with smaller individuals at hatching and a lower resistance of their shells to breakage. On the other hand, the high pCO2 level suppressed metabolism, prolonged intracapsular development, and promoted more incomplete development of the embryos. However, the combination of the two factors can mitigate--to some extent--the adverse effects of both incomplete development and lower resistance to shell breakage.

Performance of the Sysmex UF1000i system in screening for significant bacteriuria before quantitative culture of aerobic/facultative fast-growth bacteria in a reference hospital.

OBJECTIVE: To evaluate the performance of the Sysmex UF1000i automatic urine screening system in the quantitative culture of fast-growth aerobic/facultative bacteria. METHODS AND RESULTS: A standard procedure was used to recover fast-growth aerobic/facultative micro-organisms in 1225 samples, applying (Sysmex(®)) flow cytometry for parallel bacteria and leucocyte counts. According to the area under the receiver operating characteristic curve, the optimal cut-off values to detect bacteriuria >10(5) colony forming units (CFU) ml(-1) were 690/µl for bacteria and 38/µl for leucocytes (sensitivity, 92%; specificity, 65%; positive predictive value [PPV], 39%; and negative predictive value [NPV], 97%). The use of a single cut-off point of 150 bacteria µl(-1) to detect significant bacteriuria of >10(5) CFU ml(-1) or of ≥10(4) CFU ml(-1) plus leucocyturia obtained similar results (sensitivity, 89%; specificity, 54%; PPV, 31%; and NPV, 96%) and allowed 45.7% of the samples to be rapidly excluded. CONCLUSIONS: The Sysmex UF1000i system can be adapted for bacteriuria screening by the use of an appropriate cut-off point. SIGNIFICANCE AND IMPACT OF THE STUDY: This screening system significantly reduces the workload and produces very few false positives and negatives.

Long-term effects of contrasting pCO2 levels on the scope for growth in the carnivorous gastropod Concholepas concholepas.

We evaluated the effect of contrasting pCO2 levels: lower (390 µatm), moderate (700 µatm) and extreme (1000 µatm), on the scope for growth of the keystone snail Concholepas concholepas over an exposure period of 6 months. Juvenile snails were collected from rocky intertidal habitats and acclimated for 5 months to those pCO2 levels. Subsequently, three groups of snails were randomly taken (n = 7 for each treatment) and reared for an additional 1 month for each of the three pCO2 levels. Physiological traits related with energy gain and energy expenditure were quantified. The scope for growth index decreased significantly with increases in pCO2, yielding negative values throughout the experimental period for the snails exposed to 1000 µatm pCO2, probably due to the extra energy required to maintain their metabolic functions in balance. This suggests that future climate change scenarios with elevated pCO2 levels could threaten the growth and other basic functions of juvenile snails of this species.

The paralytic shellfish toxin effect on bioenergetic constituents of the fishery resource Chorus giganteus (Gastropoda: Muricidae).

Alexandrium catenella, one of the most common harmful microalgae observed in southern Chile, produces paralytic shellfish toxins, which can affect many organisms throughout the trophic chain. This research evaluated how paralytic shellfish toxins affected the principal bioenergetic constituents and fatty acids composition of the carnivorous snail Chorus giganteus. Snails were separated into a "toxic" group that was fed the toxic clam Mulinia edulis (which was previously fed A. catenella), and a "non-toxic" group, fed non-toxic clams. Both groups were kept under these conditions for 63 days. Our results indicated no difference in the ingestion rate of toxic versus non-toxic snails; however, a higher protein level was identified in toxic snails. The total lipid content proved to be no different in toxic versus non-toxic snails; although, an effect of the toxic diet on the fatty acid profile of C. giganteus was observed. High levels of essential polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in toxic snails, were identified. Our results suggest that exposure to paralytic shellfish toxins, through diet, may cause changes in the biochemical composition of C. giganteus, which may have a subsequent impact on its energetic physiology.

Evaluation of chimerism by quantitative PCR analysis of DNA polymorphism after allogeneic hematopoietic stem cell transplantation in a pediatric population with malignancies.

Relapse remains the major pitfall to success for Allo-HSCT in children with malignancies. Ninety-one patients undergoing Allo-HSCT were retrospectively reviewed. Chimerism status was evaluated at days +30, +60, and +100 in PB. VNTR-PCR and STR-PCR were used for this purpose. Thirty-one patients recurred (34%) and none survived. Thirty-two remain alive in CR (35%). Patients who achieved a CC at those days had a significant higher RFS and OS than patients who did not. Twelve patients showing PMC had an increased risk of recurrence (p=0.02. OR 7.7). In the univariate analysis, the probability of death was higher in patients who were not in first CR before transplant (p=0.008.OR 2.09) and in those receiving cells not from PB (p=0.002.OR 2.03). In the multivariate analysis, the absence of CC at day +100 was associated with a higher probability of relapse (p=0.004. OR 10.8) and death (p=0.016. OR 9.3). Serial chimerism PCR-based analyses of polymorphic DNA markers can predict relapse. Patients with PMC are at the highest risk of recurrence. Patients receiving an Allo-HSCT in first CR from PB who achieve a CC at day +100 have a better outcome.

Energetic trade-offs: Implications for selection between two bivalve prey species by a carnivorous muricid gastropod.

Active predators obtain energy and nutrients from prey through complex processes in which the energy gained must exceed the energy invested in finding and ingesting the prey. In addition, the amount of energy available will vary with the prey that are selected for consumption. The muricid gastropod Acanthina monodon inhabits rocky shores, where it routinely feeds on the mytilids Semimytilus algosus and Perumytilus purpuratus. In this study, S. algosus was highly preferred by the predator (over 90% were eaten) versus P. purpuratus (only 9% were eaten) when offered a mixed diet. The energetic cost of attacking one S. algosus individual was 91 J bivalve-1 while for P. purpuratus it was slightly higher: 95 J bivalve-1. Also, whereas A. monodon required on average 19 h to consume S. algosus, successful attacks on P. purpuratus required about 32% more time (25 h). In addition, a longer resting time was needed by the predator after preying on P. purpuratus before it initiated another attack. Moreover, the active metabolic costs associated with successfully attacking the prey increased 3.2 times over the basal metabolic costs when attacking S. algosus, but only by 2.5 times when attacking P. purpuratus. The calculations associated with preying on each species showed that the energetic gain per unit time likely accounts for the predator's preference for attacking S. algosus, even though predation on both species provided net energy gains for the predator. However, as S. algosus occurs seasonally at our study site, P. purpuratus would probably also be consumed due to its constant availability throughout the whole year.

Respiratory and desiccation constraints during encapsulated intertidal development of the marine gastropod Acanthina monodon.

Acanthina monodon commonly deposits its egg capsules in the intertidal zone. Capsule aerial exposure during low-tide can impact oxygen consumption rates (OCR) of embryos and intracapsular oxygen availability, and expose embryos to desiccation. OCR increased as embryonic development progressed, and was greater when capsules were submerged in seawater than when exposed to air. Oxygen available within the capsule was always less than that available in the immediate external environment, whether capsules were immersed or exposed. The highest internal oxygen concentrations were recorded during periods of air exposure for embryos in more advanced development stages. When exposed to air, capsules lost water the fastest when they contained early embryos, and suffered the highest mortalities following exposure. Collectively, these data suggest that, although encapsulation helps the embryos to develop across wildly fluctuating environmental conditions, the amount of stress the embryos experience will vary depending on their exact positioning within the intertidal zone.

Study of broadband near-infrared emission in Tm3+-Er3+ codoped TeO2-WO3-PbO glasses.

In this work, we report the near-infrared emission properties of Tm(3+)-Er(3+) codoped tellurite TeO(2)-WO(3)-PbO glasses under 794 nm excitation. A broad emission from 1350 to 1750 nm corresponding to the Tm(3+) and Er(3+) emissions is observed. The full width at half-maximum of this broadband increases with increasing [Tm]/[Er] concentration ratio up to a value of ~ 160 nm. The energy transfer between Tm(3+) and Er(3+) ions is evidenced by both the temporal behavior of the near-infrared luminescence and the effect of Tm3+ codoping on the visible upconversion of Er(3+) ions.

Diagnosis of paediatric anaplastic large-cell lymphoma: a historical perspective from a single institution.

INTRODUCTION: Anaplastic large-cell lymphoma (ALCL) is an infrequent childhood malignancy whose diagnosis and treatment have largely evolved since its initial description in 1985. PATIENTS AND METHODS: We retrospectively reviewed our experience in the field, and report here a single institution experience focusing on diagnostic and therapeutic milestones achieved as novel tools have been developed. This is a series of 9 children diagnosed from 1987 to 2007. RESULTS: Our first patient was diagnosed shortly alter this entity was described based on morphology and Ki-1 positivity, while the diagnostic work-up for the last two children included accurate molecular diagnosis for ALK-NPM rearrangement. Despite a wide variety of multimodal therapies used over time, only one patient died of toxicity during progression and another child relapsed and survived alter an autograft. After 156 months of median follow-up (range 4-245), 8 out of 9 children are alive, free of disease. CONCLUSIONS: Our series exemplifies the long journey travelled from the definition of a new entity only 20 years ago to the molecular characterization not only with diagnostic but also therapeutic purposes. Besides this, significant efforts are being made to recruit all European patients into a multinational collaborative trial in order to start drawing major evidence-based conclusions.

Neurosensory issues after lateralisation of the inferior alveolar nerve and simultaneous placement of osseointegrated implants.

Our aim was to evaluate neurosensory symptoms after lateralisation of the inferior alveolar nerve (IAN). We studied a retrospective case series with one-year follow up that included 139 procedures in 123 patients. After the IAN had been located it was deflected from the mandibular body and the implant placed. Sensitivity was mapped 24hours, one month, six months, and one year after the intervention by gently pressing the skin and lips with the tip of a probe. A total of 337 implants were placed in 123 patients aged between 44 and 68 years. There were 33 men and 90 women and they all recovered. The IAN was mobilised by one of two procedures, one that involves the nerve directly (transposition) and one that does not (lateralisation). During lateralisation the nerve is deflected laterally through a mandibular osteotomy, while the mental nerve and mental foramen are not manipulated. The resulting hypoaesthetic area was drawn on a graph to assess its extension. Although different techniques are available for placing implants in atrophic jaws, mobilisation of the IAN is indicated in certain cases in which other techniques are not feasible or have a high risk of complications.

Spectroscopic properties of the 1.4 microm emission of Tm3+ ions in TeO2-WO3-PbO glasses.

In this work, we report the spectroscopic properties of the infrared 3H4-->3F4 emission of Tm3+ ions in two different compositions of glasses based on TeO2, WO3, and PbO for three Tm2O3 concentrations (0.1,0.5, and 1 wt%). Judd-Ofelt intensity parameters have been determined and used to calculate the radiative transition probabilities and radiative lifetimes. The infrared emission at around 1490 nm corresponding to the 3H4-->F4 transition has two noticeable features if compared to fluoride glasses used for S-band amplifiers. On one hand, it is broader by nearly 30 nm, and on the other, the stimulated emission cross section is twice the value for fluoride glasses. Both the relative intensity ratio of the 1490 nm emission to 1820 nm and the measured lifetime of the 3H4 level decrease as concentration increases, due to the existence of energy transfer via cross-relaxation among Tm3+ ions. The analysis of the decays from the 3H4 level with increasing concentration indicates the presence of a dipole-dipole quenching process assisted by energy migration.

Trophic interactions between the toxic dinoflagellate Alexandrium catenella and Mytilus chilensis: feeding and digestive behaviour to long-term exposure.

Trophic interactions between Mytilus chilensis and the toxic dinoflagellate Alexandrium catenella were studied over a 21-day period to determine the absorption kinetics of biochemical components and the enzymatic activity of the digestive gland (i.e. amylase, cellulase complex, laminarinase and protease). For the duration of the experiment, logarithmic relationships were established for the assimilatory balance and carbohydrase activities (amylase, laminarinase and cellulase) of the mussels fed with a toxic diet. The study shows that M. chilensis was able to develop mechanisms which allow exploitation of the toxic microalgae as a food source, despite that its feeding and digestive processes were affected during the first days of contamination.