Accuracy of quantitative viral secondary standards: a re-examination.

Interlaboratory agreement of viral load assays depends on the accuracy and uniformity of quantitative calibrators. Previous work demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values in both log10 copies/mL and log10 international unit (IU)/mL, with bias from manufacturer-assigned nominal values of 0.0-0.9 log10 units (either copies or IU)/mL. Standards normalized to IU and those values assigned by dPCR rather than by real-time PCR (qPCR) showed better agreement with nominal values. The latter reinforces prior conclusions regarding the utility of using such methods for quantitative value assignment in reference materials. Quantitative standards have improved over the last several years, and the remaining bias from nominal values might be further reduced by universal implementation of dPCR methods for value assignment, normalized to IU. IMPORTANCE: Interlaboratory agreement of viral load assays depends on accuracy and uniformity of quantitative calibrators. Previous work, published in JCM several years ago, demonstrated poor agreement of secondary cytomegalovirus (CMV) standards with nominal values. This study re-evaluated this issue among commercially produced secondary standards for both BK virus (BKV) and CMV, using digital polymerase chain reaction (dPCR) to compare the materials from three different manufacturers. Overall, standards showed an improved agreement compared to prior work, against nominal values, indicating a substantial improvement in the production of accurate secondary viral standards, while supporting the need for further work in this area and for the broad adaption of international unit (IU) as a reporting standard for quantitative viral load results.

A prospective bacterial whole-genome-sequencing-based surveillance programme for comprehensive early detection of healthcare-associated infection transmission in paediatric oncology patients.

BACKGROUND: Bacterial whole-genome sequencing (WGS) and determination of genetic relatedness is an important tool for investigation of epidemiologically suspected outbreaks. AIM: This prospective cohort study evaluated a comprehensive, prospective bacterial WGS-based surveillance programme for early detection of transmission of most bacterial pathogens among patients at a paediatric oncology hospital. METHODS: Cultured bacterial isolates from clinical diagnostic specimens collected prospectively from both inpatient and outpatient encounters between January 2019 and December 2021 underwent routine WGS and core genome multi-locus sequence typing to determine isolates' relatedness. Previously collected isolates from January to December 2018 were retrospectively analysed for identification of prior or ongoing transmission. Multi-patient clusters were investigated to identify potential transmission events based on temporal and spatial epidemiological links and interventions were introduced. FINDINGS: A total of 1497 bacterial isolates from 1025 patients underwent WGS. A total of 259 genetically related clusters were detected, of which 18 (6.9%) multi-patient clusters involving 38 (3.7%) patients were identified. Sixteen clusters involved two patients each, and two clusters involved three patients. Following investigation, epidemiologically plausible transmission links were identified in five (27.8%) multi-patient clusters. None of the multi-patient clusters were suspected by conventional epidemiological surveillance. CONCLUSION: Bacterial WGS-based surveillance for early detection of hospital transmission detected several limited multi-patient clusters that were unrecognized by conventional epidemiological methods. Genomic surveillance helped efficiently focus interventions while reducing unnecessary investigations.

Field ecology of toxic Pfiesteria complex species and a conservative analysis of their role in estuarine fish kills.

Within the past decade, toxic Pfiesteria outbreaks have been documented in poorly flushed, eutrophic areas of the largest and second largest estuaries on the U.S. mainland. Here we summarize a decadal field effort in fish kill assessment, encompassing kills related to Pfiesteria (49 major kills in North Carolina estuaries since 1991 and 4 in Maryland estuaries in 1997) and to other factors such as low oxygen stress (79 major fish kills in North Carolina estuaries). The laboratory and field data considered in developing our protocols are described, including toxic Pfiesteria behavior, environmental conditions conducive to toxic Pfiesteria activity, and impacts of toxic clonal Pfiesteria on fish health. We outline the steps of the standardized fish bioassay procedure that has been used since 1991 to diagnose whether actively toxic Pfiesteria was present during estuarine fish kills. Detailed data are given for a 1998 toxic Pfiesteria outbreak in the Neuse Estuary in North Carolina to illustrate of the full suite of diagnostic steps completed. We demonstrate that our conservative approach in implicating toxic Pfiesteria involvement in fish kills has biased in favor of causes other than Pfiesteria. Data are summarized from experiments that have shown stimulation of toxic Pfiesteria strains by nutrient (N, P) enrichment, supporting field observations of highest abundance of toxic strains in eutrophic estuaries. On the basis of a decade of research on toxic Pfiesteria, we present a conceptual model of the seasonal dynamics of toxic strains as affected by changing food resources and weather patterns. We also recommend protocols and research approaches that will strengthen the science of fish kill assessment related to Pfiesteria and/or other causative factors.

Microfluorimetric analysis of a purinergic receptor (P2X7) in GH4C1 rat pituitary cells: effects of a bioactive substance produced by Pfiesteria piscicida.

Pfiesteria piscicida Steidinger & Burkholder is a toxic dinoflagellate that leads to fish and human toxicity. It produces a bioactive substance that leads to cytotoxicity of GH4C1 rat pituitary cells. Extracellular adenosine 5'-triphosphate (ATP) acting on P2X7 purinergic receptors induces the formation of a nonselective cation channel, causing elevation of the cytosolic free calcium followed by a characteristic permeabilization of the cell to progressively larger ions and subsequent cell lysis. We investigated whether GH4C1 rat pituitary cells express functional P2X7 receptors, and if so, are they activated by a bioactive substance isolated from toxic P. piscicida cultures. We tested the selective agonist 2'-3'-O-(benzoyl-4-benzoyl)-ATP (BzATP) and antagonists piridoxalphosphate-6-azophenyl-2'-4'-disulfonic acid (PPADS) and oxidized-ATP (oxATP) using elevated cytosolic free calcium in Fura-2 loaded cells, and induced permeability of these cells to the fluorescent dye YO-PRO-1 as end points. We demonstrated that in GH4C1 cells, BzATP induces both the elevation of cytosolic free calcium and the permeabilization of the cell membrane. ATP-induced membrane permeabilization was inhibited by PPADS reversibly and by oxATP irreversibly. The putative Pfiesteria toxin (pPfTx) also elevated cytosolic free calcium in Fura-2 in GH4C1 cells and increased the permeability to YO-PRO-1 in a manner inhibited fully by oxATP. This study indicates that GH4C1 cells express a purinoceptor with characteristics consistent with the P2X7 subtype, and that pPfTx mimics the kinetics of cell permeabilization by ATP.

The standardized fish bioassay procedure for detecting and culturing actively toxic Pfiesteria, used by two reference laboratories for atlantic and gulf coast states.

In the absence of purified standards of toxins from Pfiesteria species, appropriately conducted fish bioassays are the "gold standard" that must be used to detect toxic strains of Pfiesteria spp. from natural estuarine water or sediment samples and to culture actively toxic Pfiesteria. In this article, we describe the standardized steps of our fish bioassay as an abbreviated term for a procedure that includes two sets of trials with fish, following the Henle-Koch postulates modified for toxic rather than infectious agents. This procedure was developed in 1991, and has been refined over more than 12 years of experience in research with toxic Pfiesteria. The steps involve isolating toxic strains of Pfiesteria (or other potentially, as-yet-undetected, toxic Pfiesteria or Pfiesteria-like species) from fish-killing bioassays with natural samples; growing the clones with axenic algal prey; and retesting the isolates in a second set of fish bioassays. The specific environmental conditions used (e.g., temperature, salinity, light, other factors) must remain flexible, given the wide range of conditions from which natural estuarine samples are derived. We present a comparison of information provided for fish culture conditions, reported in international science journals in which such research is routinely published, and we provide information from more than 2,000 fish bioassays with toxic Pfiesteria, along with recommendations for suitable ranges and frequency of monitoring of environmental variables. We present data demonstrating that algal assays, unlike these standardized fish bioassays, should not be used to detect toxic strains of Pfiesteria spp. Finally, we recommend how quality control/assurance can be most rapidly advanced among laboratories engaged in studies that require research-quality isolates of toxic Pfiesteria spp.

Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida.

Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum.

Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: a potential target for a bioactive substance produced by Pfiesteria piscicida.

We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2',3'-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin

Species of the toxic Pfiesteria complex, and the importance of functional type in data interpretation.

We describe the two species of the toxic Pfiesteria complex to date (Pfiesteria piscicida and Pfiesteria shumwayae), their complex life cycles, and the characteristics required for inclusion within this complex. These species resemble P. piscicida Steidinger & Burkholder and also have a) strong attraction to fresh fish tissues and excreta, b) toxic activity stimulated by live fish, and c) production of toxin that can cause fish death and disease. Amoeboid stages were verified in 1992-1997 by our laboratory (various stages from toxic cultures) and that of K. Steidinger and co-workers (filose amoebae in nontoxic cultures), and in 2000 by H. Marshall and co-workers (various stages from toxic cultures), from clonal Pfiesteria spp. cultures, using species-specific polymerase chain reaction-based molecular probes with cross-confirmation by an independent specialist. Data were provided from tests of the hypothesis that Pfiesteriastrains differ in response to fresh fish mucus and excreta, algal prey, and inorganic nutrient (N, P) enrichment, depending on functional type or toxicity status. There are three functional types: TOX-A, in actively toxic, fish-killing mode; TOX-B, temporarily nontoxic, without access to live fish for days to weeks, but capable of toxic activity if fish are added; and NON-IND, noninducible with negligible toxicity in the presence of live fish. NON-IND Pfiesteria attained highest zoospore production on algal prey without or without inorganic nitrogen or inorganic phosphorus enrichment. TOX-B Pfiesteria was intermediate and TOX-A was lowest in zoospore production on algal prey with or without nutrients. TOX-A Pfiesteria spp. showed strong behavioral attraction to fresh fish mucus and excreta in short-term trials, with intermediate attraction of TOX-B zoospores and relatively low attraction of NON-IND cultures when normalized for cell density. The data for these clones indicated a potentially common predatory behavioral response, although differing in intensity distinct from a toxicity effect, in attack of fish prey. The data also demonstrated that functional types of Pfiesteria spp. show distinct differences in response to fish, algal prey, and inorganic nutrient enrichment. Collectively, the experiments indicate that NON-IND strains should not be used in research to gain insights about environmental controls on toxic strains of Pfiesteria spp.

Current progress in isolation and characterization of toxins isolated from Pfiesteria piscicida.

The isolation and partial purification of toxic substances derived from Pfiesteria piscicida Steidinger & Burkholder extracts is described. Four distinct bioassay systems were used to monitor bioactivity of the P. piscicida extracts, including a high throughput cell cytotoxicity assay and a reporter gene assay as well as assays using brine shrimp and fish. Using these bioassays to guide fractionation, we have isolated two distinct, active fractions from Pfiesteria culture medium and cell mass extracts on the basis of their solubility characteristics. We have identified and characterized a bioactive lipophilic substance from Pfiesteria-derived extracts as di(2-ethylhexyl)phthalate, a commonly used plasticizer. The source of this typically man-made substance has been identified as originating from Instant Ocean (Aquarium Systems, Mentor, OH, USA), a commercially available seawater salt mixture used to prepare our mass culture growth medium. We have developed chromatographic methodology to isolate a bioactive polar compound isolated from extracts of Pfiesteria culture and presently report the characterization of the activity of this substance. The molecular structural analysis of the polar active component(s) using mass spectrometry and nuclear magnetic resonance spectroscopy is currently under way.

Use of molecular probes to assess geographic distribution of Pfiesteria species.

We have developed multiple polymerase chain reaction (PCR)-based methods for the detection of Pfiesteria sp. in cultures and environmental samples. More than 2,100 water and sediment samples from estuarine sites of the U.S. Atlantic and gulf coasts were assayed for the presence of Pfiesteria piscicida Steidinger & Burkholder and Pfiesteria shumwayae Glasgow & Burkholder by PCR probing of extracted DNA. Positive results were found in about 3% of samples derived from routine monitoring of coastal waters and about 8% of sediments. The geographic range of both species was the same, ranging from New York to Texas. Pfiesteria spp. are likely common and generally benign inhabitants of coastal areas, but their presence maintains a potential for fish and human health problems.

Specificity of cognitive impairment from Pfiesteria piscicida exposure in rats: attention and visual function versus behavioral plasticity.

Pfiesteria piscicida is a toxic dinoflagellate that has caused massive fish kills in estuaries along the East Coast of the United States, and exposure of humans to toxic Pfiesteria has been associated with cognitive impairment. A visual signal detection task was used to determine the possible importance of attentional and visual processes in Pfiesteria effects on cognitive function. Adult female rats were trained to perform the signal detection task. After training, the rats were injected subcutaneously with fish culture water containing toxic Pfiesteria (35,600 or 106,800 cells of Pfiesteria/kg of rat body weight) or with (control) fish culture water containing no Pfiesteria. Effects of toxic Pfiesteria on maintenance of signal detection behavior were assessed for 2 weeks after treatment. Then, the signal-response contingencies were reversed. After the discrimination was reestablished on the reversed levers, the rats received a second dose of toxic Pfiesteria. The rats were again tested for 2 weeks, after which a second reversal was imposed. Pfiesteria did not affect behavior in the signal detection task during 2 weeks of prereversal testing after either exposure. However, a significant Pfiesteria-induced deficit emerged when the signal-response contingencies were reversed. These findings suggest that Pfiesteria-induced deficits emerge during periods of behavioral transition and not during performance of previously learned tasks.

Rapid neurobehavioral analysis of Pfiesteria piscicida effects in juvenile and adult rats.

The estuarine dinoflagellate Pfiesteria piscicida is known to kill fish and has been associated with neurocognitive deficits in humans. We have developed a rat model to demonstrate that exposure to Pfiesteria causes significant learning impairments. This has been repeatedly seen as a choice accuracy impairment during radial-arm maze learning. Pfiesteria-induced effects were also seen in a locomotor activity test in the figure-8 apparatus. The current studies used the short-term radial-arm maze acquisition, the figure-8 activity test, and the functional observational battery (FOB) to assess Pfiesteria-induced neurobehavioral effects in adult and juvenile rats. In study 1, the neurobehavioral potency of three different Pfiesteria cultures (Pf 113, Pf 728, and Pf Vandermere) was assessed. Ninety-six (12 per group) adult female Sprague-Dawley rats were injected subcutaneously with a single dose of Pfiesteria taken from aquarium-cultured Pfiesteria (35,600 or 106,800 Pfiesteria cells per kilogram of rat body weight). One control group (N = 12) was injected with saline and one (N = 12) with aquarium water not containing Pfiesteria. All three of the Pfiesteria samples (p < 0.05) impaired choice accuracy over the first six sessions of training. At the time of the radial-arm maze choice accuracy impairment, no overt Pfiesteria-related effects were seen using an FOB, indicating that the Pfiesteria-induced choice accuracy deficit was not due to generalized debilitation. In the figure-8 apparatus, Pfiesteria treatment caused a significant decrease in mean locomotor activity. In study 2, the neurobehavioral effects of the Pf 728 sample type were assessed in juvenile rats. Twenty-four day-old male and female rats were injected with 35,600 or 106,800 Pf-728 Pfiesteria cells per kilogram of rat body weight. As with adult females, the juvenile rats showed a significant impairment in radial-arm maze choice accuracy. No changes in locomotor activity or the FOB were detected in the juvenile rats. Furthermore, there were no differences between male and female rats in the Pfiesteria-induced choice accuracy impairment. Pfiesteria effects on choice accuracy in the radial-arm maze in rats constitute a critical component of the model of Pfiesteria toxicity, because the hallmark of Pfiesteria toxicity in humans is cognitive dysfunction. Our finding that analysis of the first six sessions of radial-arm maze testing is sufficient for determining the effect means that this test will be useful as a rapid screen for identifying the critical neurotoxin(s) of Pfiesteria in future studies.

Pfiesteria toxin and learning performance.

Pfiesteria piscicida is an estuarine dinoflagellate involved with fish kills along the east coast of the United States. We previously documented a radial-arm maze learning deficit in rats exposed to Pfiesteria that may be related to cognitive deficits seen in humans after accidental Pfiesteria exposure. The current study elucidated important behavioral parameters of this deficit. There were six dose groups. Forty (10/group) adult female Sprague-Dawley rats were injected (s.c.) with a single dose of Pfiesteria taken from aquarium-cultured Pfiesteria (35,600, 106,800, or 320,400 Pfiesteria cells/kg of rat body weight or a cell-free filtrate of the 106,800 cells/kg dose). One control group (N = 10) was injected with saline and one (N = 10) with aquarium water not containing Pfiesteria. Half of the rats in each group were tested on an 8-arm radial maze in a standard test room, and the other half were tested on the radial maze in a sound-attenuating chamber. In the standard maze room, there was a significant effect of Pfiesteria (p < 0.05) impairing choice accuracy improvement over the first six sessions of training among rats administered 106,800, 320,400, and the 106,800 cells/kg filtered sample. In contrast, there was no indication of an effect of Pfiesteria when the rats were tested on the same configuration radial maze in the sound-attenuating chamber. After 18 sessions of training in one room, the rats were switched for six sessions of testing in the other room and finally were switched back to their original room for three sessions. There was a significant Pfiesteria-induced deficit when the rats were tested in the standard test room but not when they were tested in the sound-attenuating chamber. When the Pfiesteria-exposed rats were initially switched from the sound-attenuating chamber to the standard test room they performed significantly worse than controls, whereas Pfiesteria-treated rats switched from the standard test room to the sound-attenuating chamber did not perform differently from controls. These results suggest that the Pfiesteria-induced learning impairment may result from the negative impact of distracting stimuli. At the time of the learning impairment, no overt Pfiesteria-related effects were seen using a functional observational battery and no overall response latency effects were seen, indicating that the Pfiesteria-induced choice accuracy deficit was not due to generalized debilitation. In the initial use of the figure-8 maze in this line of research, the rats in the same Pfiesteria treatment groups that showed significant deficits in the radial-arm maze showed greater declines in activity rates in a 1-h figure-8 locomotor activity test. Both the 106,800 and 320,400 Pfiesteria cells/kg groups showed significantly greater linear trends of activity decline relative to tank water-treated controls. This reflected an initial slight hyperactivity in the Pfiesteria-treated animals followed by a decrease to control levels. Pfiesteria effects in the figure-8 maze and in early radial-arm maze training may be useful in a rapid screen for identifying the critical toxin(s) of Pfiesteria in future studies.

Tobacco smoking among fourth form school students in Wellington, New Zealand, 1991-97.

OBJECTIVE: To investigate trends in smoking and associated demographic factors among fourth form (14-15 years) school students. METHOD: In 1991, all 35 secondary schools in Wellington, New Zealand, were invited to participate; 15 took part. Smoking behaviours were assessed biennially by self-report. Trends were examined among 5,834 students, using multilevel regression. RESULTS: When adjusted for sex, ethnicity and their interaction, the baseline (1991) prevalence of smoking within the past month was 18% (95% CI 10-29) rising to 28% (95% CI 15-46) in 1997. The comparable baseline prevalence of daily smoking was 8% (95% CI 3-21) rising to 15% (95% CI 4-40) in 1997. Smoking was more common among girls than boys and most common among Maori girls, for whom the adjusted odds of current and daily smoking were, respectively, 3.40 times (95% CI 2.56-4.52) and 5.00 times (95% CI 3.64-6.87) those of Europeans. School socio-economic status and sex composition added to the explanatory power of the model for daily smoking, but had negligible effect on the odds ratios for sex and ethnic group. CONCLUSIONS AND IMPLICATIONS: The suggested rise in the prevalence of smoking has implications for future adult smoking rates and health costs. Under-representation of socio-economically disadvantaged schools may have produced conservative estimates. The increased odds of smoking among girls and Maori confirm the need to develop interventions appropriate for these groups.

Are New Zealand schools smoke-free? Results from a national survey of primary and intermediate school principals.

AIMS: To document policies and practices developed by primary and intermediate schools in response to the Smoke-free Environments Act 1990 and estimate levels of staff support for legislation requiring totally smoke-free schools. METHODS: Questionnaires were mailed to a random national sample of 10% of principals of all primary and intermediate schools. RESULTS: There were 209 questionnaires returned, a response rate of 87%. While 97% of principals reported that their school had a written policy for smoking, only 58% provided a copy. Only 49% of principals said that their school smoking policy was prominently displayed. Most (82 %) claimed that school buildings were smoke-free, but only 44% said that this applied to buildings and grounds. Overall, 62% considered that school staff would support legislation that required schools to be totally smoke-free in buildings and grounds. School policies ranged from total prohibition of smoking in school buildings and grounds to the provision of designated smoking areas for staff. CONCLUSIONS: Compliance with current smoke-free legislation appears to be variable. Consistency in the meaning of smoke-free status in schools is lacking. There appears to be good staff and parent support for making all schools totally smoke-free. The best way to achieve universal, permanent and totally smoke-free status for New Zealand schools would be to amend current legislation to include specific requirements for all school premises.

Trophic controls on stage transformations of a toxic ambush-predator dinoflagellate.

The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three-year period in the Albemarle-Pamlico Estuarine System of the southeastern USA. Transformations between life-history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microtauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscicida include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life-history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish "ambush-predator" and at least one other Pfiesteria-like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re-evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient-enriched estuaries.

Sunburn and sun protection among young children.

OBJECTIVE: To examine the extent of sun exposure, sun protection and experience of sunburn among young New Zealand children on summer weekends. METHODS: In a telephone survey of 1243 respondents, those with children in the household were asked about sun exposure and protection for the youngest child in the family. Information was obtained for 285 children aged from infancy to 10 years. RESULTS: Over 90% of the children were reported to be outside on the preceding Saturday and/or Sunday; 7% of those outside experienced some degree of sunburn. The worst burning was on the face, head, neck or ears. On either day about half the children were wearing sunscreen and 60% were wearing a hat. Parental use of sun protection was the strongest predictor of sun protection among the children. CONCLUSIONS: While reports of sun protection among young children were encouraging, many children in the community are still at high risk of sunburn. Efforts to promote sun protection as a family responsibility may reduce the experience of burning among the young.