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.

Gastrointestinal and respiratory tract symptoms following brief environmental exposure to aerosols during a pfiesteria-related fish kill.

An outbreak of illness with flulike symptoms among state workers responding to a Pfiesteria bloom that resulted in fish death and distress on the Chicamacomico River on Maryland's Eastern Shore was investigated. Using case-control methodology, seven workers present at the Chicamacomico were compared to seven occupationally matched controls not present. Participants completed questionnaires assessing their exposures to water and their symptom histories and were assessed with a standard neuropsychological test battery. Three months later, the same questionnaires and neuropsychological tests were repeated. Three of the seven exposed workers cited minimal direct contact with water and four cited none. During the event, four developed burning eyes or nares and six developed a headache or sore throat. Six developed crampy abdominal pain, nausea, or diarrhea within 4 h of their exposure. In contrast, the only aforementioned symptom reported by controls was headache in two individuals. Acute and follow-up neuropsychological tests showed no consistent pattern of deficiency among the exposed. In conclusion, a flulike clinical illness was observed following exposure to a Pfiesteria-related fish kill, possibly as a result of inhalation of toxic aerosols.

Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay.

Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtained from several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses and comparison of sequences indicate that the chloroplast sequences show a higher degree of sequence divergence than the nuclear homologue. The chloroplast sequences were chosen as targets for the development of a 5'--3' exonuclease assay for detection of the organism. The assay has a very high degree of specificity and has been used to screen environmental water samples from a fish farm where the presence of this dinoflagellate species has previously been associated with fish kills. Various hypotheses for the derived nature of the chloroplast sequences are discussed, as well as what is known about the toxicity of the species.

Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates.

Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology.

Heteroduplex mobility assay-guided sequence discovery: elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools.

The newly described heterotrophic estuarine dinoflagellate Pfiesteria piscicida has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pfiesteria research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pfiesteria piscicida 18S rDNA "signature" sequences in complex pools of DNA and used those data as the basis for determination of the complete P. piscicida 18S rDNA sequence. Specific PCR assays for P. piscicida and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.

A randomized trial of surgical antimicrobial prophylaxis with and without vancomycin in organ transplant patients.

BACKGROUND: Gram-positive organisms, including vancomycin-resistant enterococci (VRE), have emerged as major pathogens on the organ transplant service at our institution. We hypothesized that our use of vancomycin as part of routine surgical prophylaxis increased the risk of VRE colonization and infection; conversely, there was concern that failure to use vancomycin prophylaxis would increase peri-operative morbidity due to gram-positive organisms. METHODS: Renal transplant recipients (n = 88) were randomized to receive either a) vancomycin/ceftriaxone or b) cefazolin; and pancreas transplants (n = 24) to receive either a) vancomycin/gentamicin or b) cefazolin/gentamicin. Stool samples or rectal swabs were obtained for culture for enterococci within 24 h of transplantation and weekly while hospitalized. RESULTS: Enterococci were isolated on stool culture from 38 (34%) of 102 patients at the time of transplantation; 4 (11%) of the isolates were VRE. The percentage of patients who subsequently acquired VRE was low (1-7% per wk) but remained constant during hospitalization. There was no association between new VRE detection and vancomycin use for either prophylactic or therapeutic purposes. Forty-four patients (39%) had a post-operative infection with 46% of these infections due to gram-positive organisms; rates were unaffected by prophylactic vancomycin use. Pancreas transplant patients who did not receive vancomycin prophylaxis had a significantly longer initial hospitalization (p = 0.03); however, differences were not statistically significant when total length of stay (LOS) within the first 90 d of transplantation was compared. CONCLUSIONS: Vancomycin surgical prophylaxis does not appear to have an effect on VRE colonization or infection, or on rates of infection with gram-positive bacteria. Elimination of vancomycin prophylaxis in renal transplant patients may be a reasonable part of an overall program to limit vancomycin usage, although as a single measure, its impact may be minimal. Vancomycin surgical prophylaxis may be of greater importance in pancreas transplants.

Pfiesteria in Maryland: preliminary epidemiologic findings.

In the fall of 1996, fish kills in Maryland rivers were attributed to the dinoflagellate, Pfiesteria piscicida. After a group of researchers established a potential link between exposure to Pfiesteria and an illness causing memory problems, state health authorities closed a portion of the Pocomoke River. To determine the extent of illness, the range of symptoms, potential risk factors for disease, and to provide information to concerned citizens, a toll-free hotline was created. All symptomatic persons who called the toll-free number were administered a standardized questionnaire. Persons who had been exposed to Pfiesteria or Pfiesteria-laden waters were more likely to have respiratory, neurologic, dermatologic, and gastrointestinal problems than those persons without exposure. Among the persons calling the hotline, many had extensive neuropsychologic testing. Of the neuropsychologic test battery, low scores on the Rey Auditory Verbal Learning Test (RAVLT), a standardized measure of learning and memory, best characterized illness related to Pfiesteria exposure. Patients with low RAVLT scores were more likely to have neurologic symptoms and skin lesions than control subjects. Low RAVLT scores were associated with fishing (OR, 9.00, 95% CI, 106, 409.87), catching fish with lesions (OR, 6.17, 95% CI 1.27, 32.10), and handling fish with lesions (OR, 5.34, 95% CI, 1.05, 29.92), but not with consumption of seafood. While preliminary, these results do suggest that some risk factors for Pfiesteria-related illness may be easy to modify and used to prevent unnecessary human exposure.

Use of renal allografts from donors positive for hepatitis B core antibody confers minimal risk for subsequent development of clinical hepatitis B virus disease.

BACKGROUND: The risk associated with transplantation of renal allografts from hepatitis B virus core antibody-positive (HBcAb(+)), hepatitis B virus surface antigen-negative (HBsAg(-)) donors is not well defined. METHODS: Over 4 years, we performed 45 kidney transplants from IgG HBcAb(+), IgM HBcAb(-), HBsAg(-) donors into recipients with a history of prior hepatitis B virus (HBV) infection or reported vaccination. We examined HBV-related outcomes in these 45 patients, in comparison with 45 recipients of allografts from HBcAb(-) donors (matched for transplant type, date, and pretransplant HBV antibodies). We sought evidence for HBV transmission by testing posttransplant sera for the presence of HBcAb, hepatitis B virus surface antibody, and HBsAg. Additionally, we analyzed alanine aminotransferase profiles and allograft survival rates for all patients. RESULTS: No patient receiving an allograft from an HBcAb(+) donor developed clinical HBV infection. No patient receiving an allograft from an HBcAb(+) donor had HBsAg detected through retrospective testing of stored sera or through prospective routine clinical evaluation and care. However, among the HBcAb(+) kidney recipients, 27% developed new HBcAb and/or hepatitis B virus surface antibody after transplant; in contrast, only 4% of control patients developed new antibody responses (relative risk=4.94; confidence interval 1.07-22.83). Among the recipients of HBcAb(+) organs, 18% developed elevated transaminases after transplant, in comparison with 36% of the controls. No association was found between "seroconverter" status and elevated alanine aminotransferase profiles in either group. CONCLUSIONS: Transplantation of renal allografts from HBcAb(+), HBsAg(-) donors was not associated with clinically detectable HBV disease or antigenemia. However, recipients had a significantly increased risk of HBV seroconversion, consistent with exposure to HBV antigen. These results suggest that HBcAb(+) kidneys can be safely used if transplanted into appropriate recipients, but highlight the need for effective HBV vaccination and vaccine-response monitoring in potential recipients.

Rapid diagnosis of Chlamydia psittaci pneumonia.

Two cases of Chlamydia psittaci pneumonia are presented. In each, a rapid diagnosis was made through the use of direct immunofluorescent antibody staining of respiratory secretions with monoclonal antibodies to chlamydial antigens. In one case the diagnosis was confirmed by the isolation of the causative organism from sputum and a pharyngeal swab. Chlamydial lipopolysaccharide was detectable in sputum from this patient in an enzyme immunoassay. Serological responses to C. psittaci, Chlamydia trachomatis, and Chlamydia pneumoniae were evaluated, and serological cross-reactivity was observed between each species. Rapid antigen detection systems for Chlamydia species that use commercially available reagents can be helpful in the evaluation of selected patient populations.