Janet M. Pruckler

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 µg/mL, a reliable lower limit of detection of 0.09 µg/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 µg/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 94.2%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.

Hospital characteristics associated with colonization of water systems by Legionella and risk of nosocomial legionnaires' disease : A cohort study of 15 hospitals

OBJECTIVEnTo investigate an increase in reports of legionnaires disease by multiple hospitals in San Antonio, Texas, and to study risk factors for nosocomial transmission of legionnaires disease and determinants for Legionella colonization of hospital hot-water systems.nnnSETTINGnThe 16 largest hospitals in the cities of San Antonio, Temple, and Austin, Texas.nnnDESIGNnReview of laboratory databases to identify patients with legionnaires disease in the 3 years prior to the investigation and to determine the number of diagnostic tests for Legionella performed; measurement of hot-water temperature and chlorine concentration and culture of potable water for Legionella. Exact univariate calculations, Poisson regression, and linear regression were used to determine factors associated with water-system colonization and transmission of Legionella.nnnRESULTSnTwelve cases of nosocomial legionnaires disease were identified; eight of these occurred in 1996. The rise in cases occurred shortly after physicians started requesting Legionella urinary antigen tests. Hospitals that frequently used Legionella urinary antigen tests tended to detect more cases of legionnaires disease. Legionella was isolated from the water systems of 11 of 12 hospitals in San Antonio; the 12th had just experienced an outbreak of legionnaires disease and had implemented control measures. Nosocomial legionellosis cases probably occurred in 5 hospitals. The number of nosocomial legionnaires disease cases in each hospital correlated better with the proportion of water-system sites that tested positive for Legionella (P=.07) than with the concentration of Legionella bacteria in water samples (P=.23). Hospitals in municipalities where the water treatment plant used monochloramine as a residual disinfectant (n=4) and the hospital that had implemented control measures were Legionella-free. The hot-water systems of all other hospitals (n=11) were colonized with Legionella. These were all supplied with municipal drinking water that contained free chlorine as a residual disinfectant. In these contaminated hospitals, the proportion of sites testing positive was inversely correlated with free residual chlorine concentration (P=.01). In all hospitals, hot-water temperatures were too low to inhibit Legionella growth.nnnCONCLUSIONSnThe increase in reporting of nosocomial legionnaires disease was attributable to increased use of urinary antigen tests; prior cases may have gone unrecognized. Risk of legionnaires disease in hospital patients was better predicted by the proportion of water-system sites testing positive for Legionella than by the measured concentration of Legionella bacteria. Use of monochloramine by municipalities for residual drinking water disinfection may help prevent legionnaires disease.

More than 10 years of unrecognized nosocomial transmission of legionnaires' disease among transplant patients.

OBJECTIVEnTo investigate a cluster of cases of legionnaires disease among patients at a hospital.nnnSETTINGnA university hospital that is a regional transplant center.nnnDESIGNnRetrospective review of microbiology and serology data from the hospital laboratories and prospective surveillance via the radiology department; a case-control study and environmental sampling within the hospital and from nearby cooling towers.nnnRESULTSnDiagnosis of seven cases of legionnaires disease in the first 9 months of 1996 led to recognition of a nosocomial outbreak that may have begun as early as 1979. Review of charts from 1987 through September 1996 identified 25 culture-confirmed cases of nosocomial or possibly nosocomial legionnaires disease, including 18 in bone marrow and heart transplant patients. Twelve patients (48%) died. During the first 9 months of 1996, the attack rate was 6% among cardiac and bone marrow transplant patients. For cases that occurred before 1996, intubation was associated with increased risk for disease. High-dose corticosteroid medication was strongly associated with the risk for disease, but other immunosuppressive therapy or cancer chemotherapy was not. Several species and serogroups of Legionella were isolated from numerous sites in the hospitals potable water system. Six of seven available clinical isolates were identical and were indistinguishable from environmental isolates by pulsed-field gel electrophoresis. Initial infection control measures failed to interrupt nosocomial acquisition of infection. After extensive modifications to the water system, closely monitored repeated hyperchlorinations, and reduction of patient exposures to aerosols, transmission was interrupted. No cases have been identified since September 1996.nnnCONCLUSIONSnLegionella can colonize hospital potable water systems for long periods of time, resulting in an ongoing risk for patients, especially those who are immunocompromised. In this hospital, nosocomial transmission possibly occurred for more than 17 years and was interrupted in 1996, after a sudden increase in incidence led to its recognition. Hospitals specializing in the care of immunocompromised patients (eg, transplant centers) should prioritize surveillance for cases of legionnaires disease. Aggressive control measures can interrupt transmission of this disease successfully.

A recurrent outbreak of nosocomial legionnaires' disease detected by urinary antigen testing : Evidence for long-term colonization of a Hospital Plumbing System

BACKGROUNDnIn 1994, a hospital reported an increase in nosocomial legionnaires disease after implementing use of a rapid urinary antigen test for Legionella pneumophila serogroup 1 (Lp-1). This hospital was the site of a previous nosocomial legionnaires disease outbreak during 1980 to 1982.nnnMETHODSnInfection control records were reviewed to compare rates of nosocomial pneumonia and the proportion of cases attributable to legionnaires disease during the 1994 outbreak period with those during the same period in 1993. Water samples were collected for Legionella culture from the hospitals potable water system and cooling towers, and isolates were subtyped by monoclonal antibody (MAb) testing and arbitrarily primed polymerase chain reaction (AP-PCR).nnnRESULTSnNosocomial pneumonia rates were similar from April through October 1993 and April through October 1994: 5.9 and 6.6 per 1,000 admissions, respectively (rate ratio [RR], 1.1; P=.56); however, 3.2% of nosocomial pneumonias were diagnosed as legionnaires disease in 1993, compared with 23.9% in 1994 (RR, 9.4; P<.001). In 1994, most legionnaires disease cases were detected by the urinary antigen testing alone. MAb testing and AP-PCR demonstrated identical patterns among Lp-1 isolates recovered from a patients respiratory secretions, the hospital potable water system, and stored potable water isolates from the 1980 to 1982 outbreak.nnnCONCLUSIONSnThere may have been persistent transmission of nosocomial legionnaires disease at this hospital that went undiscovered for many years because there was no active surveillance for legionnaires disease. Introduction of a rapid urinary antigen test improved case ascertainment. Legionella species can be established in colonized plumbing systems and may pose a risk for infection over prolonged periods.

Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov. : three unusual new Legionella species

Seven strains of Legionella-like amoebal pathogens (LLAPs) were characterized on the basis of their cultural and staining characteristics, biochemical reactions, serology, cellular fatty acids (CFAs), isoprenoid quinone composition, total DNA relatedness, analysis of 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analyses. All seven strains exhibited limited growth on buffered charcoal yeast extract alpha (BCYE) agar, required cysteine for growth and contained branched-chain CFAs and quinones typical of Legionella species. The bacilli were Gram-negative and catalase-positive. There were varying degrees of serological cross-reactions between these LLAP strains and other previously described Legionella species. Results from the various tests revealed that four LLAP strains represent three unusual new species of Legionella: Legionella drozanskii sp. nov., type strain LLAP-1T; Legionella rowbothamii sp. nov., type strain LLAP-6T; and Legionella fallonii sp. nov., type strain LLAP-10T. Three other LLAP strains, designated LLAP-7FL, LLAP-7NF and LLAP-9, were shown to be members of the species Legionella lytica. The deductions made from the phenetic characteristics of these bacteria were consistent with the phylogenetic relationships inferred from 16S rRNA and mip gene sequence analyses. This study is the first to speciate LLAP strains on the basis of data including quantitative DNA hybridization.

Epidemic Legionnaires' Disease Two Decades Later: Old Sources, New Diagnostic Methods

In July 1995 we investigated a pneumonia outbreak in a Pennsylvania town. We conducted epidemiological and molecular microbiological studies to determine the outbreak source and interrupt transmission of disease. Legionnaires disease (LD) was quickly identified by urine antigen testing, and a newly developed immunohistochemical stain confirmed nosocomial transmission to a hospital inpatient. LD was confirmed in 22 patients. Case-patients were more likely than controls to have been within 1,000 feet of the hospital (matched odds ratio, 21.0; 95% confidence interval, 2.9-368) during the 2 weeks prior to illness. Legionella pneumophila serogroup 1 (Lp-1) was isolated from hospital cooling towers (CTs) and rooftop air samples but not from hospital potable water or community CTs. Hospital CT and air Lp-1 isolates matched all five patient isolates by monoclonal antibody, arbitrarily primed polymerase chain reaction, and pulsed-field gel electrophoresis subtyping. Strategies to prevent LD must include minimizing transmission from CTs.

Lack of association between Kawasaki syndrome and Chlamydia pneumoniae infection: an investigation of a Kawasaki syndrome cluster in San Diego County.

BACKGROUNDnThe etiology of Kawasaki syndrome (KS), the leading cause of acquired coronary artery disease in children, is unknown. Recent studies have suggested that Chlamydia pneumoniae, a common respiratory pathogen associated with an increased risk of heart disease, might lead to KS.nnnOBJECTIVEnTo assess whether KS was associated with an elevated risk of having a current or antecedent infection with C. pneumoniae.nnnMETHODSnBlood, urine and pharyngeal specimens from KS patients in San Diego County, CA, during a period of high KS incidence were analyzed for evidence of recent C. pneumoniae infection by culture, PCR and serology. Specimens collected from two control groups, family members of KS patients and age-matched children attending outpatient clinics for well child visits, were similarly analyzed.nnnRESULTSnThirteen cases were identified. Forty-five outpatient controls and an average of three family members per patient were enrolled in the study. All specimens tested negative for the presence of C. pneumoniae by PCR and culture except for one blood specimen from the mother of a case-patient. Serologic analysis of patients and a subset of outpatient and family controls revealed no evidence of current C. pneumoniae infection; 4 of 13 adult family controls had IgG titers consistent with past exposure to C. pneumoniae. Case patients were no more likely than outpatient controls to have had a respiratory illness in the preceding 2 months (11 of 13 patients vs. 35 of 45 controls; odds ratio, 1.57; 95% confidence interval, 0.3 to 11.9).nnnCONCLUSIONSnWe found no evidence that C. pneumoniae infection was associated with KS.

Transforming growth factor-beta induces hemoglobin synthesis in a human erythroleukemia cell line

We have examined the effects of TGF beta 1 and TGF beta 2 on the HEL human erythroleukemia cell line. It was observed that TGF beta 1 and 2 induced hemoglobin synthesis in these cells without causing a significant negative effect on cell proliferation. The cell surface markers glycophorin A and transferrin receptor that are associated with erythroid differentiation were also increased. This cell line may provide a model system in which to study the regulation of globin gene expression by a physiological growth factor known to act on hemopoietic cells.

Phorbol Ester Induces Morphological Changes and Acidic Fibroblast Growth Factor RNA Expression in the RD/TE 671 Cell Line

The TE 671 medulloblastoma cell line was induced by phorbol-12-myristate-13-acetate (PMA) to resemble neuronal cells by both morphological and immunohistochemical criteria. Five days following PMA treatment at a concentration of 10 ng/ml, the cells acquired long cellular processes and cell growth was markedly inhibited. Immunohistochemically, PMA-treated cells stained intensely for neuron-specific enolase and were positive for neurofilaments. Northern blot analysis revealed that PMA induced the transcript for acidic fibroblast growth factor. Our data suggest that the TE 671 cell may be useful not only in the study of acidic fibroblast growth factor regulation but also as a model for embryonic tumor cell differentiation.

Potentiation of Human Natural Killer Cell Activity by Recombinant Interleukin-2 towards Multidrug-Resistant Human Epidermoid Carcinoma

Relatively little is understood about the antigen recognition and target structures used by natural killer (NK) cells. The purpose of this study was to analyze the relationship of a multidrug-resistant cell line expressing the P-glycoprotein (GP170 surface glycoprotein) derived from the parent non-drug-resistant malignant tumor cell line for susceptibility to lysis by either NK or lymphokine-activated killer cells. Our results demonstrate no significant difference in NK activity against either the non-drug-resistant or drug-resistant malignant cell line; Interleukin-2 induces a significant increase in cytolytic activity toward the multidrug-resistant cell line. These data suggest that malignant cells refractory to treatment or occurring after successful treatment are susceptible to immunotherapeutic intervention.