Biomedical subjects

B S Fields

Publications and source records attributed to B S Fields.

At least 37 records · Page 2Linked to original sources

PubMed1997

Flagella are a positive predictor for virulence in Legionella.

Pathogenesis of Legionnaires>> disease is strictly related to the ability of the legionellae to infect phagocytic cells, yet surface markers of virulence in Legionella isolates are currently unknown. Rabbit antibodies raised against purified flagella of Legionella pneumophila serogroup 1 recognized a total of 24 of 30 laboratory-maintained isolates of L. pneumophila serogroups 1-15 and 16 of 24 other Legionella species tested by rapid immunoblot and indirect immunofluorescence assay. All isolates possessing flagella detectable with these anti-flagella antibodies, regardless of species, were capable of infecting Hartmannella vermiformis. Isolates lacking immunologic cross-reactivity were shown to lack purifiable flagella. The majority of aflagellate isolates were not motile and failed to multiply intracellularly in co-culture with Hartmannella vermiformis. Some isolates characterized as aflagellate when harvested from BCYE agar were able to multiply in amoebae, and flagella were subsequently detectable by immunologic methods. These data suggest that lack of immunologic recognition of flagella in laboratory-maintained isolates of Legionella is due to their attenuation and a corresponding loss of expression of flagella. More importantly, the presence of flagella can serve as a positive predictive marker for strain virulence and is useful in determining the virulence status of Legionella isolates.

Animals↗

PubMed1997

The role of arbitrarily primed PCR in identifying the source of an outbreak of Legionnaires' disease.

An outbreak of community-acquired Legionnaires' disease (LD) occurred in Providence, R.I., in fall 1993. To find the outbreak source, exposures of 17 case patients were compared to those of 33 matched controls. Case patients were more likely than controls to have visited a section of downtown (area A) during the 2 weeks before illness (11 [65%] versus 9 [27%]; matched odds ratio, 6.5; P = 0.01). Water samples were cultured from 27 aerosol-producing devices within area A. Legionella pneumophila serogroup 1 isolates underwent monoclonal antibody (MAb) subtyping and arbitrarily primed PCR (AP-PCR). All four L. pneumophila serogroup 1 isolates available from case patients who visited area A had identical MAb and AP-PCR patterns. Among 14 environmental isolates, 5 had MAb patterns that matched the case patient isolates, but only 1 had a matching AP-PCR pattern. This investigation implicates a cooling tower in area A as the outbreak source and illustrates the usefulness of AP-PCR for identifying sources of LD outbreaks.

Disease Outbreaks↗

PubMed1997

Application of a nested, multiplex PCR to psittacosis outbreaks.

We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.

Animals↗

PubMed1997

Use of polymerase chain reaction to identify pneumococcal infection associated with hemorrhage and shock in two previously healthy young children.

A PCR assay was developed for detection of Streptococcus pneumoniae in clinical specimens including blood and paraffinized tissues. We were able to detect one organism of purified DNA or 4.5 colony-forming units in blood. The primers did not cross-react with other upper respiratory tract streptococci or with pathogens commonly found in clinical specimens. This assay was used in an investigation of an outbreak of severe illness characterized by septic shock and hemorrhage in previously healthy children. PCR detected S. pneumoniae in cerebrospinal fluid and autopsy tissues of the two infants who died. The findings from this assay indicated that PCR offers increased specificity and sensitivity over latex agglutination and counterimmunoelectrophoresis and should prove useful in the identification of additional cases of severe illness caused by S. pneumoniae.

Animals↗

PubMed1996

Outbreak of Legionnaires' disease among cruise ship passengers exposed to a contaminated whirlpool spa.

BACKGROUND: Outbreaks of travel-related Legionnaires' disease present a public-health challenge since rapid, sensitive, and specific diagnostic tests are not widely used and because detection of clusters of disease among travellers is difficult. We report an outbreak of Legionnaires' disease among cruise ship passengers that occurred in April, 1994, but that went unrecognised until July, 1994. METHODS: After rapid diagnosis of Legionnaires' disease in three passengers by urine antigen testing, we searched for additional cases of either confirmed (laboratory evidence of infection) or probable Legionnaires' disease (pneumonia of undetermined cause). A case-control study was conducted to compare exposures and activities on the ship and in ports of call between each case-passenger and two or three matched control-passengers. Water samples from the ship, from sites on Bermuda, and from the ship's water source in New York City were cultured for legionellae and examined with PCR. FINDINGS: 50 passengers with Legionnaires' disease (16 confirmed, 34 probable) were identified from nine cruises embarking between April 30 and July 9, 1994. Exposure to whirlpool spas was strongly associated with disease (odds ratio 16.2, 95% Cl 2.8-351:7); risk of acquiring Legionnaires' disease increased by 64% (95% Cl 12-140) for every hour spent in the spa water. Passengers spending time around the whirlpool spas, but not in the water, were also significantly more likely to have acquired infection. Legionella pneumophila serogroup 1 was isolated only from the sand filter in the ship's whirlpool spa. This isolate matched a clinical isolate from the respiratory secretions of a case-passenger as judged by monoclonal antibody subtyping and by arbitrarily primed PCR. INTERPRETATION: This investigation shows the benefit of obtaining a recent travel history, the usefulness or urine antigen testing for rapid diagnosis of legionella infection, and the need for improved surveillance for travel-related Legionnaires' disease. New strategies for whirlpool spa maintenance and decontamination may help to minimise transmission of legionellae from these aerosol-producing devices.

Adolescent↗

PubMed1996

The molecular ecology of legionellae.

Legionella pneumophila is the most highly characterized member of a genus of bacteria that survive as intracellular parasites of freshwater protozoa. These bacteria can also multiply intracellularly in human phagocytic cells and cause respiratory disease in humans. Comparison of the invasive strategies of L. pneumophila in mammalian and protozoan cells and study of the interactions between Legionella and protozoa should prove useful in development of strategies for the prevention of legionellosis.

Animals↗

PubMed1996

Community outbreak of Legionnaires' disease: an investigation confirming the potential for cooling towers to transmit Legionella species.

In August and September 1993, we investigated an outbreak of legionnaires' disease in Fall River, Massachusetts, that involved 11 persons; the attack rate was highest in Flint, a community of Fall River. All cases were infected with Legionella pneumophila serogroup 1 (Lp-1). A case-control study revealed that cases were more likely than matched controls to have visited sites in neighborhood A of Flint. Environmental sampling in Flint found that four of nine aerosol-producing devices sampled contained legionellae; only two, conjoined cooling towers on building A, contained Lp-1. Three independent methods of subtyping--monoclonal antibody subtyping, arbitrary primer polymerase chain reaction, and pulsed-field gel electrophoresis--revealed that Lp-1 isolates from three cases with culture-positive legionnaires' disease matched those from the cooling towers on building A. Water samples from the homes of cases with culture-positive legionnaires' disease contained no legionellae. The results of this epidemiologic and laboratory investigation indicate that the cooling towers on building A were the source of the outbreak of legionnaires' disease and confirm the importance of cooling towers in the transmission of legionnaires' disease.

Adult↗

PubMed1996

Comparison of Binax Legionella Urinary Antigen EIA kit with Binax RIA Urinary Antigen kit for detection of Legionella pneumophila serogroup 1 antigen.

The Legionella Urinary Antigen EIA kit (Binax, Portland, Maine) was compared with the EQUATE RIA Legionella Urinary Antigen kit (Binax) for its ability to detect the presence of urinary antigens to Legionella pneumophila serogroup 1. Urine specimens from patients without Legionnaires' disease (n = 33) were negative by both methods (specificity, 100%). Twenty (77%) of 26 urine specimens from patients with Legionnaires' disease positive by the radioimmunoassay kit were also positive by the enzyme immunoassay (EIA) kit. If the cutoff for a positive EIA result were lowered to a ration of > or = 2.5, 23 of 26 (88%) urine specimens would have been positive by EIA and the specificity would remain 100%. Use of the EIA kit is an acceptable method for detecting L. pneumophila serogroup 1 urinary antigens by laboratories that do not want to handle radioactive materials.

Antigens, Bacterial↗

PubMed1996

Heterogeneity of diphtheria toxin gene, tox, and its regulatory element, dtxR, in Corynebacterium diphtheriae strains causing epidemic diphtheria in Russia and Ukraine.

Diphtheria toxin (tox) and its regulatory element (dtxR) from 72 Corynebacterium diphtheriae strains isolated in Russia and Ukraine before and during the current diphtheria epidemic were studied by PCR-single-strand conformation polymorphism analysis (PCR-SSCP). Twelve sets of primers were constructed (eight for tox and four for dtxR), and three regions within tox and all four regions of dtxR showed significant variations in the number and/or sizes of the amplicons. Two to four different SSCP patterns were identified in each of the variable regions; subsequently, tox and dtxR could be classified into 6 and 12 different types, respectively. The great majority of epidemic strains from both Russia and Ukraine had tox types 3 and 4, and only in a single preepidemic strain isolated in Russia were all eight tox regions identical to those of C. diphtheriae Park-Williams No. 8 (tox type 1). Epidemic strains from Ukraine can easily be identified by dtxR type 5, while the majority of the Russian epidemic strains have dtxR of types 2 and 8. No differences in the tox regions between mitis and gravis biotype strains were observed. However, dtxR types 2, 5, and 8 were identified only in the gravis biotype, and dtxR type 1 was characteristic for the mitis biotype strains. PCR-SSCP is a simple and rapid method for the identification of variable tox and dtxR regions that allows for the clear association of tox and dtxR types with strains of distinct temporal and/or geographic origins.

Bacterial Proteins↗

PubMed1995

Reevaluation of the definition of Legionnaires' disease: use of the urinary antigen assay. Community Based Pneumonia Incidence Study Group.

Cases of Legionnaires' disease have been categorized as definitive and presumptive. The sensitivity and specificity of antibody titers of > or = 256 and of urinary antigen ratios of > or = 3 were evaluated in 68 patients with "definitive" Legionnaires' disease and in 636 patients with pneumonia who had negative cultures and did not have fourfold rises in titers of antibody to Legionella pneumophila. An acute-phase antibody titer of > or = 256 did not discriminate between cases and noncases (10% vs. 6%; P = .29). The urinary antigen assay gave a positive result in fewer than 1% of noncases but was positive in 55.9% of all cases. This assay was most sensitive (80%) in cases in which L. pneumophila serogroup 1 was isolated. We propose that the case definition for definitive Legionnaires' disease be expanded to include positive urinary antigen assays and that the category of presumptive Legionnaires' disease--based on acute-phase or standing antibody titers of > or = 256 in the nonoutbreak setting--be discarded. The urinary antigen assay will be a valuable tool in the prompt diagnosis of Legionnaires' disease.

Adult↗

PubMed1995

Association of flagellum expression and intracellular growth of Legionella pneumophila.

We examined the role of the flagella of Legionella pneumophila in the infection of amoebae and human monocyte-like cells. Insertional mutants were constructed with mini-Tn10. Ten mutants (F-) which did not react with polyclonal L. pneumophila antiflagellar antisera were identified. Ten randomly selected mutants (F+) that did react with the polyclonal antiflagellar antiserum were also identified. The infectivity of these 20 mutants in Hartmannella vermiformis and human U937 cells was characterized. Seven of the 10 F- mutants were attenuated in their ability to multiply in the amoebae during the first 3 days of coincubation and failed to multiply in U937 cells. Three of the 10 F- mutants multiplied as well as the wild-type parent strain did in amoebae and to a limited degree in U937 cells. None of the 10 F+ mutants were attenuated in either the amoebae or U937 cells. While the flagellar structure is not essential for virulence, the ability of L. pneumophila to infect amoebae and human phagocytic cells appears to be linked to flagellar expression. We believe that the attenuated F- mutants contain insertions in genes critical to both flagellum expression and the infection process.

Animals↗

PubMed1995

Comparison of Legionella pneumophila isolates by arbitrarily primed PCR and pulsed-field gel electrophoresis: analysis from seven epidemic investigations.

Arbitrarily primed PCR (AP-PCR) and pulsed-field gel electrophoresis (PFGE) subtyping were applied to clinical and environmental isolates from seven unrelated outbreaks of Legionnaires' disease. The patterns observed with each method matched patient isolates and the epidemiologically linked source of disease for each of the seven outbreaks. PFGE allowed more discrimination among various isolates, although AP-PCR usually gave comparable results. With both methods, certain patterns appeared to predominate in the comparison of the seven outbreaks. Of five clinical isolates not associated with the outbreaks, three gave profiles distinct from those observed in the outbreaks by both methods. This suggests that there are at least two predominant subtypes of Legionella pneumophila serogroup 1 associated with outbreaks. Investigations of outbreaks of legionellosis should employ either PFGE or AP-PCR in addition to monoclonal antibody analysis.

Bacterial Typing Techniques↗

PubMed1994

Protein expression by the protozoan Hartmannella vermiformis upon contact with its bacterial parasite Legionella pneumophila.

Legionella pneumophila is ingested by both human macrophages and amoebae, and it multiplies within similar endocytic compartments in both eukaryotic species. Inhibitors of eukaryotic protein synthesis, such as cycloheximide and emetine, had no effect on the uptake of L. pneumophila by macrophages but completely abolished ingestion by the amoeba Hartmannella vermiformis. Therefore, host cell protein synthesis is required for the bacterium to infect the amoeba but not human macrophages. To identify proteins expressed by H. vermiformis upon contact with L. pneumophila, we radiolabeled amoebal proteins after contact with bacteria in bacteriostatic concentrations of tetracycline to inhibit bacterial protein synthesis. We analyzed protein expression by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found that 33 amoebal proteins were induced; 12 of these were not detected in resting amoebae. Eleven other amoebal proteins were repressed; four of them became undetectable. In contrast, no phenotypic changes were observed in H. vermiformis upon contact with Escherichia coli or heat-killed L. pneumophila. An isogenic, avirulent variant of L. pneumophila, incapable of infecting either macrophages or amoebae, induced a different pattern of protein expression upon contact with H. vermiformis. Our data showed that amoebae manifested a specific phenotypic response upon contact with virulent L. pneumophila. This phenotypic modulation may be necessary for uptake of the bacteria into an endocytic compartment that permits bacterial survival and multiplication.

Animals↗

PubMed1994

Heterogeneity of rRNA gene restriction patterns of multiresistant serotype 6B Streptococcus pneumoniae strains.

Three multiresistant serotype 6B Streptococcus pneumoniae strains were isolated from the middle ear fluids of children undergoing tympanostomy in Atlanta. Because multiresistant 6B pneumococci have been reported to spread from a single clone, the three isolates were compared with 13 other multiresistant 6B pneumococci by hybridization of endonuclease-restricted DNA fragments with a digoxigenin-labeled cDNA probe complementary to 16 and 23S rRNAs (ribotyping). The ear isolates were heterogeneous, whereas six of the other pneumococcal isolates were alike, indicating a need for additional studies to determine the possibility of clonal spread.

Child↗

PubMed1993

Attachment and entry of Legionella pneumophila in Hartmannella vermiformis.

Legionella pneumophila is an intracellular parasite of Hartmannella vermiformis. Attachment to the amebae and entry of L. pneumophila were studied by two quantitative assays: One used plate counts to measure the number of bacteria attaching to amebae at 4 degrees C; the other determined the number of intracellular bacteria by use of transmission electron microscopy (TEM). The attachment assay showed that L. pneumophila are inefficient in attachment to amebae. About 0.05% of the bacteria were bound after 1 h with a 10- to 40-fold increase over the next 11 h. Attachment of both virulent and avirulent strains of L. pneumophila occurred at a similar rate. Uptake of L. pneumophila was measured by counting intracellular bacteria using TEM. Limited numbers of virulent L. pneumophila were found intracellularly before 4 h, but the numbers increased logarithmically after this time. The number of amebae containing virulent L. pneumophila increased linearly during the 12-h co-incubation. Avirulent L. pneumophila were rarely detected within amebae throughout the 12-h incubation. Results indicate that entry, not attachment, of virulent L. pneumophila is the limiting step in infection of axenically grown H. vermiformis.

Animals↗

PubMed1993

Use of polymerase chain reaction in an epidemiologic investigation of Pontiac fever.

In June 1992, 13 (38%) of 34 resort guests experienced illness that met a symptom-based case definition of Pontiac fever. Each ill guest reported using an indoor hot tub compared with 6 (29%) of 21 nonill guests (P < .001). Water samples from the indoor hot tub were culture-negative for legionellae using standard techniques, coculture with amebae, and intraperitoneal inoculation of guinea pigs. However, polymerase chain reaction (PCR) testing of the water samples indicated the presence of Legionella pneumophila. Direct fluorescent antibody testing identified the organism as serogroup 6. Seroconversion to L. pneumophila serogroup 6 occurred in 7 (64%) of 11 ill guests and none of 5 nonill guests (P = .03). These results suggest that in certain circumstances, culture of environmental samples should be supplemented with additional tests such as PCR. These results are also consistent with the concept that Pontiac fever can be caused by nonviable legionellae.

Animals↗

PubMed1993

Comparison of arbitrarily primed polymerase chain reaction, ribotyping, and monoclonal antibody analysis for subtyping Legionella pneumophila serogroup 1.

Arbitrarily primed polymerase chain reaction (AP-PCR) was used to characterize Legionella pneumophila serogroup 1. Cells from a single colony could be subtyped by AP-PCR within a few hours. The discrimination between strains of L. pneumophila serogroup 1 by AP-PCR was equivalent to that by monoclonal antibody analysis and ribotyping. Four strains representing the monoclonal antibody pattern most frequently associated with outbreaks all yielded unique amplicon patterns by AP-PCR.

Antibodies, Monoclonal↗

PubMed1992

Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages.

Legionella pneumophila is an intracellular parasite of freshwater protozoa and human macrophages. Recent studies determined that the macrophage infectivity potentiator (Mip) surface protein, a prokaryotic homolog of the FK506-binding proteins, is required for optimal infection of macrophages. To determine whether Mip is also involved in L. pneumophila infection of protozoa, we examined the ability of a strain lacking Mip to parasitize Hartmannella amoebae and Tetrahymena ciliates. After 3 days of incubation, approximately 1000-fold fewer bacteria were recovered from protozoan cocultures infected with the Mip- strain than from those cocultures infected with an isogenic Mip+ strain. The mip mutant was, however, not impaired in its ability to bind to amoebae cell surfaces, indicating that Mip is involved in bacterial resistance to intracellular killing and/or intracellular multiplication. These data suggest that L. pneumophila employs similar genes and mechanisms to infect human cells and protozoa. Furthermore, they support the hypothesis that the ability of L. pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa.

Amoeba↗