Q fever epidemiology and pathogenesis.

The lungs are a port of entry and primary infectious focus of Coxiella burnetii, the obligate intracellular contagium of the worldwide zoonosis Q fever. The infectious process and immune response are characterised by studies in cell culture and animal systems. Following endocytosis, replication exclusively occurs in the phagolysosome. Several potential virulence factors are described.

Death at home following a targeted advance-care planning process at home: the kitchen table discussion.

OBJECTIVE: To determine whether home health agency patients' preferences to die at home can be honored following a structured, professionally facilitated advance-care planning (ACP) process provided in the home. DESIGN: A case series of patients who were identified by their home health agency nurses as having a life-limiting illness and then referred for social service assessment, followed for at least 6 months, with median follow-up of 191 days. SETTING: A large, urban, home health agency owned by a not-for-profit integrated healthcare system. PARTICIPANTS: Eighty-four adult patients (median age 75, range 37-94) receiving home care services other than hospice. INTERVENTION: Formally structured social work visits at patients' homes to discuss end-of-life issues, with communication of results to home health nurses and attending physicians. Social workers performed standard psychosocial assessments, obtained patient and family preferences regarding end-of-life care, and provided education about hospice services. MEASUREMENTS: Acceptance of the ACP process, preferences for location of end-of-life care, location of care at the end of life, adequacy of timing of intervention as measured by length of life after ACP, and use of hospice services. RESULTS: Eighty-three of 84 participants (99%) were willing to complete an ACP process in the home setting. Of the 54 patients expressing a clear preference for location of end-of-life care, 46 (82%) wanted this care to be at home. Thirty-nine (46%) of the participants died within 90 days of ACP; 58 (69%) died by the end of the study. Forty-three (75%) of these deaths occurred at home or in a hospice residence. Fifty-one (61%) patients used home, residential, or nursing home-based hospice services during the study. CONCLUSION: In this series of seriously ill home health patients, most preferred to die at home and virtually all were willing to participate in a home-based ACP process. Facilitating ACP among such patients and their families was associated with end-of-life care at home. Use of hospice services was common following ACP in this population.

Genotypic and phenotypic characterization of two Swedish isolates and two prototypic strains of Coxiella burnetii.

Two Swedish isolates of Coxiella burnetii and the two prototype strains of the species, Nine Mile and Priscilla, were characterized with regard to their multiplication and cytopathic effect on BGM cells and by PCR-based amplification of repetitive element DNA and the C. burnetii-specific plasmids QpH1 and QpRS. Moreover, 1330 bp of each 16S rRNA gene were sequence-determined. All four strains multiplied at virtually the same rate and displayed the same type of vacuoles in the BGM cells. Genetic homogeneity was observed inasmuch as the 16S rDNA sequences were identical and the strains showed identical PCR amplification patterns using primers specific to enterobacterial repetitive intragenic consensus DNA sequences. The two Swedish strains and the Priscilla strain also showed identical patterns after PCR amplification of repetitive extragenic palindromic DNA sequences, whereas the Nine Mile strain demonstrated a similar, but not identical pattern. Thus, the investigated strains demonstrated very similar phenotypic and genotypic characteristics. This finding is discussed in view of the very rare occurrence of domestic Q fever in Sweden.

Early cytokine induction in mouse P388D1 macrophages infected by Coxiella burnetii.

Q-fever is caused by Coxiella burnetii, which is an obligate intracellular bacterium with a broad spectrum of host cells, including macrophages. Cytokines produced from macrophages infected by intracellular bacteria play a critical role in the expression of innate immune responses as well as in the subsequent triggering of protective acquired cell-mediated immunity. We followed the induction and secretion of the pro-inflammatory cytokines interleukin 1 alpha (IL-1alpha), tumor necrosis factor alpha (TNF-alpha), and interleukin 12 (IL-12) in the macrophage-like mouse cell line P388D1 during the initial phase of an in vitro infection by virulent C. burnetii Nine Mile. Secretion of IL-1alpha and TNF-alpha were observed within 3 h post-inoculation. IL-12, however, was not detected in cell supernatants. Two forms of C. burnetii exist, virulent phase I and avirulent phase II organisms. To determine whether the cytokine response was dependent on the form of C. burnetii, the induction of IL-1alpha and TNF-alpha in infected P388D1 cells was compared. Both cytokines were produced by macrophages early after infection with Phase I bacteria. A similar induction of TNF-alpha was observed after infection with the avirulent Phase II bacteria, but no IL-1alpha induction could be detected. As the only difference identified between the two forms of C. burnetii is the composition of their lipopolysaccharides (LPS), the ability of each of the purified LPS from the two variants to induce IL-1alpha was investigated. Purified C. burnetii LPS induced a moderate IL-1alpha response in comparison to that induced by the efficient stimulator E. coli LPS. Furthermore, there was no significant difference in action between Phase I and Phase II LPS preparations. We thus postulate that factors other than LPS differ between the two variants of C. burnetii, and these differences may account for differences in IL-1alpha induction.

The ballet of baseball: lessons of the game for hospice.

As Yogi Berra once said, "The future ain't what it used to be." In the era of rapid change in health care, hospice and palliative care programs will survive only through organizational teamwork. Using the lessons of baseball, we present a stadium-eye perspective on how programs can take the three fundamentals of baseball--pitching, batting, and fielding--and translate them into the three fundamentals of organizational teamwork--clinical, operational, and financial. The best clinicians (pitchers) are of no use if the office operations (batting) keep the patients (fans) out of the stadium and no program can survive without the financial resources (fielding.) When you come to a fork in the road, take it.

The relationship of pain and suffering in a hospice population.

Although suffering is frequently encountered in the hospice setting, few studies examine this condition. The purpose of this study was to examine the relationship between terminally ill hospice patients' pain and their physical, spiritual, and personal or family suffering. Using a tool developed to measure suffering in those categories, a convenience sample of 92 patients were asked to rate their worst pain within the last 24 hours, and to rate their suffering at the time of the interview. All items were rated on a 0-10 Numeric Intensity Scale. Pain scores and suffering scores were divided into four categories; no pain or no suffering (0), mild pain or mild suffering (1-3), moderate pain or moderate suffering (4-6), and severe pain or severe suffering (7-10). Mean scores were compared for pain and suffering. More patients experienced suffering than pain. The highest mean suffering scores occurred in the severe pain category. Correlation coefficients for each suffering and pain category were also calculated. Results indicated a statistically significant correlation only between severe pain and suffering in the categories of loss of enjoyment of life, unfinished business, and concern for loved ones. Data indicated that patients view pain and suffering as separate entities. Further research is needed to better define the relationship between pain and suffering in order to improve assessment and intervention in a hospice setting.

Effect of endocytosis inhibitors on Coxiella burnetii interaction with host cells.

The obligate intracellular rickettsia Coxiella burnetii has previously been reported to reach the intravacuolar compartment of host cells by phagocytosis. With the aim to further examine the mechanisms of C. burnetii internalisation, macrophage monolayers were treated with well characterised inhibitors of endocytosis. The treatment with two general inhibitors, colchicine and methylamine, resulted in a pronounced dose-dependent decrease of radiolabelled phase II rickettsiae retained from the intracellular fraction. A third inhibitor used, amiloride, has been reported to reduce effectively clathrin-independent pinocytic pathways. The internalisation of C. burnetii was shown to be substantially reduced also by amiloride and the effect was dependent on its concentration. The passive role of C. burnetii in the internalisation was verified by using heat-killed C. burnetii. Host cells treated with either of the three inhibitors (amiloride, colchicine and methylamine) showed a similar reduction of intracellular C. burnetii after exposure to killed as well as live organisms. The data presented indicate that different endocytic mechanisms, pinocytosis as well as phagocytosis, may mediate the uptake of C. burnetii by a host cell.

The kitchen table discussion: a creative way to discuss end-of-life issues.

Home care and hospice nurses are uniquely positioned to facilitate early discussions about patient's care wishes and goals at the end of life. This article presents a definition of advance care planning, why it is important, a model for facilitating the discussion, suggestions for overcoming barriers, and tools to assist nurses in feeling more comfortable integrating the subject into daily practice.

Genetic exchange mechanisms in Neisseria gonorrhoeae.

There are two mechanisms for genetic exchange in Neisseria gonorrhoeae. Plasmid deoxyribonucleic acid can be transferred by conjugation, which is dependent on the presence of a 24.5-megadalton plasmid in the donor cell. We have shown that chromosomal deoxyribonucleic acid can be exchanged between all colonial variants by transformation, but not by conjugation. In the nonpiliated variants, however, this exchange was dependent on the presence of the 24.5-megadalton plasmid in the recipient cell.

A survey of Q-fever in Sweden.

Coxiella burnetii, the etiological agent of Q-fever has recently been isolated from sheep in southern Sweden. In this region 24-30% of sheep farmers have been exposed to the organism as shown by serological measurements. In veterinarians, another group with high risk of exposure to C. burnetii, about 12% have antibodies to the bacteria. The seropositive veterinarians are scattered all over the country. In two non-risk groups, draftees and hospital employees, 5-7% were found to be positive. This survey showed that Q-fever is a domestic disease which is spread throughout Sweden.

Molecular cloning and expression of calcium-regulated, plasmid-coded proteins of Y. pseudotuberculosis.

A number of plasmid-associated proteins (YOPs) of Y. pseudotuberculosis are induced and expressed at high levels when the pathogen is grown at 37 degrees C in absence of Ca2+ ions. These proteins were recovered both from the outer membrane fraction and the culture supernatant. Two hours after a temperature-shift the YOPs were only found in the culture supernatant, amounting to about 5% of the total cell protein. After 4 h of incubation they were also detected in the outer membrane fraction. Separation by 2-D gel electrophoresis revealed that the YOPs could be separated into 6 different polypeptides; YOP2a (45 kDa), YOP2b (45 kDa), YOP3 (41-42 kDa), YOP4a (34 kDa), YOP4b (34 kDa) and YOP5 (26 kDa). The structural genes of all of these YOPs, except the YOP2a gene, were cloned to pBR322 and their respective genetic localization was established. It was found that the genes were not part of a common operon but scattered around plasmid plB1. Only the YOP4b protein was found to map within the Ca2+ region. The hybrid plasmid plB572 coded for a number of plasmid plB1 specific proteins, one of which showed a molecular weight of 38 kDa. This polypeptide could be precipitated by monospecific V-antiserum, showing that this protein is the V-antigen.

Deoxyribonucleic acid modifications and restriction endonuclease production in Neisseria gonorrhoeae.

Modification of gonococcal deoxyribonucleic acid (DNA) was investigated, and the relationship with endonuclease production was explored. Both chromosomal and plasmid DNA from different gonococcal strains, irrespective of their plasmid content, was poorly cleaved by the restriction endonucleases HaeII, HaeIII, SacII, and BamHI. The fragment pattern of the Tn3 segment present on the 7.2-kilobase gonococcal resistance plasmid, when compared to its known DNA sequence, allowed us to conclude that the HaeIII and BamHI resistance was due to modification of these sites. A comparison of the fragment pattern of the resistance plasmid, when isolated from Escherichia coli or Neisseria gonorrhoeae, revealed that the resistance of HaeII must also be due to modification of its recognition sequence. Isoschizomers of HaeII and HaeIII can be found in isolates of N. gonorrhoeae (NgoI and NgoII, respectively). A new restriction endonuclease in gonococci, NgoIII, with a specificity similar to SacII, is reported here. High-pressure liquid chromatography of gonococcal DNA showed the presence of 5-methylcytosine. It is suggested that the methylation of cytosine residues in the HaeII (NgoI), HaeIII (NgoII), and SacII (NgoIII) recognition sites is the basis for the resistance of gonococcal DNA to cleavage by these enzymes. This methylation may be part of a host restriction modification system. In two out of five gonococcal strains the sequence -GATC- was modified. One strain unable to modify this sequence was a spontaneous mutant of a strain carrying such a modifying function.

Contribution of a TEM-1-like beta-lactamase to penicillin resistance in Neisseria gonorrhoeae.

Two beta-lactamase-producing strains of Neisseria gonorrhoeae were studied. The substrate profile, molecular weight, and isoelectric point of their beta-lactamases were similar to those of the TEM-1 enzyme produced by many gram-negative bacilli. The gonococcal beta-lactamase was cell bound during exponential growth and was most likely located in the periplasm. Penicillin hydrolysis was efficient in intact cells, suggesting that the cell-bound beta-lactamase was freely accessible to benzylpenicillin. Both beta-lactamase-producing strains of N. gonorrhoeae contained an additional multicopy plasmid with a mass of 3.3 megadaltons (Mdal). A spontaneous penicillin-susceptible revertant lacked both beta-lactamase activity and the 3.3-Mdal plasmid, providing evidence for plasmid-mediated penicillin resistance. During a shift from GC medium to rich MOPS medium, growth of the penicillin-susceptible revertant in contrast to that of the plasmid-carrying strain was markedly impaired, suggesting a physiological effect due to the presence of the 3.3-Mdal plasmid.

Genetic basis for colonial variation in Neisseria gonorrhoeae.

When the piliated colony types of Neisseria gonorrhoeae, which predominate in recent isolates, were nonselectively subcultured in vitro, they gave rise to large numbers of nonpiliated, avirulent colonial variants. Evidence is presented to show that most of this variation occurs after active growth has ceased and that the variation is sensitive to the action of deoxyribonuclease. We suggest that this variation is a result of transformation. A second variation in colonial morphology involved differing levels of "colony opacity-associated proteins" in the outer membrane. This variation was also inhibited by the presence of deoxyribonuclease, but the genetic basis for it is not as yet clear.

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii.

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.

Specific detection of Coxiella burnetii through partial amplification of 23S rDNA.

A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.