Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein.

Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.

Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a).

Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.

Characterization of envelope glycoprotein gp41 genotype and phenotypic susceptibility to enfuvirtide at baseline and on treatment in the phase III clinical trials TORO-1 and TORO-2.

Enfuvirtide (T-20) is the first entry inhibitor approved for treatment of HIV infection and acts by inhibiting conformational changes in the viral envelope protein gp41 that are necessary for fusion of the virus and host cell membranes. Here we present genotypic and phenotypic data on viral envelopes obtained at baseline (n = 627) and after 48 weeks of enfuvirtide treatment (n = 302) from patients in the TORO (T-20 versus Optimized Regimen Only)-1 and -2 phase III pivotal studies. The amino acid sequence at residues 36-45 of gp41 was highly conserved at baseline except for polymorphism of approximately 16% at position 42. Substitutions within gp41 residues 36-45 on treatment were observed in virus from 92.7% of patients who met protocol defined virological failure criteria and occurred in nearly all cases (98.8%) when decreases in susceptibility to enfuvirtide from baseline of greater than 4-fold were observed. Consistent with previous observations, a wide range of baseline susceptibilities (spanning 3 logs) was observed; however, lower in vitro baseline susceptibility was not significantly associated with a decreased virological response in vivo. Virological response was also independent of baseline coreceptor tropism and viral subtype.

MD-2 binds to bacterial lipopolysaccharide.

Many LPS binding proteins have been described, but the exact nature of the LPS receptors that signal cells remains unclear. MD-2 is a molecule that is found in association with Toll-like receptor 4, which has been shown to be a receptor for LPS. We have produced human MD-2 in baculovirus and tested it for LPS binding. MD-2 binds the lipid A region of LPS without the need for LPS binding protein. These data suggest that MD-2 may be binding LPS as part of the TLR4 receptor complex.

Isolation and characterization of two chitinase-encoding genes (cts1, cts2) from the fungus Coccidioides immitis.

Two chitinase (CTS)-encoding genes (cts) from Coccidioides immitis (Ci), a respiratory fungal pathogen of humans, were cloned and sequenced. Both the genomic and cDNA sequences are presented. The transcription start points and poly(A)-addition sites were confirmed. The cts1 gene contains five introns and a 1281-bp ORF which translates a 427-amino-acid (aa) protein of 47.4 kDa. The cts2 gene contains two introns and a 2580-bp ORF which translates a 860-aa protein of 91.4 kDa. The deduced CTS1 protein showed highest homology to the Aphanocladium album and Trichoderma harzianum CTS (74% and 76%, respectively), while CTS2 showed highest homology to the CTS of Saccharomyces cerevisiae (Sc) and Candida albicans (47% and 51%, respectively). The putative N-terminal sequence of the mature CTS1 protein also showed 89% homology to the reported N-terminal sequence of a 48-kDa complement fixation antigen (CF-Ag) of Ci which has demonstrated chitinase activity. The CF-Ag is a clinically important antigen used in serodiagnosis of this fungal disease. CTS2 showed several of the conserved features of the Sc CTS, including putative catalytic and Ser/Thr-rich domains, and a C-terminal Cys-rich region. We propose that CTS1 and CTS2 of Ci are members of two distinct classes of fungal chitinases, an observation not previously reported for a single fungus.

Isolation and characterization of the urease gene (URE) from the pathogenic fungus Coccidioides immitis.

The urease (URE)-encoding gene from Coccidioides immitis (Ci), a respiratory fungal pathogen of humans, was cloned, sequenced, chromosome-mapped and expressed. Both the genomic and cDNA sequences are reported. The transcription start point and poly(A)-addition site were confirmed. The URE gene contains eight introns and a 2517-bp ORF that translates a 839-amino-acid (aa) protein of 91.5 kDa and pI of 5.5, as deduced by computer analysis of the nucleotide sequence. The translated protein revealed eight putative N-glycosylation sites. The deduced URE showed comparable levels of homology to reported URE of the jack bean plant (Canavalia ensiformis; 71.8%) and URE of several genera of bacteria (Bp, 71.7%; Hp, 68.3%; Ka, 71.6%; Pm, 71.9%). The URE gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern hybridization. Expression of a 1687-bp fragment of the URE gene in E. coli resulted in the production of a 63-kDa recombinant protein that was recognized in an immunoblot by antiserum raised against the Ka URE homolog. This is the first report of a fungal URE gene.

Cloning and expression of a gene encoding a T-cell reactive protein from Coccidioides immitis: homology to 4-hydroxyphenylpyruvate dioxygenase and the mammalian F antigen.

The gene which encodes a previously described T-cell reactive protein (TCRP) of the human fungal pathogen Coccidioides immitis (Ci) was cloned and sequenced. Both the genomic and cDNA sequences were determined. The transcription start point was confirmed. The tcrP gene has three introns and a 1197-bp ORF which translates to a 399-amino-acid (aa) protein (45.2 kDa). The predicted protein has approx. 50% aa sequence identity and 70% similarity to mammalian 4-hydroxyphenylpyruvate dioxygenase (HPPD) proteins and mammalian F-antigens. Expression of the Ci tcrP in Escherichia coli resulted in production of a deep brown pigment, consistent with E. coli expression of the bacterial HPPD homolog from Shewanella colwelliana. The TCRP is likely the Ci form of HPPD.

The hsp60 gene of the human pathogenic fungus Coccidioides immitis encodes a T-cell reactive protein.

A heat shock protein-encoding gene (hsp60) from the human respiratory fungal pathogen, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped, expressed and immunolocalized in parasitic cells. Both the genomic and cDNA sequences are presented. The transcription start point and poly (A) addition site were confirmed. The hsp60 gene contains two introns and a 1782-bp ORF which translates a 594-amino acid (aa) protein of 62.4 kDa and pI of 5.6. The translated protein revealed two potential N-glycosylation sites. The deduced HSP60 showed 78-83% aa sequence similarity to reported fungal HSP60 proteins. The hsp60 gene was mapped to chromosome III of Ci and was shown to be a single copy gene by Southern and Northern hybridization. Expression of a 1737-bp cDNA fragment of the hsp60 gene in E. coli resulted in production of a recombinant protein. Amino acid sequence analysis of the recombinant protein confirmed that it was encoded by the Ci hsp60 gene. Antiserum raised in mice against the isolated recombinant protein immunolocalized HSP60 in the cytoplasm and wall of parasitic cells of Ci. The recombinant HSP60 was used to immunize BALB/c mice and was shown to induce proliferation of T cells isolated from lymph nodes of these animals. The hsp60 gene of Ci is the first reported heat-shock protein gene of this human pathogen.

Treatment of coccidioidomycosis with ketoconazole: an evaluation utilizing a new scoring system.

The evaluation of the response of patients with coccidioidomycosis to any therapeutic modality is a major challenge. A numerical scoring system was devised to quantitate separately the severity of disease on clinical presentation, the findings on chest film, bone scan, gallium scan, serology and skin test with coccidioidin and spherulin. The scoring system was used to evaluate the response to treatment with ketoconazole of seven patients with infiltrate pulmonary coccidioidomycosis; 20 patients with chronic cavitary coccidioidomycosis; and 40 patients with disseminated coccidioidomycosis. Dissemination included the soft tissue in 15, bone in 15, synovium in 11 and skin in 18. In all categories clinical severity scores improved dramatically. Radiographic scores showed similar improvement in cases of infiltrative pulmonary coccidioidomycosis but showed no change in cavitary coccidioidomycosis. Serology scores improved significantly (-2 or more) in one of seven infiltrative pulmonary cases, three of twenty chronic cavitary cases and twenty-three of forty disseminated cases. Among those with adequate mycology followup, cultures converted to negative in two of three infiltrative pulmonary coccidioidomycosis; seven of fourteen chronic cavitary coccidioidomycosis; and sixteen of twenty-two with disseminated disease. Unfortunately, when ketoconazole was discontinued or interrupted, symptoms recurred in four of twenty (20 percent) with chronic cavitary and ten of forty (25 percent) of disseminated cases. The disease in two patients progressed while on ketonconazole. One of those developed meningitis.

Aggressive bladder carcinoma in an adolescent. Report of a case with immunohistochemical, cytogenetic, and flow cytometric characterization.

Malignant bladder neoplasms of urothelial origin are rare among children; fewer than 125 cases have been reported. Typically, these tumors are single papillary lesions of low grade and stage that have an excellent prognosis following surgical excision. A grade III transitional cell carcinoma of the bladder occurred in a 14-year-old boy who had no urinary tract malformation, carcinogenic exposure, or family history of cancer. Immunohistochemical stains of the tumor were positive for cytokeratin and high-molecular-weight keratin. The tumor tissue failed to stain with an antibody to the patient's blood group [anti-ABO(H)] but was positive for the Thomsen-Frieden-reich antigen. Flow cytometry of the tumor cells demonstrated a diploid or near-diploid DNA content. A karyo-type of the tumor showed a modal chromosome number of 46 with one reciprocal translocation between chromosomes 17 and 22 and a nonreciprocal translocation between chromosomes 18 and 22. The tumor was unique because of its highly aggressive nature and its diploid chromosome number. This case represents the first indepth characterization of a transitional cell carcinoma in a pediatric patient by flow cytometry and cytogenetics, as well as a variety of immunohistochemical studies including ABO(H) blood group and Thomsen-Freidenreich antigens.

Bacteremic nosocomial pneumonia. A 7-year experience in one institution.

STUDY OBJECT: To describe the epidemiology, microbiology, and outcome of nosocomial pneumonia with secondary bloodstream infection. DESIGN: Prospective cohort study. SETTING: Tertiary care Canadian teaching hospital. PATIENTS: Inpatients. MEASUREMENT: All inpatient blood cultures were concurrently monitored over an 89 month period. Following chart review, patients experiencing nosocomial bloodstream infection due to pneumonia were identified. A standardized definition of pneumonia was used. RESULTS: One hundred forty-nine episodes occurred in 145 patients, 0.66/1,000 hospital admissions, 8.4% of all nosocomial bloodstream infections. No change in rate occurred in the study period. Fifty-four percent of episodes developed in one of seven ICUs. Staphylococcus aureus was the most frequently identified etiologic organism (27%). The ICU and non-ICU cases did not differ in etiology. No organism became more prevalent during the study period. Twenty percent of patients died within 1 week of first positive culture; episodes associated with Pseudomonas species had a much higher mortality rate (45%) than other infections (14%) (p = 0.002). The ICU and non-ICU infections had a similar mortality rate. CONCLUSION: Pneumonia is an important cause of nosocomial bloodstream infection, but it is not increasing in frequency or changing in etiology in our institution. The ICUs are a major contributor to this problem but have the same case short-term mortality rate and microbial etiology as non-ICU cases. Cases associated with Pseudomonas have a much higher mortality rate.

Effect of surgeon's diagnosis on surgical wound infection rates.

To determine the impact of a surgeon's diagnosis of surgical wound infections on infection rates, during a 6-month period we prospectively examined patients undergoing surgical wound surveillance for any of four services (orthopedic surgery, general surgery, neurosurgery, or cardiovascular surgery). Criteria were judged as standardized if the infection control practitioner observed pus, redness, or drainage associated with positive culture or if a diagnosis of deep-seated infection was made. Surgeon's diagnosis was judged as a nonstandardized criterion. Using the Centers for Disease Control's criteria, we identified 113 surgical wound infections in 3024 patients undergoing surgical procedures in the four services. Of these, 95 (84%) met objective criteria (pus observed in 53%; drainage, redness, and positive culture in 20%; and deep-seated infection in 11%). In 18 patients (16%), the nonstandardized criterion alone was used for diagnosis. There was wide variation in use of the nonstandardized criterion, ranging from 5% of orthopedic infections to 21% of cardiovascular surgery infections and 40% of neurosurgical infections. For individual surgeons with at least one wound infection, the range of surgeon's diagnosis was up to 67%. We conclude that a surgeon's diagnosis can have a major impact on surgical wound infection rates; this impact is not borne equally among surgical services or individual surgeons.

Bacteremia due to transplantation of contaminated cryopreserved pancreatic islets.

OBJECTIVE: To report two cases of pancreatic islet transplantation-related septicemia, and the results of an investigative protocol to identify potential sources of contamination. DESIGN: Case series. SETTING: University hospital clinical investigational islet transplantation program. RESULTS: The last two of our first seven islet transplantation recipients developed Enterobacter cloacae septicemia within hours of islet infusion. Both had received thawed cryopreserved islet infusions. No source of infection apart from islets could be identified. Pancreas harvesting and islet isolation protocols provided multiple opportunities for contamination. Environmental cultures during a mock islet isolation procedure failed to identify a source of Enterobacter. Previously cryopreserved islet lots were thawed and submitted for culture, 14/47 grew micro-organisms including E. cloacae in four instances. Following revision of protocols for aseptic handling of islets during processing and cryopreservation 55 consecutive pancreata undergoing processing were evaluated; 7 grew micro-organisms on arrival and in 3 cases these persisted through to cryopreservation. CONCLUSION: Two of seven islet transplantation recipients developed septicemia, likely related to infusion of contaminated cryopreserved islets. Using existing technology, for isolating islets from donor pancreata, recipients will remain at risk for this complication. Prevention should entail strict adherence to aseptic technique, and, possibly, use of surveillance microbial cultures during the islet isolation process.

Identification of antigens of Coccidioides immitis which stimulated immune T lymphocytes.

T-cell mediated immune response to coccidioidomycosis has been shown to be the principal mechanism of resistance to this respiratory fungal disease in experimental animals. In this study, a Coccidioides immitis antigen-specific murine T-cell line was used to identify macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T-cell line was selected on the basis of its strong positive response to a soluble conidial wall fraction (SCWF), which had previously been shown to be reactive in humoral and cellular immunoassays. An antigen-specific T-cell line rather than T-cell clones was used to identify multiple antigens. The T-cell immunoblot method was employed first to identify immunoreactive sub-fractions of the SCWF, and then to identify T-cell fusion proteins (FPs) obtained from a cDNA expression library constructed in lambda gt11. The library was screened with anti-SCWF. The nucleotide sequence of a 0.2 kilobase cDNA insert encoding a FP which elicited vigorous T-cell response was determined. A construct of this insert was subcloned into the pET expression vector system and 6.5-kilodalton (kDa) recombinant protein (RP) expressed in Escherichia coli was isolated. The RP and FP were shown to be homologous on the basis of identify of their amino acid sequences. Antibody raised in guinea pigs against the RP recognized a 59-kDa native protein of the mycelial culture filtrate produced by three separate strains of C. immitis, and reacted with the cell wall of arthroconidia as detected by immunofluorescence microscopy. In this study we have identified and partly characterized a potentially important T-cell stimulating antigen of C. immitis.

Predominance of staphylococcal organisms in infections occurring in a burns intensive care unit.

To assess the sites, incidence, and bacteriology of infections in intensive care burn patients, a prospective survey of all admissions to a tertiary care institution burn unit was carried out over a 12-month period. One hundred and sixteen patients were admitted, 106 with a diagnosis of thermal burns. Forty patients developed 90 infections. Only two deaths occurred, one in a patient with sepsis. In order of frequency, pneumonia, burn infection, UTI and primary bacteraemia were most common. Staphylococcal species accounted for a majority of infections at all body sites except UTI (47 per cent of all infections, including 11 of 14 bacteraemic infections). Staph. aureus sepsis was more common in those carrying the organism on admission. Strain typing of paired admission and subsequent clinical isolates in 19 patients with Staph. aureus sepsis indicated that eight (42 per cent) became infected with a strain they carried on admission. Further reductions in septic complications of burns in our center would be best directed at staphylococcal species, particularly Staph. aureus. Both eradication of carrier state, and prevention of acquisition of Staph. aureus strains could be explored.

Increased risk of bacteremia in patients hemodialyzed through central catheters.

As part of an ongoing prospective survey of nosocomial bacteremias, patients developing bacteremia while undergoing in-centre hemodialysis were observed over a 23 month period. Thirty-six episodes of bacteremia occurred in 30 patients: every episode was directly attributable to hemodialysis. In 28 of the 36 episodes (78%), there was evidence of inflammation with or without drainage of pus at the hemodialysis access site. Staphylococcus aureus accounted for 76% of the bacteremic isolates. Patients hemodialyzing through central venous catheters had a far higher incidence of bacteremia (0.01 per dialysis run) than patients hemodialyzing through vascular grafts (0.0005 per dialysis run).

A multistrain cluster of methicillin-resistant Staphylococcus aureus based in a native community.

Since 1986 the authors' hospital has experienced increased numbers of methicillin-resistant Staphylococcus aureus (MRSA) isolates linked to residents of a native Indian community infected or colonized on admission. A survey of 422 consecutive persons from that community admitted to hospital over a three year period identified 21 (4.9%) carrying MRSA. In a case control study of 34 carriers compared to noncarriers from the community, only prior hospitalization within the past 12 months was identified as being significantly associated with the carrier state, but a specific hospital associated with this risk was not identified. A study of subsets of MRSA isolates in these patients revealed multiple strains present, identified by antibiograms, phage typing profiles and plasmid analysis. Community-based clusters of MRSA have only rarely been previously identified.

Long term trends in the occurrence of nosocomial blood stream infection.

OBJECTIVE: To determine trends in the occurrence of nosocomial blood stream infection at the University of Alberta Hospital. METHODS: A prospective survey of nosocomial blood stream infection was conducted; cases from August 1986 to December 1996 were reviewed. Cases were detected by a review of positive blood cultures reported by the microbiology laboratory. Centers for Disease Control and Prevention definitions of nosocomial infection were used to categorize isolates as nosocomial, community acquired or contaminant. RESULTS: There were 2389 cases; primary bacteremia was the most common source (57%), followed by urinary tract, respiratory tract and surgical site sources (10% each). The nosocomial blood steam infection rate rose progressively from 6.0/1000 admissions and 4.59/10,000 patient days in 1986 to 11.2/1000 admissions and 14.31/10,000 days in 1996 (P<0.01); 48% of the total increase in rate occurred between 1995 and 1996. Significant increases occurred between 1986 and 1996 in primary infections (from 3.2 to 7.5/1000 admissions, P<0.01) and infections from all secondary sources (from 2.5 to 3.8/1000 admissions, P=0.01). Coagulase-negative staphylococci (27%), Staphylococcus aureus (19%) and enterococci (9%) were the most common microbial causes. Aerobic Gram-negative bacilli accounted for 28% and candida for 6%. Coagulase-negative staphylococci, enterococci and candida all became more prevalent as causes of infection over the study period. CONCLUSIONS: The nosocomial blood stream infection rate in the hospital has nearly doubled in the past 10 years, largely due to increased primary bacteremia.

Reduction in surgical wound infection rates associated with reporting data to surgeons.

Several studies have shown that wound infection (surgical site infection [ ssi ]) rates fall when surgeons are provided with data on their performance. Since 1987, the authors have been performing concurrent surveillance of surgical patients and confidentially reporting surgeon-specific ssi rates to individual surgeons and their clinical directors, and providing surgeons with the mean rates of their peers. The program has been gradually refined and expanded. Data are now collected on wound infection risk and report risk adjusted rates compared with the mean for hospitals in the United States National Nosocomial Infections Surveillance (nnis) data bank. Since inception through to December 1993, ssi rates have fallen 68% in clean contaminated general surgery cases (relative risk [rr] 0.36, 95% ci 0.2 to 0.6, P=0.0001), 64% in clean plastic surgery cases (rr 0.35, 95% ci 0.06 to 1.8), 72% in caesarean section cases (rr 0.23, 95% ci 0.03 to 1.96) and 42% in clean cardiovascular surgery cases (rr 0.59, 95% ci 0.34 to 1.0). In clean orthopedic surgery the ssi rate remained stable from 1987 through 1992. In 1993 a marked increase was experienced. Reasons for this are being explored. Overall there was a 32% decrease in ssi rate between the index year and 1993 or, in percentage terms, 2.8% to 1.9% (rr 0.65, 95% ci 0.51 to 0.86, P=0.002). ssi surveillance should become standard in Canadian hospitals interested in improving the quality of surgical care and reducing the clinical impact and cost associated with nosocomial infection.

The impact of health care restructuring on nosocomially acquired blood stream infections.

OBJECTIVE: To assess the impact of the health care restructuring, which occurred in Alberta in 1995, on the occurrence of nosocomial blood stream infection and risk factors for these infections at the University of Alberta Hospital. PATIENTS AND METHODS: Changes in patient population, hospital bed numbers, admissions and hospital days for 1993 and 1994 (1993/94) were compared with those for 1996 and 1997(1996/97). Central venous catheter (CVC) use in intensive care units (ICU), days of total parenteral nutrition (TPN) and hemodialysis were compared for the two time periods. Prospectively collected data obtained by monitoring blood culture results on nosocomial blood stream infections in 1993/94 were compared with those obtained in 1996/97. RESULTS: Hospital bed number fell by 10% between 1993/94 and 1996/97. Annual admissions fell by 19% and patient days by 17%. Some services markedly increased patient days (neurosurgery 49%, nephrology 30%, orthopedic surgery 24%), and others markedly reduced patient days (obstetrics and gynecology 99%, ophthalmology 100%, adult medicine 41%, general paediatrics 38%). ICU use of CVCs increased by 41%, TPN days increased by 25% and hemodialysis runs increased by 9%. Annual nosocomial blood stream infections increased by 31% and the annual rate per 10,000 patient days increased by 60%. TPN-related blood stream infection rates and ICU CVC infection rates did not change from 1993/94 to 1996/97. CONCLUSIONS: Hospital restructuring has been associated with a 31% increase in nosocomial blood stream infection number and a 60% increase in rate. The increase has been associated with a change in patient populations and increases in risk factors for blood stream infection.