A longitudinal study of Acinetobacter in three Australian hospitals.

Acinetobacter has recently risen in prominence as a nosocomial pathogen, particularly due to increasing antibiotic resistance. The aim of this study was to describe changes in rates and antibiotic susceptibility patterns of Acinetobacter in three Melbourne hospitals. This was a retrospective review of microbiology records over five years. The rates of new clinical isolates of Acinetobacter per 10 000 discharges per quarter were calculated. Other information collected included antibiotic susceptibility patterns, age, gender, length of stay and ward [intensive care unit (ICU) or non-ICU]. Rates increased substantially at two hospitals, but not at the third. Increasing numbers at one hospital were associated with antibiotic resistance. Most first isolates were identified while the patient was in the ICU. Many isolates were from respiratory specimens, although a significant proportion was from blood. This study documents the establishment of Acinetobacter as a nosocomial pathogen in two Melbourne hospitals and serves as a warning for the future.

Genomic typing of Pseudomonas aeruginosa isolates by comparison of Riboprinting and PFGE: correlation of experimental results with those predicted from the complete genome sequence of isolate PAO1.

The whole genomic typing of 21 isolates of Pseudomonas aeruginosa from 15 intensive care unit (ICU) patients was performed by pulsed-field gel electrophoresis (PFGE using SpeI) and Riboprinting (using EcoRI and PvuII), and then the results were compared with predictions made from the whole genome sequence of P. aeruginosa PAO1. The analysis of electronic images from PFGE and Riboprinting by GelComparII demonstrated similar discrimination between PFGE and Riboprinting with PvuII enzyme; however, Riboprinting by EcoRI had reduced banding patterns and was shown to be of lower discrimination than PvuII. When analyzing isolates from patients, both PFGE and Riboprinting using PvuII enzyme gave equivalent results, with the exception of two isolates that were closely related by PvuII Riboprinting and unrelated by PFGE. These discrepancies in typing results can be explained and adjusted for by comparisons with the rrn properties and the SpeI restriction fragments predicted from the whole genome of P. aeruginosa PAO1. Properties of the rrn operon that need to be taken into account include: (i) restriction enzyme sites that produce one or two fragments for each rrn operon; (ii) genomic variability in ISR sequence length; (iii) different enzymes need to be used to determine differences in rrn operon copy number from Riboprints; and (iv) choice of a restriction enzyme that produces riboprinter bands derived from rrn operon regions that are highly variable within the genome and between isolates. This knowledge has ramifications for PFGE and Riboprinter design and analysis so that for each new species to be typed comparisons can be made using the whole genome sequence.

A comparison of polymerase chain reaction and phenotyping for rapid speciation of enterococci and detection of vancomycin resistance.

This study aimed to ascertain the ability of the microbiology laboratory to detect and identify catalase-negative Gram-positive cocci with particular reference to vancomycin-resistant enterococci (VRE). Twenty-seven reference strains and 42 prospectively collected catalase-negative Gram-positive cocci were screened by agar dilution breakpoint susceptibility and linked biochemical methods in routine use. Ability to speciate organisms was then compared using: (i) a multiplex polymerase chain reaction, designed to detect gene sequences specific to Enterococcus faecalis and E. faecium, and vancomycin resistance (van) genes; (ii) a commercial "API 20 strep" (iii) an algorithm using individual tests from a commercial API 20 strep strip; and (iv) the same algorithm utilising traditional phenotyping methods. All vancomycin resistant catalase-negative Gram-positive cocci were detected by an agar dilution screening plate containing 4 micrograms/ml of vancomycin. Polymerase chain reaction (PCR) detected all enterococci with van genes, speciated all vancomycin-sensitive E. faecalis and E. faecium isolates and excluded non-enterococcal vancomycin-resistant catalase-negative Gram-positive cocci. Algorithm-based methods speciated 41 of the 42 study isolates (98%). The API 20 strep correctly identified only 25 (60%) of these organisms, 38 of which were vancomycin-sensitive E. faecalis. VRE are detected by current screening methods for vancomycin-resistant catalase-negative Gram-positive cocci in this laboratory. API 20 strep, currently used to speciate catalase-negative Gram-positive cocci, is less reliable and should be replaced. Algorithm-based phenotyping by either method tested is more reliable for speciation than API 20 strep in its recommended form. Compared to the other methods tested, PCR is a rapid, accurate and inexpensive method of detecting and speciating vancomycin-resistant enterococci and it provides important extra information impacting on clinical therapy and infection control.

Quantitative detection of human cytomegalovirus DNA in lung transplant recipients.

Human cytomegalovirus (HCMV) disease remains a major cause of morbidity and mortality after lung transplantation. Currently, routine diagnostic tests for HCMV are inefficient and insensitive or nonspecific for HCMV disease. We describe an efficient, highly sensitive, quantitative polymerase chain reaction (PCR) assay for HCMV using competitive PCR and fluorescently labeled primers, and we have used this to measure HCMV DNA load in donor and recipient tissues of six lung transplant recipients at the time of transplantation, and 2 wk after transplantation when clinically stable. Total DNA yield was adequate for analysis in transbronchial biopsy, bronchoalveolar lavage, and peripheral blood leukocytes, but the endobronchial biopsy specimens did not consistently produce sufficient DNA for analysis. There was a large intersubject and intrasubject variability between tissues in HCMV DNA load, with a tendency for greater levels in lung tissue compared with BAL or peripheral blood cells. All six HCMV IgG seronegative donors or recipients were found to have HCMV DNA present. One of the three seronegative matched transplant recipients developed histopathologically proven HCMV disease, and HCMV DNA levels were shown to increase at that time point and subsequently decrease with ganciclovir treatment. This assay will allow prospective studies to confirm the predictive value of HCMV DNA load in donor and recipient tissues for HCMV disease.

Bartonella (Rochalimaea) quintana causing fever and bacteremia in an immunocompromised patient with non-Hodgkin's lymphoma.

A 48-yr-old man with stage IV non-Hodgkin's lymphoma, became neutropenic following chemotherapy and developed a fever. His blood cultures were processed to enhance the yield of fastidious bacteria. A slow-growing, capnophilic Gram-negative rod was isolated. The febrile episode was treated with cefotaxime, imipenem and vancomycin and resolved. The bacterial isolate was identified as Bartonella (Rochalimaea) quintana by 16S-rDNA gene sequencing. The isolate showed 99.8% sequence homology with the type strain. This is the first isolation of Bartonella (Rochalimaea) quintana from a bacteremic patient in Australia. This bacterium is a fastidious Gram-negative rod requiring prolonged culture for its isolation. Patients with culture-negative pyrexia, especially immunocompromised patients, may need to be investigated for infection with this agent.

Mice lacking both macrophage- and granulocyte-macrophage colony-stimulating factor have macrophages and coexistent osteopetrosis and severe lung disease.

Mice deficient in granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF, CSF-1) were generated by interbreeding GM-CSF-deficient mice generated by gene targeting (genotype GM-/-) with M-CSF-deficient osteopetrotic mice (genotype M-/-, op/op). Mice deficient in both GM-CSF and M-CSF (genotype GM-/-M-/-) are viable and have coexistent features corresponding to mice deficient in either factor alone. Like M-CSF-deficient mice, they have osteopetrosis and are toothless because of failure of incisor eruption. Like GM-CSF-deficient mice, they have a characteristic alveolar-proteinosis-like lung pathology, but it is more severe than that of GM-CSF-deficient mice and is often fatal. In particular, in GM-/-M-/- mice the accumulation of lipo-proteinaceous alveolar material is more marked, and bacterial pneumonic infections are more prevalent and more extensive, particularly involving Gram-negative bacteria. Neutrophilia consistently accompanies pulmonary infections, and some older GM-/-M-/- mice have polycythemia. Survival of GM-/-M-/- mice is significantly reduced compared with mice deficient in either factor alone, and all GM-/-M-/- mice have broncho- or lobar-pneumonia at death. These observations indicate that in vivo, M-CSF is involved in modulating the consequences of GM-CSF deficiency in the lung. Interestingly, GM-/-M-/- mice have circulating monocytes at levels comparable with those in M-CSF-deficient mice and the diseased lungs of all GM-/-M-/- mice contain numerous phagocytically active macrophages, indicating that in addition to GM-CSF and M-CSF, other factors can be used for macrophage production and function in vivo.

Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology.

Mice homozygous for a disrupted granulocyte/macrophage colony-stimulating factor (GM-CSF) gene develop normally and show no major perturbation of hematopoiesis up to 12 weeks of age. While most GM-CSF-deficient mice are superficially healthy and fertile, all develop abnormal lungs. There is extensive peribronchovascular infiltration with lymphocytes, predominantly B cells. Alveoli contain granular eosinophilic material and lamellar bodies, indicative of surfactant accumulation. There are numerous large intraalveolar phagocytic macrophages. Some mice have subclinical lung infections involving bacterial or fungal organisms, occasionally with focal areas of acute purulent inflammation or lobar pneumonia. Some features of this pathology resemble the human disorder alveolar proteinosis. These observations indicate that GM-CSF is not essential for the maintenance of normal levels of the major types of mature hematopoietic cells and their precursors in blood, marrow, and spleen. However, they implicate GM-CSF as essential for normal pulmonary physiology and resistance to local infection.

The optimal dosage of ceftazidime for severe lower respiratory tract infections.

The optimal dosage of antibiotics for the treatment of patients with severe lower respiratory tract infections (LRTI) has not been determined but may be lower than is commonly administered at present. We have compared the efficacy of a low dosage of ceftazidime (1 g tds) with the more usual dosage (2 g tds) in a prospective, randomized study of the treatment of LRTI in seriously ill patients. Fifty patients on an Intensive Care Unit received one or the other regimen for 5 days; the demographic characteristics of the two groups were comparable. There was no significant difference in terms of clinical and microbiological response rates between the two regimens. Overall, clinical resolution was documented for 86% of patients, there was no change in 8% and 6% deteriorated. Microbiological eradication was achieved in 52% of patients from whom a pathogen was isolated (46% of all patients). We conclude that ceftazidime 1 g tds is effective treatment for severe LRTI in hospitalized patients.

Efficacy of a low dose of cefotaxime in serious chest infections.

The optimal antibiotic dosage in serious chest infections is not established and commonly used regimens may well be excessive. We have compared the efficacy of a low dose of cefotaxime (2 g every 12 h) with a more usual dose (2 g every 8 h) in a prospective, randomized study of the treatment of chest infections in the seriously ill. Fifty intensive care unit patients received either regimen for five days. The two groups appeared demographically comparable. Clinical resolution occurred in 86 percent, no change occurred in 4 percent, and deterioration occurred in 10 percent. Microbiologic clearance occurred in 52 percent of those in whom a pathogen was isolated (46 percent of patients). There was no significant difference in clinical or microbiologic response between the two regimens. It is concluded that cefotaxime in a dose of 2 g twice daily is effective in the treatment of serious chest infections.

Uncertain relevance of gastric colonization in the seriously ill.

We have studied the temporal relationship for the same micro-organisms between gastric colonization and both nasopharyngeal colonization and major clinical infections in 100 consecutive, long-stay, intensive care patients. 67% of patients developed positive gastric cultures, mainly with aerobic Gram-negative bacilli and C. albicans; 33% developed positive nasopharyngeal cultures with similar organisms, but in only 8% was the same organism previously cultured from the stomach; 48% of patients developed infections, mainly respiratory, but commonly with different organisms. The presence of a positive gastric culture was not associated with gastric pH, bleeding, severity of illness, or mortality. The results fail to confirm that an ascending migration of organisms from the stomach is.frequent or that there is a relationship between gastric colonization and clinical infections. Firm therapeutic recommendation in these areas may be premature.

Detection of cytomegalovirus by the polymerase chain reaction. A simple, rapid and sensitive non-radioactive method.

Cytomegalovirus (CMV) is an important pathogen in persons who are immunocompromised or who have received organ transplants. The rate of symptomatic CMV infection is relatively higher after liver transplantation, and CMV hepatitis may be difficult to differentiate from rejection or cholangitis without recourse to liver biopsy and tissue culture. In this study we have used the polymerase chain reaction (PCR) to amplify and detect CMV-DNA sequences directly from the saliva and urine of liver transplant recipients. The procedure is simple, rapid and specific, and was found to be as sensitive as tissue culture for the diagnosis of CMV.

An Australia-wide epidemic of Pseudomonas pickettii bacteraemia due to contaminated "sterile" water for injection.

Nineteen cases of Pseudomonas pickettii bacteraemia and one case of Pseudomonas cepacia bacteraemia were identified in an Australia-wide outbreak of nosocomial sepsis associated with contaminated water for injection. The contamination was limited to one batch of commercially produced water for injection. Four different organisms were identified (three biotypes of P. pickettii and one of P. cepacia). However, P. pickettii biotype 1 appeared to be relatively more virulent than the other biotypes as it was the only identified organism in blood cultures in nearly all cases of sepsis. The ampoules of "sterile" water were each contaminated with approximately 10(3) organisms per millilitre. The lack of an Australian central reporting system for bacteraemia delayed the recognition of this outbreak.

Quantitative considerations of T-cell activation and self tolerance.

We have reviewed evidence for the hypothesis that T-cell activation by antigen is an all or none process which is triggered when signal strength exceeds a certain threshold. Signal is generated by multivalent interaction between T-cell antigen receptors (TCR) and antigenic epitopes on the surface of antigen-presenting cells (APC) or target cells. Therefore total signal strength (Stot) will depend upon the concentration of TCR (and other accessory molecules that bind to cell surface ligands, e.g. CD4 and CD8) on T cells, the concentration of antigen on APC or targets and the affinity of interaction between receptors (a broad term incorporating TCR plus CD4 and CD8) and antigen. This hypothesis means that T-cell self tolerance is quantitatively determined by self-antigen concentrations on cell surfaces. Implications for a variety of immunological phenomenona are reviewed.

The cytotoxic response to murine cytomegalovirus: requirements for the generation of MCMV-specific target cells.

The preparation of target cells susceptible to lysis by murine cytomegalovirus (MCMV)-immune T cells in vitro was investigated and found to be dependent upon target cell type, culture conditions, virus adsorption protocol and virus preparation. Optimally sensitive MCMV-infected targets were obtained by preculture of mouse embryo fibroblasts (MEF) in 3% vol/vol concanavalin-A-stimulated spleen cell supernatant (CSS)-supplemented medium, adsorption of salivary gland stock MCMV under 800 g centrifugation and at least 4 h further incubation at 37 degrees before addition of T cells. In contrast, salivary gland stock MCMV did not cause thioglycollate-induced peritoneal exudate cells to be susceptible to MCMV-specific T cell-mediated lysis, whereas tissue culture-passaged stock MCMV was successful. The preparation of MCMV-infected target cells is discussed in terms of the need for H-2 and viral antigen expression for T cell recognition.

Concanavalin A-induced splenocyte supernatant causes a preferential increase in H-2K over H-2D and vice versa depending on culture conditions.

Mouse embryo fibroblasts (MEF) from BALB/c mice were exposed to the culture supernatant (CS) from concanavalin A-stimulated splenocytes for 16 h. This caused an increase in the amount of Kd and Dd antigens expressed on the cell surface of the MEF. However, on MEF cultures at 37 degrees C and subcultured daily for the first 5 days after embryonic trypsinization, CS caused the K antigens to increase significantly more than the D antigens, while on MEF cultured for 24 days with a combination of 37 degrees C and room temperature, CS invariably caused the D antigens to increase more than the K antigens. In another protocol involving MEF culture at 37 degrees C and subculture every 4 days, either response was possible when the MEF were exposed to CS. We propose that cells may differentially control K and D antigen increase and discuss how this may be useful in the eradication of viral infection in the animal.

The cytotoxic response to murine cytomegalovirus. III. Lymphokine release and cytotoxicity are dependent upon phenotypically similar immune cell populations.

Murine cytomegalovirus (MCMV)-immune lymph node (LN) cells were generated following 4 days culture of draining popliteal LN cells removed after bilateral hind footpad inoculation of mice with 4 X 10(2) p.f.u. MCMV. This cell population expressed both cytotoxic activity against MCMV-infected mouse embryo fibroblasts (MEF) and the capacity to release lymphokine upon stimulation with MCMV-infected MEF. Cytotoxicity and lymphokine release were Class I, H-2-restricted, virus-specific and dependent upon Thy1. 2+, Lyt2+ cells. Mixing of 5 X 10(5) MCMV-immune T cells with an excess of syngeneic MCMV-infected MEF stimulators produced detectable levels of the lymphokine interleukin-3 by 1 h, with a maximum rate of its release between 1 and 6 h and plateau levels by 14 h. The capacity to stimulate virus-specific MCMV-immune T cells was acquired by MEF at 1 h after MCMV infection with maximum stimulator ability attained by 8 h. In the presence of a fixed number of MCMV-immune T cells, and titration to excess of syngeneic 3 h or 18 h MCMV-infected MEF, lower interleukin-3 plateau levels were obtained with 3 h than with 18 h MCMV-infected stimulators. This suggested that fewer T cells responded to 3 h than to 18 h MCMV-infected MEF.

The cytotoxic response to murine cytomegalovirus. I. Parameters in vivo.

Combined in vivo and in vitro protocols for the generation of anti-murine cytomegalovirus (anti-MCMV) cytotoxic responses were investigated using BALB/c mice and syngeneic mouse embryo fibroblast target cells. Injections of doses of MCMV from 10(2.6) up to 10(6.1) p.f.u. into the hind footpad, harvest of draining popliteal lymph node cells after 6 to 8 days, followed by 4 days culture of these cells gave similar and optimal cytotoxic activity against MCMV-infected target cells. After injection of 10(4.6) p.f.u. into the hind footpad, MCMV was detectable at about 10(3) to 10(4) p.f.u. per popliteal node after 3 days, but became undetectable in most animals by 6 days. Cell numbers in the draining popliteal lymph node increased following MCMV inoculation into the hind footpad, but the extent of the increase was inversely related to virus dose and bore no relationship to the anti-MCMV cytotoxic potential of the cells. Lymph node cells applied directly to target cells upon harvest from MCMV-infected mice never gave detectable anti-MCMV cytotoxic activity at 2, 4, 6, 8, 10, 12, 17 or 19 days post-infection, but lysis of uninfected syngeneic targets was obtained 4 to 8 days post-infection.