Rotavirus detection using ultrasound enhanced latex agglutination and turbidimetry.

Application of a non-cavitating ultrasonic standing wave to suspended microparticles brings the particles into close approximation and has been used previously to enhance the performance of several diagnostic agglutination tests. The sensitivity of rotavirus detection by ultrasound enhanced latex agglutination was compared with conventional test-card agglutination. Application of ultrasound gave a 32-fold improvement in the sensitivity of detection of rotavirus antigen in buffer compared with the test card method. A novel turbidimetric approach was used to measure agglutination occurring following the test-card procedure (in place of visual examination) and following exposure of commercial rotavirus latex reagents to a 4.5 MHz ultrasonic field (in place of microscopy). The sensitivity enhancement over the conventional method achievable through ultrasonic exposure was comparable whether agglutination measurements were made visually or turbidimetrically and demonstrates the potential for turbidimetry in combination with the ultrasonic method. Turbidimetry offers an alternative to visual assessment that may be more easily incorporated into automated systems.

Targeted prophylaxis with amphotericin B lipid complex in liver transplantation.

The purpose of this study is to prospectively evaluate a strategy in which prophylaxis with amphotericin B lipid complex at 3 different dosages was targeted to liver transplant recipients at high risk for the development of invasive fungal infection (IFI). High risk was defined as a postoperative requirement for prolonged (>/=5 days) intensive care unit (ICU) treatment. Consecutive high-risk patients were administered prophylaxis with amphotericin B lipid complex from day 5 after orthotopic liver transplantation (OLT) until ICU discharge or death. The first 10 eligible patients were administered 5 mg/kg/d, the next 10 patients were administered 2.5 mg/kg/d, and a final 10 patients were administered 1 mg/kg/d. Drug safety and efficacy were assessed before each dosage reduction. During the study period, 130 adult patients underwent 137 OLTs. Thirty patients fulfilled the entry criteria and were administered prophylaxis with amphotericin B lipid complex. No patient developed proven IFI during prophylaxis. Cultures from normally sterile sites (blood and abdominal drain fluid) always showed negative results. All fungal isolates were sensitive in vitro to amphotericin B. There was no significant difference in colonization scores among the groups of patients administered different dosages of amphotericin B lipid complex. No death, serious adverse reaction, or nephrotoxicity was attributed to amphotericin B lipid complex. We conclude that prophylaxis with amphotericin B lipid complex targeted to patients requiring prolonged ICU treatment after OLT appears to be well tolerated and may prevent IFI. Our current policy is to use amphotericin B lipid complex, 1 mg/kg/d, as antifungal prophylaxis in this high-risk group.

Respiratory viruses in adult liver transplant recipients.

BACKGROUND: The contribution of respiratory viruses to respiratory disease in adult liver transplant (LT) recipients has not been studied. We performed a prospective audit to document the incidence of respiratory syncytial viruses ([RSVs], parainfluenza virus, influenza virus, and adenovirus) after LT, and to determine their contribution to respiratory disease in this setting. METHODS: Consecutive adult recipients were followed for 8 months after LT. Throat swabs were collected weekly for up to 12 weeks after LT, and virological surveillance was performed using conventional techniques (direct immunofluorescence and cell culture). A polymerase chain reaction assay for RSV was subsequently performed on selected specimens. Clinical data, including episodes of respiratory disease, were also recorded. RESULTS: During the study period, 51 patients received 53 LT. Five patients died, but no viruses were isolated from these patients at any stage. A total of 323 swabs were examined by conventional techniques and yielded 35 viral isolates (10.8%). Herpes simplex virus (type 1) accounted for 33 isolates, none of which were associated with respiratory disease. Two of 323 swabs (0.62%), in 2 patients, yielded respiratory viruses (both RSV); both patients had self-limiting, mild, upper respiratory tract symptoms. In these 2 patients, the polymerase chain reaction assay was more sensitive than conventional techniques and was able to detect extended RSV excretion. Of 51 recipients, 31 (61%) were always negative for viruses. Of 51 recipients, 10 developed respiratory failure, but no respiratory viruses were isolated from any of these patients. CONCLUSIONS: Respiratory viruses are rarely isolated from adult recipients after LT and are not associated with serious morbidity or with mortality. Routine surveillance for respiratory viruses in this patient population is not justified on the basis of this study.

Fungal infection and liposomal amphotericin B (AmBisome) therapy in liver transplantation: a 2 year review.

We reviewed the use of liposomal amphotericin B in 30 patients receiving therapy following liver transplantation over a 2 year period. Five of these patients were treated for presumed invasive aspergillosis: four of them died despite therapy, each having combined renal and respiratory failure at the time of diagnosis of presumed aspergillosis. Post-mortem examination of three of these patients confirmed the diagnosis of aspergillosis. Twenty-five patients were treated empirically; 11 died and supportive evidence for invasive fungal infection following commencement of therapy was found in only one case. Following liver transplantation, the use of liposomal amphotericin B following confirmation of aspergillus infection or for empirical therapy is of uncertain value, and strategies based on selective prophylaxis for high-risk cases may be preferable.

Fungal colonisation and fluconazole therapy in acute liver disease.

BACKGROUND/AIMS: Fungal infection, particularly with Candida spp., has been identified as an important cause of morbidity and mortality in patients with acute liver failure. Fungal colonisation of superficial mucosal sites usually precedes invasive infection. We investigated colonisation patterns in patients with acute liver disease receiving fluconazole therapy in order to investigate the possibility of emergence of fluconazole-resistant C. albicans or other species. METHODS: During a 6-month study period, we studied all patients referred to our unit with acute liver disease by twice-weekly sampling and mycological analysis of specimens from superficial mucosal and other sites as appropriate. Patients were treated with prophylactic antimicrobials including 100 mg fluconazole daily in accordance with our usual protocol. RESULTS: Twenty-two patients with acute liver disease were studied, eight of whom underwent transplantation. Eighteen patients were colonised by fungi at presentation, and six developed secondary colonisation during fluconazole therapy. Four of these patients (all transplanted) became colonised by resistant species; one of these was Aspergillus fumigatus, which led to death. There were no other invasive fungal infections identified during the study period, and no fluconazole-resistant C. albicans were identified. CONCLUSIONS: Resistance to fluconazole is unlikely to develop in C. albicans during short-term fluconazole prophylaxis in acute liver disease, and in this study we did not find evidence of invasive disease from other Candida spp. during fluconazole therapy. However, in patients at particularly high risk, other strategies are required to prevent infection with Aspergillus spp.

Plasmid-mediated vanB glycopeptide resistance in enterococci.

Enterococcus faecium, which was highly resistant to vancomycin (MIC 256 mg/liter), but susceptible to teicoplanin (MIC 2 mg/liter), caused two distinct episodes of infection on a renal unit in the United Kingdom. Pulsed field gel electrophoresis (PFGE) indicated that a single strain caused the first episode, while the second episode, which occurred 1 year later, involved multiple strains, all of which were distinct from the original strain. Vancomycin resistance in all but one of these strains was mediated by transferable plasmids that carried the vanB glycopeptide resistance gene. Transfer either of resistance plasmids or the vanB resistance determinant itself to different strains occurred during the second episode. Plasmid-mediated vanB resistance has not been widely documented. A retrospective study of a reference collection revealed two other vanB-encoding plasmids from an E. faecalis and an E. faecium referred from two further UK centers. Although restriction analysis indicated no similarity between the plasmids from the three different centers, all contained a 2.1-kb EcoRV fragment that hybridized with a probe for the vanB gene. This suggests that there has been dissemination of a conserved glycopeptide resistance determinant, of which vanB is a part.

Therapeutic progress. IV: Treatment and prophylaxis of Pneumocystis carinii infection.

Co-trimoxazole presently remains the first choice for prophylaxis and treatment of Pneumocystis carinii infections. The high incidence of adverse reactions experienced by patients taking co-trimoxazole has led to a number of trials comparing it with other antipneumocystis agents. Adjuvant therapy with corticosteroids may benefit patients with severe P. carinii pneumonia. This paper reviews the standard treatments for P. carinii pneumonia, some of the newer agents such as atovaquone, recently licensed in the U.K., and a variety of novel agents being assessed for treatment and prophylaxis. Current recommendations may change over the new few years.

Comparison of cell-wall teichoic acid with high-molecular-weight extracellular slime material from Staphylococcus epidermidis.

Extracellular high-mol.-wt material was separated from liquid cultures of Staphylococcus epidermidis. This material contained protein c. 20% w/w and polysaccharide c. 80% w/w. The polysaccharide was isolated by gel and ion-exchange chromatography and contained glycerol phosphate, glucose, N-acetylglucosamine, and D-alanine. Cell-wall teichoic acid was isolated from strain RP-62A and had a similar composition.

Radiochemical assay to measure the biofilm produced by coagulase-negative staphylococci on solid surfaces and its use to quantitate the effects of various antibacterial compounds on the formation of the biofilm.

A firmly adherent mass of slime plus organisms (biofilm) accumulates on the sides of culture tubes when some strains of coagulase-negative staphylococci are grown in a chemically-defined medium containing [14C]glucose. This mass was washed (to remove labelled medium) and then counted after adding scintillation fluid. Organisms from the liquid culture were also washed and counted to check that [14C]glucose had been utilised to label the bacteria. Nine strains were examined in this way, and the results were compared with those obtained with four older techniques for recognising slime production or adherent bacteria. The new method is quick, and has advantages of reproducibility and good discrimination between strains; there was a 15-fold difference in counts in the biofilm between slime-producing and non-producing strains respectively. With the new radiolabel assay, the effects of several antibacterial compounds on the build-up of the biofilm were investigated with four slime-producing strains. Tunicamycin, chloramphenicol and 5-fluorouracil, at levels below their minimum growth-inhibitory concentrations, each greatly diminished biofilm formation; several other drugs had less effect.

Antimicrobial susceptibility testing of mycoplasmas by ATP bioluminescence.

The susceptibility of 72 mycoplasmas to a range of antimicrobial agents was assessed in a 6-h ATP bioluminescence system. ATP was assayed with the Amerlite Analyser. Correlation with conventionally determined MICs was excellent for erythromycin and tetracycline even at 3 h. However, for ciprofloxacin, correlation was poor unless incubation was extended to 6 h.

Enterococcal meningitis in an HIV positive haemophilic patient.

A 25 year old, human immunodeficiency virus (HIV) seropositive, severe haemophilic patient was treated for suspected Pneumocystis carinii infection with high dose intravenous cotrimoxazole and subsequently with prednisolone. When he improved he was discharged on oral treatment only to return two days later, extremely unwell, with headaches, fever, sweats, tachycardia and hypotension. A lumbar puncture showed modest neutrophil pleocytosis but despite empirical antibiotic treatment with intravenous benzylpenicillin and cefuroxime he continued to deteriorate. Culture of cerebrospinal fluid subsequently grew Enterococcus faecalis that was resistant to trimethoprim and sensitive to ampicillin, rifampicin, and vancomycin. After a change in treatment to intravenous ampicillin and rifampicin he dramatically improved. Enterococcal meningitis is rare in adults but important to recognise and treat appropriately in view of its high mortality and relative resistance to antibiotics. In our case the combination of HIV infection and previous treatment with antibiotics or steroids, or both, were probable predisposing factors.

Isolation and composition of the extracellular slime made by coagulase-negative staphylococci in a chemically defined medium.

Slime isolated after growth of four strains of coagulase-negative staphylococci on chemically defined medium plus agar was rich in galactose. However, when sterile agar plates were extracted with saline, high-molecular-weight material with similar properties was obtained that also was galactose-rich. Most of the dry weight attributed to slime, and probably all the galactose, originated from agar. Slime isolated by gel and ion-exchange chromatography from liquid culture in the same medium contained glycerol phosphate, glucose (no galactose), glucosamine, alanine, uronate, an unidentified component, and protein. Separation of protein from carbohydrate was achieved by affinity chromatography. [14C]glucose in the medium labeled the carbohydrate polymer; [14C]amino acids chiefly labeled extracellular proteins. Slime from bacteria grown on medium solidified with silica gel or on dialysis membrane above an agar surface showed essentially the same composition and behavior after purification as the material isolated from liquid culture.

A chemically defined medium for slime production by coagulase-negative staphylococci.

In a chemically defined medium in which the principal constituents were glucose, 18 amino acids, two purines and six vitamins, eight strains of coagulase-negative staphylococci grew as rapidly and heavily as in tryptic soy broth. Slime formation was slightly better in the defined medium than in tryptic soy broth.

Synergy between penicillin and gentamicin against enterococci.

The role of active uptake in aminoglycoside activity against penicillin-treated enterococci was studied by viable counts and ATP determinations. Penicillin and gentamicin gave synergistic bactericidal and post-antibiotic effects (PAEs) which were partially reduced by sodium azide, an electron transport inhibitor, and totally blocked in the presence of both sodium azide and EDTA, which chelates divalent cations. EDTA and gentamicin showed marked synergy in both 'killing curve' and PAE experiments. This synergy was completely inhibited by sodium azide. The data indicate that the activity of gentamicin against enterococci that have been damaged by penicillin or EDTA is energy-dependent. This is consistent with present theories of gentamicin uptake via transportation drive by a protonmotive force.