Fortuitous vasculitis

A 43-year-old man with a cardiac device for dilated cardiomyopathy presented with fever, night sweats, and weight loss. Investigations revealed pancytopenia, acute renal failure, abnormal lung function, and raised inflammatory markers. A renal biopsy demonstrated pauci-immune necrotizing crescentic glomerulonephritis. He was diagnosed with pulmonary-renal antineutrophil cytoplasmic antibody-negative systemic small vessel vasculitis. He commenced immunosuppression with prednisolone and cyclophosphamide with recovery from pancytopenia and improvement in renal function 3 months later. Subsequently, a bone marrow culture grew Mycobacterium fortuitum. Isolation on repeat peripheral mycobacterial blood cultures prompted treatment with ciprofloxacin and clarithromycin. Four months later, he presented with neutropenic sepsis, influenza A/H1N1, and Aspergillus flavus pneumonia. Despite treatment he deteriorated. A transthoracic echocardiogram revealed a vegetation on the right ventricular pacing wire. The device was removed. The vegetation revealed acid and alcohol fast bacilli on Ziehl-Neelsen staining and grew M. fortuitum on culture, sensitive to ciprofloxacin and clarithromycin. Despite device removal and antimicrobial therapy, the patient succumbed to treatment-related complications. The association between glomerulonephritis and endocarditis is well known; however, this is the first case to our knowledge describing pauci-immune necrotizing crescentic glomerulonephritis in the context of M. fortuitum endocarditis. Clinicians should maintain a high index of suspicion for endocarditis in patients with a cardiac device who present with fever and pauci-immune necrotizing crescentic glomerulonephritis. Patients should be investigated with mycobacterial blood cultures, at least three sets of standard blood cultures and transthoracic and transesophageal echocardiography. Clinicians should beware the perils of immunosuppression in the face of an occult sepsis.     

Developmental and Behavioral Effects of Toe Clipping on Neonatal and Preweanling Mice with and without Vapocoolant Anesthesia

Toe clipping is used to identify and genotype preweanling mice, but the procedure generates concerns relevant to pain and distress. The few pertinent studies available evaluated mice between postnatal days (PND) 3 and 7, advocate the use of toe clipping in mice PND 7 or younger, and identify handling as the most distressing aspect of the procedure. Because both toe and tail clipping may be necessary in older mice to obtain sufficient DNA for genotyping, we surmised that performing these procedures concurrently to minimize handling would be beneficial. We also examined reflex development until PND 21 and adult behavior at 8 to 10 wk of age in mice toe clipped at PND 7 or 17 and the benefits of using topical vapocoolant anesthesia. C57BL/6J pups at PND 7 and 17 were assigned to 1 of 4 groups: 1) clipping of digit 3 of contralateral fore- and hindpaws; 2) toe clipping after topical vapocoolant anesthesia; 3) unclipped, unsprayed controls; and, 4) unclipped and vapocoolant-sprayed. Compared with unanesthetized pups, those sprayed with vapocoolant vocalized and struggled more when handled and had more bleeding, erythema, and swelling, which persisted for as long as 12 h after toe clipping. Reflex development, anxiety, locomotion, and motor coordination were not different among groups or with regard to the age of toe clipping. No tissue reaction was noted microscopically in paws collected at 10 wk of age. We conclude that the use of vapocoolant cannot be recommended due to its harmful effects and that toe clipping at PND 7 or 17 does not significantly affect the long-term welfare of mice.Abbreviations: PND, postnatal day; SHIRPA, SmithKline Beecham Pharmaceuticals, Harwell, MRC Mouse Genome Centre and Mammalian Genetics Unit, Imperial College School of Medicine at St Mary''s, Royal London Hospital, St Bartholomew''s and the Royal London School of Medicine Phenotype AssessmentToe clipping of juvenile mice constitutes the amputation of individual digits of the forepaw or hindpaw.^1,17,18 This technique is used for the purposes of early identification of preweanling mice at ages when other permanent identification techniques are limited and to obtain nucleic acid for genotyping.^25,27 Once personnel are trained appropriately, the additional advantages of toe clipping are that it is a fast and easy technique to apply and it provides the ability to assign a unique number when identifying large numbers of mice. The use of toe clipping is a contentious issue because it is presumed by some to cause pain and distress.^20,28A number of UK-based animal welfare organizations, including the British Veterinary Association''s Animal Welfare Foundation, the Fund for the Replacement of Animals in Medical Experiments, the Royal Society for the Prevention of Cruelty to Animals and the Universities Federation for Animal Welfare established a Joint Working Group on Refinement to address key issues in the generation of genetically modified mice.²⁸ Their recommendations regarding tissue biopsy indicated that because the removal of any tissue may result in pain, suffering, and distress, care must be taken to select the most appropriate biopsy method under suitable conditions and at the appropriate age. The group suggested that the least invasive method of tissue collection should be used and that the minimal amount of tissue sample collected. Their position on toe clipping was that it should not be used routinely as a method of identification or for the purpose of genotyping because of its assumed potential to cause pain and to affect the animal''s ability to grip and groom; that toe clipping should only be used as a last resort in situations where no alternative method of identification or genotyping is possible; and that when used, only a single toe from one hindpaw should be removed under local anesthesia.²⁸ However at the time the group was convened, there were no objective studies published to evaluate methods of toe clipping or its effects.Subsequently, 2 studies evaluated removal of the distal phalanx for toe clipping in mouse pups at postnatal days (PND) 3 and 7.^6,30 These studies concluded that toe clipping at these ages does not appear to have any significant short- or long-term adverse physiologic or behavioral effects on mice;^6,30 that it may be preferable to other methods of permanent identification such as ink tattooing and transponder implantation;⁶ and that the short period of handling, rather than the procedure of toe clipping itself, was the most stressful component of the procedure.³⁰ Stress was assessed by measuring serum corticosterone levels of euthanized clipped, nonclipped and nonhandled pups at PND 7.³⁰The Guide for the Care and Use of Laboratory Animals,¹⁹ referencing these studies, indicates that toe clipping should be reserved for cases where identification is necessary at a young age and when other methods of identification are insufficient or not feasible; that, when used for combined identification and genotyping of neonatal mice, it may be preferable to perform toe clipping prior to PND 7 because it appears to have few adverse effects on behavior or well-being at this age; and that the use of age-appropriate anesthesia and analgesia is recommended. No detailed suggestions for anesthesia or analgesia are provided.¹⁹The Federation of European Laboratory Animal Science Associations established 2 separate working groups that published reports on rodent identification and genotyping methods in early 2013. The Federation''s working group on animal identification discourages the use of ‘nonrefined’ toe clipping (defined as the arbitrary removal of the digit) for identification and instead advocates the use of distal phalanx removal. In view of earlier studies,^6,30 the working group''s recommendation also states that a maximum of one toe per paw should be clipped and that the procedure should take place between PND 5 to 7.⁹ The working group on genotyping recommends that when both identification and genotyping are required, a single method that achieves both aims should be used.⁴ This group also recommends the use of distal phalanx biopsy in place of nonrefined toe clipping under the same conditions outlined by the working group on animal identification and that tail biopsy only be used when a combined method of identification and genotyping does not provide a sufficiently large DNA sample for the proposed genotyping assay, such as Southern blot analysis.⁴The previous studies conducted on the procedure^6,30 did not evaluate toe clipping in mice beyond PND 7. Clipping at such an early age can be impractical due to the small size of the digits. Another limitation of this early time point is that the collection of both toe and tail biopsies is often necessary to obtain sufficient DNA for multiple genotyping assessments by Southern blot analysis. In their intramural program, the NIH currently recommends performing tail biopsy on mice from PND 10.²⁶Therefore, we sought to measure the developmental and long-term behavioral effects of toe clipping at PND 7 and 17. PND 7 was selected as the lower age limit to test the reproducibility of the previous studies, whereas PND 17 was selected as the upper limit because this age is the maximum at which, in light of previous work,¹⁶ our institutions currently allow tail biopsy to be performed without the use of general anesthesia. In addition, because genotyping results would be available by weaning if conducted on or before PND 17, mice of the incorrect genotype could be culled at or before weaning. Our hypothesis was that toe clipping at PND 17 would be no more detrimental to the development and long-term behavior of mice than would be toe clipping at PND 7.We also evaluated the use of a vapocoolant as a method of pain reduction that might improve animal welfare. Typically, anesthesia of neonatal rodents is achieved by three principal methods: injectable anesthesia, inhalant anesthesia, or hypothermia. Injectable agents, such as ketamine or pentobarbital, are not ideal due to their narrow margin of safety, and their use can result in prolonged recovery.¹⁰ The inhalant anesthetic agent isoflurane has been shown to be safe in neonates as young as PND 3.³² However, the practicality of isoflurane anesthesia for this relatively quick procedure is questionable, because this method of anesthesia requires specialized equipment such as a precision vaporizer and the means to scavenge waste gases, and exposure to the gas may be more stressful than the procedure itself. In addition, isoflurane anesthesia does not significantly improve the welfare of mice that undergo tail biopsy between weaning age and PND 31 compared with sham-controls.¹⁵ Hypothermic anesthesia is achieved by placing altricial neonates on, but not in direct contact with, ice. The average duration to reach torpor is 15 min, with a 20 to 30 min recovery period when mice are rewarmed in an incubator. Apart from the lengthy duration of recovery given the short duration of toe clipping, this method is associated with the development of ventricular fibrillation, tissue hypoxia, and metabolic acidosis during the rewarming stage.³⁴ We elected to evaluate application of a vapocoolant to provide short-acting topical anesthesia immediately prior to toe clipping. This method is used by some members of the research community when performing tail biopsy and is recommended in the NIH''s Intramural Guidelines for the Genotyping of Mice and Rats.²⁶     

Does contemporary vancomycin dosing achieve therapeutic targets in a heterogeneous clinical cohort of critically ill patients? Data from the multinational DALI study

Introduction

The objective of this study was to describe the pharmacokinetics of vancomycin in ICU patients and to examine whether contemporary antibiotic dosing results in concentrations that have been associated with favourable response.

Methods

The Defining Antibiotic Levels in Intensive Care (DALI) study was a prospective, multicentre pharmacokinetic point-prevalence study. Antibiotic dosing was as per the treating clinician either by intermittent bolus or continuous infusion. Target trough concentration was defined as ≥15 mg/L and target pharmacodynamic index was defined as an area under the concentration-time curve over a 24-hour period divided by the minimum inhibitory concentration of the suspected bacteria (AUC_0–24/MIC ratio) >400 (assuming MIC ≤1 mg/L).

Results

Data of 42 patients from 26 ICUs were eligible for analysis. A total of 24 patients received vancomycin by continuous infusion (57%). Daily dosage of vancomycin was 27 mg/kg (interquartile range (IQR) 18 to 32), and not different between patients receiving intermittent or continuous infusion. Trough concentrations were highly variable (median 27, IQR 8 to 23 mg/L). Target trough concentrations were achieved in 57% of patients, but more frequently in patients receiving continuous infusion (71% versus 39%; P = 0.038). Also the target AUC_0–24/MIC ratio was reached more frequently in patients receiving continuous infusion (88% versus 50%; P = 0.008). Multivariable logistic regression analysis with adjustment by the propensity score could not confirm continuous infusion as an independent predictor of an AUC_0–24/MIC >400 (odds ratio (OR) 1.65, 95% confidence interval (CI) 0.2 to 12.0) or a C_min ≥15 mg/L (OR 1.8, 95% CI 0.4 to 8.5).

Conclusions

This study demonstrated large interindividual variability in vancomycin pharmacokinetic and pharmacodynamic target attainment in ICU patients. These data suggests that a re-evaluation of current vancomycin dosing recommendations in critically ill patients is needed to more rapidly and consistently achieve sufficient vancomycin exposure.     

The Durban World Congress Ethics Round Table Conference Report: I. Differences between withholding and withdrawing life-sustaining treatments

Introduction

Withholding life-sustaining treatments (WHLST) and withdrawing life-sustaining treatments (WDLST) occur in most intensive care units (ICUs) around the world to varying degrees.

Methods

Speakers from invited faculty of the World Federation of Societies of Intensive and Critical Care Medicine Congress in 2013 with an interest in ethics were approached to participate in an ethics round table. Participants were asked if they agreed with the statement “There is no moral difference between withholding and withdrawing a mechanical ventilator.” Differences between WHLST and WDLST were discussed. Official statements relating to WHLST and WDLST from intensive care societies, professional bodies, and government statements were sourced, documented, and compared.

Results

Sixteen respondents stated that there was no moral difference between withholding or withdrawing a mechanical ventilator, 2 were neutral, and 4 stated that there was a difference. Most ethicists and medical organizations state that there is no moral difference between WHLST and WDLST. A review of guidelines noted that all but 1 of 29 considered WHLST and WDLST as ethically or legally equivalent.

Conclusions

Most respondents, practicing intensivists, stated that there is no difference between WHLST and WDLST, supporting most ethicists and professional organizations. A minority of physicians still do not accept their equivalency.     

DALI: Defining Antibiotic Levels in Intensive care unit patients: a multi-centre point of prevalence study to determine whether contemporary antibiotic dosing for critically ill patients is therapeutic

ABSTRACT: BACKGROUND: The clinical effects of varying pharmacokinetic exposures of antibiotics (antibacterials and antifungals) on outcome in infected critically ill patients are poorly described. A large-scale multi-centre study (DALI Study) is currently underway describing the clinical outcomes of patients achieving pre-defined antibiotic exposures. This report describes the protocol. METHODS: DALI will recruit over 500 patients administered a wide range of either beta-lactam or glycopeptide antibiotics or triazole or echinocandin antifungals in a pharmacokinetic pointprevalence study. It is anticipated that over 70 European intensive care units (ICUs) will participate. The primary aim will be to determine whether contemporary antibiotic dosing for critically ill patients achieves plasma concentrations associated with maximal activity. Secondary aims will compare antibiotic pharmacokinetic exposures with patient outcome and will describe the population pharmacokinetics of the antibiotics included. Various subgroup analyses will be conducted to determine patient groups that may be at risk of very low or very high concentrations of antibiotics. DISCUSSION: The DALI study should inform clinicians of the potential clinical advantages of achieving certain antibiotic pharmacokinetic exposures in infected critically ill patients.     

TUMOR INDUCTION BY IMMUNOLOGICALLY ACTIVATED MURINE LEUKEMIA VIRUS

A graft-vs.-host reaction (GVHR) was induced in young male CAF_I and CB6F₁ mice by the administration of BALB/cJ spleen cells. A proportion of such mice subsequently developed lymphoreticular rumors. Cell-free extracts (CFEs) prepared from the reticular tissues of CAF₁ mice killed at intervals after the induction of the GVHR were tested for their capacity to produce the same tumors in a litter of syngeneic mice inoculated at birth. 12 of 29 (41.4%) such extracts were positive, causing lymphoreticular tumors in one or more littermate recipients. The positive CFEs came from donors killed at all stages of the GVHR, from tumor-bearing mice as well as from non-tumor-bearing mice. However, whereas less than 30% of CFEs from mice killed within 12 mo of GVHR induction were oncogenic, the incidence of oncogenic extracts from mice killed 12–15 mo after GVHR induction rose to 75%. None of the CFEs prepared from nine normal uninjected male CAF₁ mice killed between the ages of 8 and 18 mo transmitted tumors to recipients. CFEs prepared from CAF₁ mice with the GVHR were tested for infectious murine leukemia virus (MuLV) using the XC assay and also for complement-fixing (CF) group-specific MuLV antigen. Substantial titers of B-tropic MuLV and CF antigen were detected in at least half the extracts from mice killed 11–14 mo after GVHR induction. During the first few months of GVHR induction, MuLV titers were low and CF antigen was absent. Neither infectious MuLV nor CF antigen were detected in CFEs prepared from normal control mice. Serially passed CFEs originating from a CB6F₁ GVHR-induced RCN caused similar tumors in successive generations of syngeneic recipient mice. These lymphoreticular tumors were shown to contain infectious MuLV, CF MuLV antigen, and C-type particles. These data together provide evidence that MuLV is activated during the GVHR and that it is responsible for the eventual development of lymphoreticular tumors.     

Ciprofloxacin pharmacokinetic profiles in paediatric sepsis: how much ciprofloxacin is enough?

OBJECTIVE: To determine the pharmacokinetic profile of ciprofloxacin 20 mg/kg per day (10 mg/kg administered intravenously 12 hourly) in paediatric patients with severe sepsis. DESIGN: Open and prospective. SETTING: Tertiary referral multi-disciplinary ICU. PATIENTS: Twenty patients (two groups - group A: 3 months-1 year; group B 1-5 years). INTERVENTIONS: Timed blood samples were taken for pharmacokinetics after the first dose (D(0)), as well as day 2 (D(2)) and then between days 6-8. MEASUREMENTS AND RESULTS: Ciprofloxacin serum levels were measured by high performance liquid chromatography. Demographic and clinical data and all adverse events were noted. Standard pharmacokinetic variables were calculated by non-compartmental methods. Peak concentrations (C(max)) for group A were D(0) 6.1+/-1.2 mg/l, D(2) 9.0+/-1.8 mg/l and D(7) 5.8+/-1.3 mg/l and, for group B, 7.4+/-1.3 mg/l, 7.8+/-1.6 mg/l and 6.4+/-1.3 mg/l, respectively, for the study periods. Concentration 12 h after the start of infusion (C(min)) for all periods were 0.2 mg/l or less. Areas under the curve (AUC, 12 h) were group A: 15.6+/-1.3, 19.2+/-1.63 and 14.1+/-1.4 mg/h per l, and group B: 15.9+/-1.3, 18.0+/-1.7 and 13.2+/-1.26 mg/h per l. One patient presenting with seizures, initially controlled, had another convulsion and a further patient developed seizures whilst on ciprofloxacin. C(max) in these patients were higher than the average C(max). The convulsions of both patients were easily controlled. No other drug-related serious adverse events occurred. No arthropathy was noted. Three patients died of their underlying disease. CONCLUSIONS: There was no accumulation of drug even after 7 days of administration. Our C(max) and AUC were lower than that achieved in a similar adult pharmacokinetic study. To achieve end points of area under the inhibitory curve (AUIC) of 100-150 mg/h per l, 10 mg/kg ciprofloxacin eight hourly would be required for some resistant ICU organisms.