Current Opinion in Infectious Diseases Was Launched in 1988

August 2007, Volume 20, Issue 4,pp.345-448

Editorial introductions vii Editorial introductions. Nosocomial and hospital-related infections 345 Advances in pathogenesis and management of sepsis. Ismail Cinel; R Phillip Dellinger 353 Diagnosis of intravascular catheter infection. Philippe Eggimann 360 Prevention of intravascular catheter infection. Philippe Eggimann 370 Neonatal immune responses to coagulase-negative staphylococci. Tobias Strunk; Peter Richmond; Karen Simmer; Andrew Currie; Ofer Levy; David Burgner 376 Clostridium difficile: changing epidemiology and new treatment options. Ed J Kuijper; Jaap T van Dissel; Mark H Wilcox 384 Glycopeptide-resistant enterococci: deciphering virulence, resistance and epidemicity. Rob JL Willems; Marc JM Bonten 391 Measuring the impact of multidrug resistance in nosocomial infection. Stijn Blot; Pieter Depuydt; Koenraad Vandewoude; Dirk De Bacquer Infections of the immunocompromised host 397 Improving the tools in the fight against cytomegalovirus or strengthening David to defeat Goliath. José G Montoya 399 The pathogenesis and clinical management of cytomegalovirus infection in the organ transplant recipient: the end of the 'silo hypothesis'. Robert H Rubin 408 Laboratory diagnosis of cytomegalovirus infection and disease in immunocompromised patients. W Lawrence Drew 412 Late-onset cytomegalovirus disease in patients with solid organ transplant. Pascal R Meylan; Oriol Manuel

419 Prophylaxis and treatment of cytomegalovirus disease in recipients of solid organ transplants: current approach and future challenges. Yoram A Puius; David R Snydman 425 Cytomegalovirus-associated allograft rejection in heart transplant patients. Luciano Potena; Hannah A Valantine Current World Literature Bibliography 432 Current World Literature.

Editorial introductions

Current Opinion in Infectious Diseases was launched in 1988. and Infection, Infection, Journal of Medical Microbiology, It is part of a successful series of review journals whose Reviews in Medical Microbiology, Infection Genetics and unique format is designed to provide a systematic and Evolution and Journal of Microbiological Methods.Heisa critical assessment of the literature as presented in the member of several national and international advisory many primary journals. The field of infectious diseases is boards and EU-supported research networks in the fields divided into 12 sections that are reviewed once a year. of microbiology, hospital infection and antimicrobial Each section is assigned a Section Editor, a leading resistance. He has served as Scientific Officer and Pre- authority in the area, who identifies the most important sident for the European Society for Clinical Microbiology topics at that time. Here we are pleased to introduce the and Infectious Diseases, and chaired the European Study Section Editors for this issue. Group on Epidemiological Markers, the Belgian Infec- tion Control Society and the Belgian Infectious Disease Advisory Board Section Editors

Marc J. Struelens Jose´ G. Montoya

Dr. Struelens completed Dr. Montoya is originally his medical degree from from Cali, Colombia and the Universite´ Libre de completed his medical Bruxelles (ULB) in 1980. degree with honors at the After obtaining a Diploma Universidad del Valle. He of Tropical Medicine at trained in Internal Medicine Prince Le´opold Institute at Tulane University in New of Tropical Medicine in Orleans. Following his resi- Antwerp, he worked for dency, he completed his 3 years as Research Asso- fellowship in Infectious Dis- ciate at the International eases at Stanford University Centre for Diarrhoeal Dis- in Palo Alto under the eases Research, Bangladesh. mentorship of Dr. Jack S. He then returned to Brussels Remington. He is currently where he specialised at ULB Erasme Hospital and was Associate Professor of Medicine and Associate Chief for later appointed Assistant then Associate Professor in Clinical Affairs for the Division of Infectious Diseases Clinical Pathology and Hospital infection Control. He and Geographic Medicine at Stanford University School obtained a PhD in Microbiology at the same university of Medicine. He is also the Director of the Toxoplasmosis with a thesis on the molecular epidemiology of noso- Serology laboratory at the Palo Alto Medical Foundation comial infections. He is currently Professor of Medical Research Institute in Palo Alto and the co-director of the Microbiology at the Faculty of Medicine of the ULB, Immunocompromised Host service at Stanford Univer- Director of the Department of Microbiology, Erasme sity Medical Center that sees all infectious complications Hospital, and Head of the Infectious Diseases Epide- in immunocompromised patients. He has authored miology Unit, School of Public Health, ULB. He has and co-authored over 50 publications in peer-reviewed published over 180 peer-reviewed articles and 20 book journals along with more than 10 book chapters and has chapters and served as editor for international journals given over 100 presentations at CME programs. He has including Microbial Drug Resistance, Clinical Microbiology received more than 10 school-wide teaching awards.

Purpose of review Abbreviations The rationale for therapeutic targets in sepsis has arisen CRP C-reactive protein from the concept of pathogenesis. This review focuses on CVP central venous pressure MAP mean arterial pressure recent advances in pathogenesis of sepsis that can aid in PAMP pathogen-associated molecular patterns management of sepsis patients. PARP poly(ADP-ribose) polymerase PPR pattern recognition receptor Recent findings rhAPC recombinant human activated protein C Cellular survival in sepsis is related to the magnitude of the RIG-I retinoic-acid-inducible gene I ROS/RNS reactive oxygen and/or reactive nitrogen species stimulus, the stage of the cell cycle and the type of microbe. SSC Surviving Sepsis Campaign While phenotypic modification of the endothelium TLR toll-like receptor ScvO2 central venous oxygen saturation (procoagulant and proadhesive properties, increased SvO2 mixed venous oxygen saturation endothelial permeability, endothelial apoptosis and changes in vasomotor properties) leads to vasoplegia as a ß 2007 Lippincott Williams & Wilkins direct correlate to septic shock mortality, phenotypic 0951-7375 changes in the epithelium cause activation of the virulence of the opportunistic pathogens and loss of mucosal barrier function, the latter causing a vicious circle in severe sepsis. Introduction Early identification of sepsis with protocolized screening, Sepsis is the systemic maladaptive response of the host triggering evidence-based protocolized care, is anticipated organism to the invasion of normally sterile tissue, fluid or to reduce sepsis morbidity and mortality. Current treatment body cavity by pathogenic or potentially pathogenic of sepsis includes early antibiotic therapy, early aggressive microorganisms. The culmination of complex inter- goal-directed resuscitation targeting tissue hypoperfusion, actions between the infecting microorganism and the steroids (for refractory shock), activated protein C (for high host immune, inflammatory and coagulation responses risk of death) and maintaining support of organ systems. influences the outcome in sepsis. Until recently, sepsis Summary was regarded as a condition of hyperinflammation and A better understanding of pathogenesis of sepsis has led to hypercoagulation resulting in cellular damage and macro- specific proven management tools that are likely to improve circulation/microcirculation derangement. Dysregulation clinical outcome once incorporated into protocolized care. of the immune response favoring a shift to an anti- inflammatory phenotype and phenotypic modulations Keywords of cells which can activate the virulence of the opportu- lactate, pathogenesis of sepsis, protocolized care, severe nistic pathogens, however, may be equally important sepsis, steroids, Surviving Sepsis Campaign [1,2 ]. Phenotypic modification of the endothelium including changes in procoagulant and proadhesive pro- Curr Opin Infect Dis 20:345–352. ß 2007 Lippincott Williams & Wilkins. perties, increased endothelial permeability, endothelial cell apoptosis and changes in vasomotor properties leads Robert Wood Johnson School of Medicine, University of Medicine and Dentistry of to vasoplegia, which is directly related to septic shock New Jersey, Department of Critical Care Medicine, Cooper University Hospital, Camden, New Jersey, USA mortality. Correspondence to R. Phillip Dellinger, MD, Head, Department of Critical Care Medicine, Cooper University Hospital, One Cooper Plaza, 393 Dorrance, Camden, Pathogenesis of sepsis NJ 08103, USA Tel: +1 856 342 2657; fax: +1 856 968 8306; The innate immune system is an evolutionally conserved e-mail: [email protected] host defense mechanism against pathogens [3]. Innate

Current Opinion in Infectious Diseases 2007, 20:345–352 immune responses are initiated by pattern recognition receptors (PRRs), which recognize speciﬁc structures of microorganisms.

Pattern-recognition receptors and pathogen-associated molecular patterns The initiation of the response during sepsis or in response to sterile tissue injury involves three families of PRRs [4]: toll-like receptors (TLRs), nucleotide-oligomerization domain (NOD) leucine-rich repeat proteins and retinoic-

345

Figure 1 Pathogenic mechanisms leading to organ dysfunction PRRs. Organ dysfunctions in severe sepsis can be seen as the clinical manifestation of a TLR-mediated dysregulation of the immune response to pathogens. Pathogenic mechanisms leading to organ dysfunction TNF-α The lipopolysaccharide of Gram-negative bacilli binds to IL-1 HMGB-1 Protease activation lipopolysaccharide-binding protein, CD14 complex. The Heparan sulfate Hyaluronic acid peptidoglycan of Gram-positive bacteria and the lipopo- Fibronectin Heat shock lysaccharide of Gram-negative bacteria bind to TLR-2 proteins Insult Fibrinogen 24 hr Surfactant A and TLR-4, respectively. Binding of TLRs activates PAMPs LPS, LTA, PGN, Flagellin Endogenous activators intracellular signal-transduction pathways that lead to the activation of cytosolic nuclear factor-kb (NF-kb). PMN, Monocyte, Lymphocyte, Dendritic cells Endothelium, Epithelium, VSMCs RBCs Platelets Activated NF-kb moves from the cytoplasm to the ibition PRRs inh TLRs NOD-LRRs RLHs nucleus, binds to transcription sites and induces acti- NADPH oxidase Myeloperoxidase β Caspase-1 iNOS vation of a set of genes, as well as enzymatic activation Release of TLR NF-k Inflammasome COX-2 of a cellular protease. TLRs induce pro-interleukin-1b Proinflammatory cytokines Anti-inflammatory cytokines Enzymes production and prime NOD-like receptor-containing

Adhesion molecules RAGE expr. ROCK expr. PARP-1 expr. PPAR Expr. multiprotein complexes, termed ‘inﬂammasomes’,to Cell signaling with respond to bacterial products and products of damaged cytokines Inflammation vs Anti-inflammation and ROS/RNS Coagulation vs Anti-coagulation cells [3]. This results in caspase-1 activation and the Potential imbalances between Oxidant vs Anti-oxidant Apoptotic vs Anti-apoptotic subsequent processing of pro-interleukin-1b to its active

Vasodilation Capillary perm. Edema RBC deformability form. Negative regulation of TLRs and TLR-induced Ischemia/Reperfusion Hypoxia/Reoxygenation programmed cell death have also taken place [5].

MPT, Cyt C release Mitochondrial dysfunction Bcl-2 Cell signaling with reactive oxygen and/or O2 Delivery/Consumption mismatch Mitoptosis ++Apoptosis Necrosis nitrogen species Endothelial dysfunction Epithelial dysfunction Reactive oxygen and/or reactive nitrogen species (ROS/ Microcirculatory failure RNS) exert several beneficial physiological cellular func- Multiorgan failure tions such as intracellular signaling (several cytokines, growth factors and hormones use them as second mes- Pathogenic mechanisms during sepsis or in response to sterile tissue sengers) and redox regulation. ROS/RNS are produced injury can lead to multiorgan failure. COX-2, cyclooxygenase; Cyt C, by the nicotinamide adenine dinucleotide phosphate- cytochrome C release; HMGB-1, high mobility group box-1; IL-1, inter- oxidase complex, and represent a defense mechanism leukin 1; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; LTA, lipoteichoic acid; MPT, mitochondrial permeability; NOD-LRR, against invading microorganisms. Despite their import- nucleotide-oligomerization domain leucine-rich repeat protein receptors; ance in innate immunity as a defense mechanism against PARP-1, poly(ADP ribose) polymerase-1; PGN, peptidoglycan; PPAR, invading pathogens, an overwhelming production of peroxisome proliferator-activated receptor; PRRs, pattern recognition receptors; RAGE, receptor for advanced glycation end-products; RBC, ROS/RNS or a deficit in antioxidant systems can result red blood cell; RIG-I-like helicases, retinoic-acid-inducible gene I (RIG-I)- in oxidative/nitrosative stress, which is the key element like helicases; ROCK, RhoA/Rho kinase; ROS/RNS, reactive oxygen in the cascade of deleterious processes in sepsis [7]. and nitrogen species; TLRs, toll-like receptors; TNF-a, tumor necrosis factor a; VSMCs, vascular smooth muscle cells. Superoxide anion (O2 ) and peroxynitrite (ONOO ) play key roles in the pathogenesis of hemodynamic instability and organ dysfunction during septic shock. ONOO can acid-inducible gene I (RIG-I)-like helicases, as shown in cause DNA strand breakage, triggering the activation of Fig. 1. TLRs with 13 distinct receptors are capable of poly(ADP-ribose) polymerase (PARP). PARP plays a role sensing organisms ranging from bacteria to fungi, proto- in the repair of strand breaks in DNA, and its activation zoa and viruses, and play a major role in human innate results in a substantial depletion of nicotinamide adenine immunity [5]. Gram-positive and Gram-negative bac- dinucleotide, thus leading to cell dysfunction. It has been teria, viruses and fungi have unique cell-wall molecules shown that PARP inhibitors have beneficial effects known as pathogen-associated molecular patterns against oxidative and nitrosative stress-induced organ (PAMPs), also termed ‘microbial-associated molecular dysfunctions in endotoxemia [8,9]. Recently, the poten- patterns’. These molecules are common to pathogenic, tial role of PARP activation has been demonstrated in the nonpathogenic and commensal bacteria [6]. PAMPs bind pathogenesis of myocardial contractile dysfunction to PRRs, that is TLRs, on the surface of immune cells. associated with human septic shock [10]. Cytoplasmic PRRs have, however, been identified to detect pathogens that have invaded cytosols [5]. Obser- Several important antioxidant defense systems are based vations suggest that specific host immune response to around glutathione, which in the reduced form is the each pathogen is mediated by various sets of PAMPs and most important intracellular antioxidant within human

cells. Replacement of glutathione stores with glutamine various microparticle formations in oxido-inflammatory in sepsis has been demonstrated to exert a beneficial states may participate in the mechanism of vascular effect in the prevention of organ damage [11]. Another endothelial injury and resultant organ dysfunction. element of the defense system is formed by chaperones Evidence is emerging that microparticles defined as or heat shock proteins. It has recently been demonstrated deleterious partners in sepsis play an important role in that glutamine’s protection against sepsis is dependent coagulation, inflammation and endothelial dysfunction on HSP70 expression [12]. [19]. Levels of microparticles and their interactions with leukocytes have however been shown to negatively Coagulation and inflammation correlate with organ dysfunction in severe sepsis [20]. Sepsis is characterized by exacerbated coagulation, impaired anticoagulation and decreased fibrin removal. Mitochondrial dysfunction With systemic inflammation, interleukin-6 release trig- Although microvascular blood flow abnormalities have gers tissue factor upregulation and tumor necrosis factor- been described in experimental and human sepsis, it is a suppresses the natural anticoagulants, combining to unlikely that these alone explain the pathogenesis of organ produce a tendency towards coagulation activation in dysfunctions seen in severe sepsis [21]. The concept of sepsis [13]. These derangements are implicated in the sepsis-induced abnormalities in oxygen utilization at the generation of microcirculation thrombosis, with depo- mitochondrial level is supported by findings of elevated sition of microclots and obstruction of microcirculation, tissue oxygen tension and decreased oxygen consumption, impairing blood flow and contributing to tissue hypoper- together with functional and biochemical derangements fusion and organ dysfunction. The consumption of associated with minimal cell death in sepsis and septic protein C in sepsis may play a pivotal role in the associ- shock [22,23]. Growing evidence suggests that pertur- ation of inflammation and coagulation. Baseline protein C bations of key mitochondrial functions, including mito- levels are an independent predictor of sepsis outcome, chondrial permeability transition, play a critical role in and day 1 changes in protein C, regardless of baseline septic organ dysfunction [24]. In humans, skeletal levels, are also a predictor of outcome [14]. muscle mitochondrial dysfunction has been demonstrated to relate to severity of sepsis and poor outcome [25]. Endothelial dysfunction and microparticles Vascular endothelium plays an important role in regulat- Apoptosis ing immune and inflammatory responses to pathogens. Apoptosis (programmed cell death) of immune effector Endothelium dysfunction and impaired microvascular cells is a hallmark of sepsis. Sepsis induces extensive function in sepsis are increasingly recognized as key lymphocyte and dendritic cell apoptosis that alters characteristics contributing to organ dysfunction and immune responsiveness, resulting in decreased clearance death. Sepsis induces phenotypic modulations of the of invading organisms [1,26]. The profound decrease in endothelium through direct or indirect interaction the numbers of T and B cells impairs the adaptive between the endothelial layer and components of the immune response. The loss of cells of the adaptive bacterial wall, inducing various host-derived factors from immune system also impairs the innate immune response endothelial cells. On the molecular level, endothelial because of the important cross-talk between the innate dysfunction is caused by reduced nitric oxide bioavail- and adaptive immune system. Additionally, the uptake of ability, which is, in turn, regulated by genes such as nitric apoptotic cells has an anti-inflammatory/immunosuppres- oxide synthase, phosphatidylinositol 3-kinase and AKT sive effect through the induction of anergy and T-helper- [15]. On the cellular level, endothelial dysfunction is 2 cell responses on surviving immune cells. Three based on a progressive loss of endothelial cells deter- independent autopsy studies of adult, pediatric and mined by the degree of apoptosis of endothelial cells [16]. neonatal patients who died of sepsis showed profound The microvasculature contributes to inflammation apoptosis-induced depletion of CD4þ T cells and B through altered leukocyte recruitment and impaired per- cells [23,27,28]. These findings were similar to animal fusion [17]. It has been demonstrated that early micro- studies showing increased lymphocyte and epithelial cell circulatory perfusion indices in severe sepsis and septic apoptosis [29,30]. Moreover, clinical studies of patients shock are more impaired in nonsurvivors compared with with sepsis demonstrate that the degree of apoptosis of survivors [18]. circulating lymphocytes correlates with sepsis severity and predicted fatal outcome in septic shock patients, Microparticles shed during cell activation or apoptosis suggesting the importance of apoptosis as a biomarker have procoagulant and proinflammatory properties. [29,31,32]. Microparticles can be derived from circulating cells (platelets, leukocytes and erythrocytes) as well as cells that Management of sepsis compose the vessel wall, mainly endothelial cells, macro- Early identification of sepsis with the help of protocolized phages and smooth muscle cells. The orchestration of screening, triggering evidence-based protocolized care, is

anticipated to reduce sepsis morbidity and mortality. Patients may develop fever, leukocytosis or elevated The evidence for best clinical practice for resuscitation, CRP without infection. Measurement of procalcitonin management of infection and intensive care unit sup- has been shown to be superior to CRP in detecting portive care has been synthesized by the Surviving Sepsis significant infection compared with clinical signs [39]. Campaign (SSC), and published as evidence-based Several studies have underscored the value of pro- guidelines for the management of severe sepsis and calcitonin in identifying infectious processes, charac- septic shock [33]. terizing the severity of the underlying illness [39], guiding therapy [40] and risk stratification [41]. It has Protocolized screening been reported that procalcitonin increase for 1 day Efforts have been made to reduce the time needed to (1.0 ng/ml) is an independent predictor of 90-day sur- diagnose sepsis in order to reduce mortality from sepsis- vival [42]. Optimizing antimicrobial dosing, especially related multiple organ dysfunction. Protocolized screen- avoiding underdosing, is an important goal to achieve ing is very important, especially in the early phase of effectiveness of therapy and prevent the risk of devel- sepsis, and can also help to identify critically-ill patients opment of resistant microbial side effects and treatment who are at a high risk of mortality [34]. One such costs. A reduced use of antimicrobial therapy has been approach is that recommended by the SSC using the demonstrated when treatment was guided by procalcito- sepsis bundle performance improvement program, which nin in patients with suspected lower respiratory tract is based on selected recommendations from the SSC infection without affecting outcome [40]. bundles for the management of severe sepsis and septic shock [33]. Bundles represent performance indicators Antibiotic therapy which, when achieved in a timely manner, are anticipated Selecting initial antibiotics that cover the infecting organ- to improve clinical outcome. Protocolized screening ism is a high priority in sepsis. It has been demonstrated should be employed throughout the hospital and should that administration of an antimicrobial effective for iso- not be delayed pending intensive care unit admission. lated or suspected pathogens within the first hour of documented hypotension was associated with a survival Lactate rate of 79.9%, and each hour of delay in antimicrobial The conventional view in severe sepsis or septic shock is administration over the ensuing 6 h was associated with that most of the lactate that accumulates in the circulation an average decrease in survival of 7.6% [43]. In multi- is due to cellular hypoxia and the onset of anaerobic variate analysis [including Acute Physiology and Chronic glycolysis. There is increasing evidence that sepsis is Health Evaluation (APACHE) II score and therapeutic accompanied by a hypermetabolic state, with enhanced variables], time to initiation of effective antimicrobial glycolysis and hyperlactatemia [35]. This should not therapy has been shown to be the single strongest pre- be rigorously interpreted as an indication of hypoxia. dictor of outcome. After appropriate cultures have been The link between Naþ/Kþ-ATPase pump activity and obtained, intravenous antibiotic therapy should be muscle lactate formation has been shown in human septic started within the first hour of recognition of severe shock [36]. Although serum lactate concentrations may sepsis. Establishing a supply of premixed antibiotics in lack precision as a measure of tissue metabolic status, an emergency department or critical care unit for such levels equal to or greater than 4.0 mEq/l support aggres- urgent situations is an appropriate strategy for enhancing sive resuscitation. Persistence of an elevated lactate level the likelihood that antimicrobial agents will be infused can be due to consistent overproduction related to a promptly. Cycling or rotating antibiotics has been advo- persistence of the initiator mechanism as well as lowering cated to reduce the risk of emergence and selection of of lactate clearance due to hepatic dysfunction. In septic bacterial resistance, although the frequency of cycles is shock, hyperlactatemia is mainly related to increased unclear [44]. production with lactate clearance similar to healthy subjects [37]. Irrespective of its mechanism of formation, Early aggressive goal directed resuscitation targeting hyperlactatemia remains an excellent prognostic marker tissue hypoperfusion in sepsis. Aggressive resuscitation of a patient with sepsis-induced tissue hypoperfusion (hypotension persisting after initial Future role of biomarkers fluid challenge or serum lactate of at least 4 mmol/dl) Diagnosis of infection is difficult in critically-ill patients. should begin as soon as recognized [33,34,45]. Data from Markers of inflammation such as C-reactive protein the Sepsis Occurrence in Acutely Ill Patients (SOAP) (CRP) and white blood cell count have proved less than study showed that fluid balance was the most important ideal in identifying critically-ill patients who need anti- predictor of mortality [46]. Assessing fluid responsive- microbial therapy, as sensitivity and specificity for bac- ness, however, is complicated. A fundamental component terial infection is low. Patients with liver dysfunction may of the fluid challenge technique is the monitoring of not mount an adequate CRP response to infection [38]. cardiac filling pressures, as hydrostatic pressures are

the primary determinant of edema formation. End-dias- under-resuscitated. Without a measurement of central tolic volumes represent ventricular preload better than venous oxygenation, these subjects may be mistriaged. In filling pressures. The goal of a fluid challenge must be a reference to the correlation between ScvO2 and SvO2, the clinically relevant end point, such as an increase in relationship seems strong (with a correlation coefficient of arterial pressure, a decrease in heart rate, or an improve- 0.8 in several studies) [51]. ment in peripheral perfusion. Rate of fluid administration needs reduction with rising filling pressures and no The Saline versus Albumin Fluid Evaluation (SAFE) improvement in tissue perfusion. trial showed no benefit of albumin over crystalloid resus- citations [52]. There are, however, multiple types of The validity of central venous pressure (CVP) measure- colloids (e.g. gelatins and dextrans), and it is not clear ments in patients with sepsis is widely debated. It is whether the results of the study can be extrapolated to all commonly accepted that a very low CVP is indicative of these compounds. low intravascular volumes and supports the administration of fluids (crystalloids or colloids) for volume Source control expansion and improvement in tissue hypoperfusion. Source control is defined as therapy targeting a focus of An elevated CVP does not however always correlate with infection that is unlikely to be cleared with antibiotics adequate intravascular volume. Despite these limita- alone. Source control is an essential component of the tions, CVP measurement in conjunction with other early management of severe sepsis. When a focus of measurements is often utilized to assess and guide resus- infection that requires source control is identified, source citation in patients with sepsis as more sophisticated control measures should be instituted as soon as possible monitoring tools are usually not available during the following initial resuscitation. critical early hours of resuscitation. In mechanically ventilated patients or patients with known preexisting Steroids for refractory shock decreased ventricular compliance, a higher target CVP The CORTICUS study, which is an international, multi- of 12–15 mmHg is recommended to account for the center, randomized trial of corticosteroids in sepsis impediment to filling (Fig. 2). Similar consideration (n ¼ 499 analyzable patients), showed no benefit in intent may be warranted in circumstances of increased abdomi- to treat mortality or shock reversal [53]. Steroids did nal pressure. The pulmonary artery catheter allows produce earlier reversal of shock which was unrelated measurements of intracardiac pressures, determination to adrenocorticotropic hormone stimulation test results. of cardiac output (through thermodilution), and mixed Superinfection and new sepsis/septic shock occurred venous oxygen saturation (SvO2) which can be useful in more frequently in the steroid group. Steroids were not diagnosing different causes of shock as well as monitoring associated with increased incidence of polyneuropathy. disease progression and response to therapeutic interven- These results suggest that hydrocortisone therapy cannot tions. Studies randomizing critically-ill patients to treat- be recommended as routine adjuvant therapy for septic ment with or without pulmonary artery catheter have not shock. If systolic blood pressure remains less than shown any significant difference in outcome [47,48]. 90 mmHg despite appropriate fluid and vasopressor therapy, hydrocortisone at 200 mg/day for 7 days in four The determinants of SvO2 include cardiac output, oxy- divided doses or by continuous infusion should be con- gen demand, hemoglobin and arterial oxygen saturation. sidered [54]. Normal SvO2 is 70–75%. Following resuscitation of sepsis, SvO2 may be elevated secondary to maldistribu- Activated protein C for high risk of death tion of flow defined as blood returning to the venous Realization of the links between the coagulation system circulation without opportunity for oxygen transfer. and the immune response to sepsis led to the develop- Patients with sepsis, however, frequently present with ment of recombinant human activated protein C (rhAPC) a low SvO2. Although a normal or high SvO2 does not [55]. After the rhAPC Worldwide Evaluation in Severe always indicate adequate resuscitation, a low SvO2 should Sepsis (PROWESS) trial, the Food and Drug Adminis- trigger aggressive interventions to increase oxygen deliv- tration (FDA) approved rhAPC for adults who had severe ery to the tissues and minimize sepsis-induced tissue sepsis and a high risk of death (such as an APACHE II hypoperfusion. An association between good clinical out- score 25) in November 2001. The Administration of come in septic shock and mean arterial pressure (MAP) of Drotrecogin alpha (activated) in Early Stage Severe at least 65 mmHg as well as SvO2 no less than 70% have Sepsis (ADDRESS) trial, designed with the purpose of been demonstrated [49]. Recently, it has also been shown prospectively studying the effect of rhAPC in severe that SvO2 runs 5–7% lower than central venous oxygen sepsis patients with a clinical assessment of low risk of saturation (ScvO2) in shock [50]. MAP is not necessarily a death, supported the FDA labeling that rhAPC was not of marker of adequate resuscitation. Some patients, despite utility in severe sepsis patients with a clinical assessment a normal MAP, have low ScvO2 levels and are clearly of low risk of death (defined by an APACHE II score

Figure 2 Resuscitation protocol for severe sepsis

SEPSIS-INDUCED HYPOPERFUSION Clinical picture of sepsis PLUS: SBP <90 mmHg or MAP <65 mmHg OR Lactate >4 mmol/l

Supplemental O2 ± ETI with mechanical ventilation (if necessary). Target SaO2 of>95% Begin fluid resuscitation (initial bolus of at least 20 ml/kg crystalloid or colloid equivalent)†

SBP remains <90 mmHg or MAP remains <65 mmHg or initial lactate >4 mmol/l

CVP <8 mm Hg Boluses crystalloid or Insert CVP colloid equivalent catheter

CVP 8--12 mmHg CVP 12--15 (if mechanically ventilated) Vasopressors MAP <65 (norepinephrine or MAP dopamine preferred)

• Administer stress dose steroids MAP >65 • Consider for drotrecogin α <70% Transfuse if HCT ScvO †† <30 2

YES NO Dobutamine

MAP <65 mmHg >70% and vasopressors still required? MAP> 65 mmHg • Consider for and vasopressors YES drotrecogin α still required?

YES NO NO Resuscitation complete. Achieve ALL Establish re-evaluation goals? intervals.

†In circumstances where MAP is judged to be critically low, vasopressors may be started at any point in this algorithm. †† If pulmonary artery catheter is used, a mixed venous O2 saturation is an acceptable surrogate and 65% would be the target.

Cooper University Hospital Protocol for initial resuscitation of sepsis-induced tissue hypoperfusion (targets adapted from Rivers’ [45] early goal directed therapy and Surviving Sepsis Campaign bundles performance improvement program). CVP, central venous pressure; ETI, endotracheal intubation; HCT, hematocrit; MAP, mean arterial pressure; SBP, systolic blood pressure.

below 25 or single-organ failure) [56]. A decline in protein and apoptotic stress, may afford protection for the vicious C levels in patients with severe sepsis and septic shock circle leading to severe sepsis. The SSC guidelines have has been recently proposed as a population at high risk for been an important advance in promoting optimal care. death [14]. High risk of death due to sepsis-induced organ Protocolized care is very important, especially in the early dysfunction determined at the bedside by a seasoned phase of severe sepsis. Large trials studying the effects of critical care clinician with an understanding and knowl- interventions based on molecular knowledge are most edge of severe sepsis and rhAPC clinical results, while likely to lead to the development of effective treatment weighing risk/benefit ratio in that patient, however, is the strategies in sepsis. optimal method for determining need for rhAPC administration [57,58]. Genetically-engineered variants of APC References and recommended reading have been also designed with greater antiapoptotic Papers of particular interest, published within the annual period of review, have been highlighted as: activity and reduced anticoagulant activity relative to of special interest wild-type APC [59]. of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 432–435). Sustained support of organ system dysfunction 1 Hotchkiss RS, Nicholson DW. Apoptosis and caspases regulate death and Maintaining support of organ systems in severe sepsis is inflammation in sepsis. Nat Rev Immunol 2006; 6:813–822. important. Recommendations to achieve this goal 2 Patel NJ, Zaborina O, Wu L, et al. Recognition of intestinal epithelial include: glycemic control; semi-recumbent position to HIF-1alpha activation by Pseudomonas aeruginosa. Am J Physiol Gastro- intest Liver Physiol 2007; 292:G134–G142. prevent ventilator-associated pneumonia; use of daily This study describes the microbial virulence strategy in hypoxic conditions. spontaneous breathing trial to evaluate for ventilation 3 Creagh EM, O’Neill LA. TLRs, NLRs and RLRs: a trinity of pathogen discontinuation and a standardized weaning protocol; use sensors that co-operate in innate immunity. Trends Immunol 2006; 27:352– 357. of sedation protocols; use of sedation scores and retitrate 4 Uematsu S, Akira S. Toll-like receptors and innate immunity. J Mol Med 2007; daily to the minimum necessary dose; avoidance of 84:712–725. neuromuscular blockers if at all possible and, if necessary, 5 Liew FY, Xu D, Brint EK, O’Neill LA. Negative regulation of toll-like receptor- intermittent dosing preferred; and use of deep vein mediated immune responses. Nat Rev Immunol 2005; 5:446–458. 6 Granucci F, Foti M, Ricciardi-Castagnoli P. Dendritic cell biology. Adv thrombosis prophylaxis. In addition, although adequate Immunol 2005; 88:193–233. nutrition has not been demonstrated in clinical trials to 7 Matejovic M, Krouzecky A, Rokyta R Jr, et al. Effects of combining inducible alter outcome in septic patients, it is generally considered nitric oxide synthase inhibitor and radical scavenger during porcine bacteremia. Shock 2007; 27:61–68. worthy of achieving [34]. 8 Cinel I, Buyukafsar K, Cinel L, et al. The role of poly(ADP-ribose) synthetase inhibition in preventing endotoxemia-induced intestinal epithelial apoptosis. Surviving Sepsis Campaign performance Pharmacol Res 2002; 46:119–127. 9 Taner S, Cinel I, Ozer L, et al. Poly(ADP-ribose) synthetase inhibition reduces improvement program bacterial translocation in rats after endotoxin challenge. Shock 2001; 16: The use of standardized decision support tools assists in 159–162. standardizing assessment and interventions in a specific 10 Soriano FG, Nogueira AC, Caldini EG, et al. Potential role of poly(adenosine 50-diphosphate-ribose) polymerase activation in the pathogenesis of myocar- patient population [60]. The SSC has developed per- dial contractile dysfunction associated with human septic shock. Crit Care formance improvement bundles with associated database Med 2006; 34:1073–1079. software and educational tools in order to integrate the 11 Doruk N, Buyukakilli B, Cinel I, et al. The effect of preventive use of alanyl- glutamine on diaphragm muscle function in cecal ligation and puncture- SSC guidelines into clinical practice and measure per- induced sepsis model. J Parenter Enteral Nutr 2005; 29:36–43. formance [61]. Improvement in process of care should 12 Singleton KD, Wischmeyer PE. Glutamine’s protection against sepsis and lead to better outcomes. Evaluation of process change lung injury is dependent on heat shock protein 70 expression. 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Endothelial microparticles as markers vided a mechanism to produce and measure improved of endothelial dysfunction. Front Biosci 2004; 9:1118–1135. clinical performance effectively [62,63]. 17 Azevedo LC, Janiszewski M, Soriano FG, Laurindo FR. Redox mechanisms of vascular cell dysfunction in sepsis. Endocr Metab Immune Disord Drug Targets 2006; 6:159–164. Conclusion 18 Trzeciak S, Dellinger RP, Parrillo JE, et al., Microcirculatory Alterations in Mortality rates remain high in severe sepsis, and despite Resuscitation and Shock Investigators. Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: relationship recent therapeutic breakthroughs much remains to be to hemodynamics, oxygen transport, and survival. Ann Emerg Med 2007; 49: done to advance our understanding and treatment of 88–98. This was a study of the early microcirculatory perfusion derangements in patients sepsis. Early correction of tissue hypoperfusion and with severe sepsis/septic shock and the microcirculation difference between hypoxia, as well as modulation of oxidative/nitrosative survivors and nonsurvivors.

19 Fujimi S, Ogura H, Tanaka H, et al. Activated polymorphonuclear leukocytes 41 Luyt CE, Guerin V, Combes A, et al. Procalcitonin kinetics as a prognostic enhance production of leukocyte microparticles with increased adhesion marker of ventilator-associated pneumonia. Am J Respir Crit Care Med 2005; molecules in patients with sepsis. J Trauma 2002; 52:443–448. 171:48–53. 20 Soriano AO, Jy W, Chirinos JA, et al. Levels of endothelial and platelet 42 Jensen JU, Heslet L, Jensen TH, et al. Procalcitonin increase in early identi- microparticles and their interactions with leukocytes negatively correlate with fication of critically ill patients at high risk of mortality. Crit Care Med 2006; organ dysfunction and predict mortality in severe sepsis. Crit Care Med 2005; 34:2596–2602. 33:2540–2546. 43 Kumar A, Roberts D, Wood KE, et al. Duration of hypotension prior to initiation 21 Elbers PE, Ince C. 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Purpose of review Introduction To review the distinction between catheter-related and Infections associated with the use of intravascular catheter-associated infections and to report the recent catheters or devices represent 10–20% of all nosocomial advances in the methods used for their diagnosis. infections. They may complicate the stay of up to 10% of Recent findings intensive care unit (ICU) patients. Almost all patients The distinction between device-associated and device- staying in an ICU require at least one intravascular device related infections affects the effective benchmarking of the for fluid/drug administration, and approximately half are rates of both types of infection. Numerous microbiological central venous catheters (CVCs) [1]. According to data methods have been described to diagnose these infections. from the National Nosocomial Infections Surveillance Studies comparing the performance of microbiological system, it is estimated that at least 48 600 ICU patients methods that avoid the removal of the intravascular device develop a CVC-related bloodstream infection every have recently suggested that they may be effective in daily year in US ICUs (approximately five episodes per life. 1000 catheter-days). These infections, mostly caused Summary by coagulase-negative staphylococci, Staphylococcus The present review summarizes recent advances in the aureus, Enterococcus species, and Candida species, are methods currently available to diagnose intravascular associated with considerable morbidity (prolonged catheter-related infections and their performance at the length of stay and increased costs) and mortality [2]. bedside. Although debated by experts with regard to magnitude, the attributable mortality of these infections may Keywords correspond to 5000–15 000 deaths directly caused by catheter, catheter-related infection, diagnosis, nosocomial catheter-related infections; the benchmarking of rates is infection currently included in the assessment of quality of care in many institutions [3]. Curr Opin Infect Dis 20:353–359. ß 2007 Lippincott Williams & Wilkins. The diagnosis of infections attributable to the use of Department of Adult Intensive Care Medicine and Burn Centre, Centre Hospitalier intravascular catheters or devices is the subject of intense Universitaire Vaudois (CHUV), Lausanne, Switzerland clinical research. There is, however, no consensus on a Correspondence to Philippe Eggimann, MD, Department of Adult Intensive Care Medicine and Burn Centre, Centre Hospitalier Universitaire Vaudois (CHUV), BH true gold standard, and the accuracy of numerous micro- 08-609, Bugnon 46, CH-1011 Lausanne, Switzerland biological methods has generated vigorous debate among Tel: +41 21 314 2923; fax: +41 21 314 3045; e-mail: [email protected] experts [4]. In addition, the variability in the definitions used over the past decades has not simplified the under- Current Opinion in Infectious Diseases 2007, 20:353–359 standing of the literature [5]. Abbreviations CVC central venous catheter In this context, the distinction between device-associated ICU intensive care unit and device-related infections proposed in the 2002 guidelines for the prevention of intravascular catheter-related ß 2007 Lippincott Williams & Wilkins 0951-7375 infections provided a useful tool [6]. Infection rates vary according to the type of surveillance. In studies designed to study complications associated with the use of intravascular devices, epidemiological definitions frequently result in higher infection rates. In studies dedicated to device surveillance, systematic microbiological investigation allows the determination of infection rates directly related to the colonization or infection of the device [7].

Diagnosis of infections associated with or related to vascular access Before reviewing the methods available to diagnose intravascular catheter-related infections, it is important

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to summarize the different definitions commonly used in primary bloodstream infection is considered indirect the literature. evidence of catheter-associated bloodstream infection. Comparisons between infection rates in different types Definitions for vascular access-associated of ICUs are more accurate when infections are reported and related infections as incidence densities associated with the use of intra- Infections linked to the use of intravascular devices vascular devices. According to this method, widely include exit-site infections, catheter colonization and diffused by the National Nosocomial Infections Surveil- both catheter-associated and catheter-related infections lance system and using epidemiological definitions, [6,8–15] (Table 1). catheter-associated infections range between 2.3 and 16.8 episodes per 1000 CVC-days [1]. This may over- Catheter-associated infections include primary blood- estimate the rate of infections related to intravascular stream infections and clinical sepsis, which are epide- devices, but is probably more representative of daily life. miologically associated with the use of intravascular This method allows the benchmarking of rates of infec- devices [6,7]. It should be emphasized that in the tion after eventual adjustment for the case mix without absence of device culture, defervescence after the the need for sophisticated laboratory work-up. Although removal of an implicated catheter from a patient with included in some reports, secondary bacteraemia, which

Table 1 Deﬁnitions of infections potentially related to vascular accessa Type of infection Criteria

Exit-site infection Clinically documented: a clinical infection (erythema, tenderness, induration or purulent discharge) at the skin insertion site Microbiologically documented: a positive (semi) quantitative catheter culture in the presence of clinical signs of infection at the insertion site Catheter colonization In the absence of clinical signs of infection at the insertion site, growth of microorganisms on the device according to microbiological criteria from quantitative (technique [8], sonication [9], vortexing technique [10]) or semi-quantitative (roll-plate technique [11]) cultures. Positive blood culture Microorganism potentially pathogen cultured from one or more blood culturesb Bloodstream infection Positive blood culture with at least one of the following clinical signs or symptoms: Fever (> 100.48F; > 388C) or hypothermia (< 98.68F; < 378C) Chills Low blood pressure (systolic blood pressure 90 mmHg or decrease > 40 mmHg from baseline) In the absence of catheter culture, defervescence after removal of an implicated catheter from a patient with primary bloodstream infection is considered as indirect evidence of catheter-associated bloodstream infection Primary bloodstream infection Laboratory-confirmed bloodstream infection or clinical sepsis occurring without documented distal source of infection Secondary bloodstream infection Laboratory-confirmed bloodstream infection occurring in the presence of another documented infection Clinical sepsis Requires one of the following signs with no other recognized cause: Fever (> 100.48F; > 388C) or hypothermia (< 98.68F; < 378C) Low blood pressure (systolic blood pressure 90 mmHg or decrease > 40 mmHg from baseline) Oliguria (< 20 ml/h) and the presence of all of the following conditions: Blood cultures not performed or no organism detected in blood No apparent infection at another body site Physician institutes therapy for sepsis Catheter-associated bloodstream infection Primary bloodstream infection or clinical sepsis in the presence of an intravascular device Catheter-related bloodstream infection Laboratory-confirmed bloodstream infection in a patient with an intravascular access with at least one positive blood culture obtained from a peripheral vein, with clinical manifestations of infection (fever, chills or hypotension) and no apparent source of the bloodstream infection except the vascular access, and with one of the microbiological methods described in Table 2: A positive semi-quantitative culture (> 15 cfu/catheter segment) with the same organism [11] A positive quantitative culture (> 103 cfu/catheter segment) with the same organism [8–10] Paired quantitative blood cultures with a 5 : 1 ratio device versus peripheral [12] Differential period of device culture versus peripheral blood culture positivity of > 2 h [13] cfu, Colony-forming units. a Adapted from [6,12,14,15]. b One of the following: Common skin contaminant (diphtheroids, Bacillus spp., Propionibacterium spp., coagulase-negative staphylococci, or micrococci) cultured from twoormorebloodculturesdrawnonseparatesets. Common skin contaminant (diphtheroids, Bacillus spp., Propionibacterium spp., coagulase-negative staphylococci, or micrococci) cultured from one or more blood culture from a patient with a vascular access, and the physician institutes appropriate antimicrobial therapy. Positive antigen test on blood and signs and symptoms with positive laboratory results are not related to an infection at another site.

is related to another documented focus of infection, The definition is sensitive but non-specific. The impact should not be considered as being catheter related. of clinical sepsis is very close to that of a microbiologically documented episode. In a prospective surveillance study Catheter-related infections include colonization of of nosocomial infections in 1068 patients who stayed in the device by microorganisms, exit-site infection and a medical ICU for more than 48 h, Hugonnet et al. [7] microbiologically confirmed device-related bloodstream analysed 109 episodes of bloodstream infections, infection. In the absence of a gold standard reference including 32 episodes of microbiologically documented technique, microbiological criteria are the subject of catheter-related infections and 77 of clinical sepsis. intense clinical research, and their clinical relevance is Exposure to central lines and arterial lines, censored currently widely discussed among experts [4]. Maki et al. atthetimeofthefirst episode of bloodstream infection, [16] recently extracted the risk of bloodstream infec- was similar in patients with a microbiologically docu- tions associated with different intravascular devices from mented episode and those with clinical sepsis, but was a systematic review of 200 published prospective studies. significantly lower in patients without bloodstream Using microbiologically based criteria, they showed that infection. The median ICU length of stay was longer all types of intravascular devices are at risk of device- in patients with microbiologically documented blood- related bloodstream infections. As rates of infections are stream infections (15.5 days; range 4–67) and clinical likely to be used for benchmarking, they showed that sepsis (14.0 days; range 3–48) than among patients with- expressing the risks of device-related bloodstream infec- out bloodstream infection (4 days; range 2–134; both tions per 1000 device-days allows for more meaningful P < 0.001). The hospital mortality rates in patients with- estimates of risk than measuring bloodstream infections out a bloodstream infection, with a microbiologically per 100 devices. Peripheral and midline intravenous confirmed bloodstream infection, and with clinical sepsis catheters are associated with the lowest rates of infection were 22.7, 32.1, and 39.7%, respectively (P ¼ 0.01). (0.1%, 0,4% and 0.5, 0.2 per 1000 device-days, respect- These data strongly suggest that clinical sepsis and ively). The rates are slightly higher for arterial catheters primary bloodstream infection microbiologically related used for haemodynamic monitoring (0.8%, 1.7 per 1000 to intravascular devices have the same impact. device-days) and peripherally inserted CVCs in hospitalized patients (2.4%, 12.1 per 1000 device-days). If confirmed by large multicentre clinical trials, these data According to these data, the rates are higher for non- may justify the aggressive strategy currently applied at impregnated CVCs inserted in critically ill patients the bedside in many ICUs, where suspect intravascular (4.4%, 2.7 per 1000 device-days). The highest rates devices are removed or exchanged over a guidewire in are reported for short-term non-cuffed and non-tunneled all cases of clinical sepsis associated with severe sepsis or haemodialysis catheters (8.0%, 4.8 per 1000 device- septic shock developing without another obvious source days), for intra-aortic balloon pumps (3.0%, 7.3 per of infection. This technique may increase the likelihood 1000 device-days), and for left ventricular assist devices of infection of the new catheter, but reduces the rate of (21.6%, 2.1 per 1000 device-days). complications associated with CVC insertion in a new site [19]. Removal of the exchanged device with further Clinical diagnosis of infections associated insertion at a new site is then only required in the with vascular access presence of a positive culture of the exchanged device Except for some exit-site infections, the clinical diag- [20]. nosis of infections related to vascular access is difficult. Most clinical signs are insensitive, non-specificorlate, Only approximately a quarter to one third of these such as septic thrombophlebitis, endocarditis or septic episodes will be demonstrated to be caused by a micro- emboli. Accordingly, they are clinically suspected when biologically documented infection of the intravascular clinical sepsis develops without other obvious sources device, and experts suggested that ‘... nontunneled of infection. CVCs should not be routinely removed in patients with mild to moderate disease’ [21,22]. The concept of ‘clinical sepsis’ is included in the surveillance definitions proposed by the Centers for Disease Rijinders et al. [23] studied the impact of a clinical Control and Prevention for primary bloodstream infec- algorithm designed to avoid catheter removal in ICU tions to take into account sepsis episodes in which no patients developing clinical sepsis. Of 140 patients poten- pathogen has been cultured from blood [6]. This entity tially eligible, 80 (55%) were excluded for haemodynamic that is used for epidemiological purposes is relatively instability, confirmed bacteraemia or local signs of infec- close to the definition of the syndromes of systematic tion at the insertion site. During the 10 days after inflammatory response, severe sepsis and septic shock inclusion, only 16 CVCs (38%) were removed in the in response to an inflammatory or infectious process ‘watchful waiting’ arm (32 patients) compared with 38 [17,18]. (100%) in the control group (32 patients), P < 0.01.

A catheter-related bloodstream infection developed in data, the authors concluded that the Gram stain and three (1%) compared with two (1%) patients, respect- acridine-orange leukocyte cytospin test are simple and ively, but in 47 (25%) of those excluded before random- rapid methods for the diagnosis of catheter-related ization. This preliminary result confirms that some CVCs bloodstream infection, which compare favourably with may be maintained, and further studies should now other methods. confirm the usefulness of this approach. In a prospective cohort study of 125 CVCs suspected of In this context, microbiological techniques likely to pro- causing catheter-related bloodstream infection, Catton vide early laboratory confirmation of the clinical suspicion et al. [36] compared the performance of three methods of device-related infection should be improved to avoid that allowed the device to remain in situ. The sensitivities unnecessary intravascular device removal or exchange. of the endoluminal brush, of quantitative culture blood cultures, and of the differential time to positivity were Microbiological diagnosis of infections 100, 89, and 72%, respectively, with corresponding spe- related to vascular access cificities of 89, 97, and 95%, respectively. Blood could be A large majority of primary bloodstream infections directly aspirated from only 74% of all lumens; however, originates from infected vascular access, but a microbio- the authors concluded that the differential time to posi- logical confirmation of an infection of the device is tivity was the most simple technique to perform. As a required to be scored as an intravascular access-related result of the high specificity of the method, they recom- bloodstream infection. mended its use as a first-line approach, with the endoluminal brush technique reserved for cases in which Microbiological methods blood cannot be obtained from the device. Many microbiological methods have been described to diagnose intravascular access-related infections, but in In a prospective cohort study of 204 CVCs suspected the absence of a true gold standard there is currently no of causing catheter-related bloodstream infections in consensus of opinion on which method to use. These critically ill patients, Bouza et al. [37] compared the methods may be divided schematically into those requir- performance of three methods that allowed the device to ing study of the catheter itself and those that avoid remain in situ. The sensitivity and specificity of cultures removing the device. They are the subject of intense of swabs from the insertion site and from the hub were clinical research, and meta-analyses on the performance 78.6 and 92.0%, respectively; for differential quantitative of some of these methods have recently appeared in the blood cultures, 71.4 and 97.7%, respectively; and for literature [8–13,15,21,24–35] (Table 2). the differential time to positivity, 96.4 and 90.3%, respectively. From these data, the authors argued that Of particular interest is the fact that paired qualitative convenience in different medical contexts, the use of blood cultures drawn from the device and venipunctures resources, and expertise should determine the choice of a and cultures of swabs obtained from the skin insertion technique. As a result of the ease of performance, low site or from the hub, which are less sophisticated from a cost, and wide availability, they recommended combin- microbiological point of view but are also cheaper, are ing semiquantitative superficial cultures and peripheral characterized by a high specificity and have the highest vein blood cultures for the screening of devices sus- negative predictive value. This may explain partly why pected of causing infection, and to use differential more sophisticated microbiological methods with high quantitative blood cultures as a confirmatory method. sensitivity and the highest positive predictive value and accuracy are currently not widely used. Those studies suggested that the choice of a precise microbiological method, or of the eventual combinations Comparison of methods of some of them, should be made according to technical Some microbiological techniques have been carefully availability and should be integrated in strategies dis- compared in a few prospective studies. cussed between clinicians and microbiologists in order to provide useful information at the bedside. In addition, In a prospective cohort study on 128 CVCs suspected of economic considerations, such as cost-effectiveness, may causing catheter-related bloodstream infection, Kite et al. also be taken into account. [30] compared the performance of four methods that allowed the device to remain in situ. The sensitivity of Recommendations of experts the Gram stain and acridine-orange leukocyte cytospin Experts have proposed algorithms taking into account test was 96% and the specificity was 92%. By comparison, most of these difficulties to help clinicians in the the tip-roll, tip-flush, and endoluminal-brush methods diagnosis of intravascular access-related infections. had sensitivities of 90, 95, and 92%, respectively, with Worthington and Eliott [4] suggested obtaining for every specificities of 55, 76, and 98%, respectively. From these patient two sets of paired blood cultures drawn through

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Intravascular catheter infection Eggimann 357 [12,15] method [12,15] method [12,15] value positive predictive value if performed on specimen obtained by endoluminal brushing; 4th most accurate test [15] most automated blood culture systems 78 The least accurate method 91 The most accurate 87 The 2nd most accurate 71 Good negative predictive 98 Arrhythmias, embolization 95 The lowest sensitivity and 89 2nd most accurate test [15] 89 5th most accurate test [15] 97 Accuracy may be improved 100 The most accurate test [15] 87 Currently available with – – – – – – – – – – – 96 72 88 87 89 85 100 67 100 84 65 78 93 83 98 83 96 89 84 98 92 79 – – – – – – – – – – – Sensitivity (%) Speci fi city (%) Comments 78 81 79 92 96 80 80 74 51 84 86 infections fi eld 10-fold higher – 120 min before those and concentration of microorganisms from the device 5 cellular monolayer in aof minimum 100 high-power than from the peripheral venipuncture turns positive obtained from venipuncture 1000 cfu 15 cfu 100 cfu 100 cfu Criteria for positivity Any growth Any growth Positive cultures from both sites Any growth Any growth Any microorganism within the Blood culture drawn through the device through the 30] – obtained through the obtained through the of the removed device al techniques used for the diagnosis of catheter-related obtained through the a segment of the removed l blood and examined under m 27] 34] – – 4 cm distal tip segment of the removed fl ushed with broth [8] or sonicated [9] – device in broth media [24] is or vortexed in brothincubated [10] that is further device is rolled acrossincubated an overnight agar [11] plate and passed down the internaldevice lumen distal to tip the [28 [25 from 50 device and from avenipuncture separate [12,15,21] device and from a[32 separate venipuncture device [12,15] device [12,15] ultraviolet light [12,15,30,31] blood cultures obtained fromand the from device a separatecontinuously venipuncture monitored until growthmicroorganisms of [12,13,15] Concomitant conventional qualitative Culture of sonicated and vortexed brush Description of methods A3 Semiquantitative cultures on agar plate Staining with acridine orange of a slide culture and of the hub on blood drawn through the device Semi-quantitative catheter segment Qualitative catheter segment culture Incubation of Quantitative catheter segment culture A distal tip segment Culture of swabs of skin insertion site Endoluminal brushing Acridine-orange leukocyte cytospin Paired quantitative blood culture Paired blood cultures Paired qualitative blood cultures Paired blood cultures Unpaired quantitative blood culture Blood cultures obtained Unpaired qualitative blood culture Blood cultures Differential time to positivity Table 2 Summary of the most common microbiologic Type of technique Methods requiring device removal Methods not requiring device removal cfu, Colony-forming units.

the device and peripherally from venipuncture. A suffi- the prevention of vascular access-related or associated cient volume of blood collected per set (20–30 ml) and infections. inoculated into both aerobic and anaerobic media should allow the identification of 99% of detectable bacterae- mias. In cases in which clinical judgement mandates the References and recommended reading Papers of particular interest, published within the annual period of review, have removal of the device, quantitative cultures should pro- been highlighted as: vide information likely to confirm the diagnosis. If of special interest of outstanding interest the intravascular access is not removed, the differential Additional references related to this topic can also be found in the Current time to positivity is then recommended as the first-line World Literature section in this issue (p. 436). method, followed by quantitative blood cultures. Alter- 1 National Nosocomial Infections Surveillance (NNIS). System report, data natively, if only qualitative blood cultures are available, summary from January 1992 through June 2004, issued October 2004. Am J Infect Control 2004; 32: 470–485. the authors strongly recommend performing additional 2 Warren DK, Quadir WW, Hollenbeak CS, et al. Attributable cost of catheter- tests, such as culture of the device, to improve the associated bloodstream infections among intensive care patients in a sensitivity of the method. In any cases of positive micro- nonteaching hospital. Crit Care Med 2006; 34:2084–2089. After adjusting for potential confounders in a cohort of 41 catheter-associated biological cultures, the authors recommend applying bloodstream infections, these infections resulted in an attributable cost of more strict criteria in the presence of coagulase-negative US$11 971, ICU length of stay of 2.41 days, and hospital length of stay of 7.54 days. staphylococci likely to reflect only contamination [4]. 3 Wenzel RP, Edmond MB. Team-based prevention of catheter-related infections. N Engl J Med 2006; 355:2781–2783. The International Sepsis Forum Consensus Conference In a well-documented editorial, after new estimations of the epidemiology of catheter-related infection in the United States, the authors recommend to the on Definitions of Infection in the Intensive Care Unit medical community to adhere to such concepts as caring behaviour that con- suggested taking into account risk factors likely to sistently lead to safety and comfort for patients. increase the probability of an infection being related to 4 Worthington T, Elliott TS. Diagnosis of central venous catheter related infection in adult patients. J Infect 2005; 51:267–280. an intravascular access in its management. Removal of 5 Linares J. Diagnosis of catheter-related bloodstream infection: conservative the device is strongly recommended in the presence of techniques. Clin Infect Dis 2007; 44:827–829. severe sepsis or septic shock with episodes of hypoten- An editorial about the difficulties in diagnosing catheter-related infections. 6 O’Grady NP, Alexander M, Dellinger EP, et al. Guidelines for the prevention of sion when the catheter is flushed, with the catheter in intravascular catheter-related infections. Centers for Disease Control and place for more than 7 days or inserted in non-sterile Prevention. Morb Mortal Wkly Rep 2002; 51:1–29. conditions, or with evidence of exit-site infection. In 7 Hugonnet S, Sax H, Eggimann P, et al. Clinical sepsis: epidemiology of an the absence of bacteraemia but positive culture of the unrecognized entity. Emerg Infect Dis 2004; 10:76–81. 8 Cleri DJ, Corrado ML, Seligman SJ. Quantitative culture of intravenous tip of the device, the hub or the exit-site, the infection is catheters and other intravascular inserts. J Infect Dis 1980; 141:781–786. scored as a possible clinical catheter-related sepsis. It is 9 Sherertz RJ, Raad II, Belani A, et al. Three-year experience with sonicated scored as a catheter-related sepsis with bacteriological vascular catheter cultures in a clinical microbiology laboratory. J Clin Microbiol confirmation in the presence of bacteraemia with com- 1990; 28:76–82. 10 Brun-Buisson C, Abrouk F, Legrand P, et al. Diagnosis of central venous mon skin commensals and positive culture of the tip or catheter-related sepsis. Arch Intern Med 1987; 147:873–877. exit site with the same microorganism [18]. 11 Maki DG, Weise CE, Sarafin HW. A semiquantitative culture method for identifying intravenous-catheter-related infection. N Engl J Med 1977; 296: 1305–1309. 12 Siegman-Igra Y, Anglim AM, Shapiro DE, et al. Diagnosis of vascular catheter- Conclusion related bloodstream infection: a meta-analysis. J Clin Microbiol 1997; 35: The distinction between device-associated and device- 928–936. related infections has improved our ability to diagnose 13 Blot F, Nitenberg G, Chachaty E, et al. Diagnosis of catheter-related bacteremia: a prospective comparison of the time to positivity of hub-blood clinical infections at the bedside, and has clarified the versus peripheral-blood cultures. Lancet 1999; 354:1071–1077. situations in which further microbiological diagnostic 14 Garner JS, Jarvis WR, Emori TG, et al. CDC definitions for nosocomial methods should be performed. Despite the usefulness infections. Am J Infect Control 1988; 16:128–140. of the recently proposed algorithms, however, they all 15 Safdar N, Fine JP, Maki DG. Meta-analysis: methods for diagnosing intravascular device-related bloodstream infection. Ann Intern Med 2005; include some simplifications, and none has been vali- 142:451–466. dated in prospective clinical trials. In addition, the poten- 16 Maki DG, Kluger DM, Crnich CJ. The risk of bloodstream infection in adults tial impact of concomitant systemic antibiotic treatment with different intravascular devices: a systematic review of 200 published prospective studies. Mayo Clin Proc 2006; 81:1159–1171. or the use of antiseptic/antimicrobial-coated devices on A comprehensive determination of the absolute (expressed as episodes per 100 the accuracy of microbiological techniques remains to devices) and relative risks (expressed as episodes per 1000 device-days) of microbiologically documented bloodstream infections associated with the use of be determined. various types of intravascular device, extracted and pooled from a systematic review of 200 published studies over four decades. Accordingly, precise diagnostic criteria should be clearly 17 Bone RC, Balk RA, Cerra FB, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. the ACCP/SCCM discussed and defined in each institution by a close consensus conference committee. American College of Chest Physicians/ collaboration between clinicians and microbiologists. Society of Critical Care Medicine. Chest 1992; 101:1644–1655. 18 Calandra T, Cohen J. The international sepsis forum consensus conference on They should then be used for eventual benchmarking definitions of infection in the intensive care unit. Crit Care Med 2005; 33: and further integrated into global strategies targeted at 1538–1548.

19 Cook D, Randolph A, Kernerman P, et al. Central venous catheter replace- 29 Tighe MJ, Kite P, Fawley WN, et al. An endoluminal brush to detect the infected ment strategies: a systematic review of the literature. Crit Care Med 1997; central venous catheter in situ: a pilot study. BMJ 1996; 313:1528–1529. 25:1417–1424. 30 Kite P, Dobbins BM, Wilcox MH, McMahon MJ. Rapid diagnosis of central- 20 Timsit JF. Scheduled replacement of central venous catheters is not neces- venous-catheter-related bloodstream infection without catheter removal. Lan- sary. Infect Control Hosp Epidemiol 2000; 21:371–374. cet 1999; 354:1504–1507. 21 Mermel LA, Farr BM, Sherertz RJ, et al. Guidelines for the management of 31 Rushforth JA, Hoy CM, Kite P, Puntis JW. Rapid diagnosis of central venous intravascular catheter-related infections. Clin Infect Dis 2001; 32:1249– catheter sepsis. Lancet 1993; 342:402–403. 1272. 32 Martinez JA, DesJardin JA, Aronoff M, et al. Clinical utility of blood cultures 22 Brun-Buisson C. Suspected central venous catheter-associated infection: drawn from central venous or arterial catheters in critically ill surgical patients. can the catheter be safely retained? Intensive Care Med 2004; 30:1005– Crit Care Med 2002; 30:7–13. 1007. 33 Beutz M, Sherman G, Mayfield J, et al. Clinical utility of blood cultures drawn 23 Rijnders BJ, Peetermans WE, Verwaest C, et al. Watchful waiting versus from central vein catheters and peripheral venipuncture in critically ill medical immediate catheter removal in ICU patients with suspected catheter-related patients. Chest 2003; 123:854–861. infection: a randomized trial. Intensive Care Med 2004; 30:1073–1080. 34 Tanguy M, Seguin P, Laviolle B, et al. Hub qualitative blood culture is useful for 24 Druskin MS, Siegel PD. Bacterial contamination of indwelling intravenous diagnosis of catheter-related infections in critically ill patients. Intensive Care polyethylene catheters. JAMA 1963; 185:966–968; 966–968. Med 2005; 31:645–648. 25 Cercenado E, Ena J, Rodriguez-Creixems M, et al. A conservative procedure 35 Marik PE. Fever in the ICU. Chest 2000; 117:855–869. for the diagnosis of catheter-related infections. Arch Intern Med 1990; 150:1417–1420. 36 Catton JA, Dobbins BM, Kite P, et al. In situ diagnosis of intravascular catheter-related bloodstream infection: a comparison of quantitative culture, 26 Atela I, Coll P, Rello J, et al. Serial surveillance cultures of skin and catheter differential time to positivity, and endoluminal brushing. Crit Care Med 2005; hub specimens from critically ill patients with central venous catheters: 33:787–791. molecular epidemiology of infection and implications for clinical management and research. J Clin Microbiol 1997; 35:1784–1790. 37 Bouza E, Alvarado N, Alcala L, et al. A randomized and prospective study of 3 procedures for the diagnosis of catheter-related bloodstream infection with- 27 Bouza E, Munoz P, Burillo A, et al. The challenge of anticipating catheter tip out catheter withdrawal. Clin Infect Dis 2007; 44:820–826. colonization in major heart surgery patients in the intensive care unit: are A prospective comparison of three microbiological methods avoiding the removal of surface cultures useful? Crit Care Med 2005; 33:1953–1960. the intravascular device in 204 suspicions of CVC-related infection. The authors 28 Kite P, Dobbins BM, Wilcox MH, et al. Evaluation of a novel endoluminal brush recommended combining semiquantitative superficial cultures and peripheral vein method for in situ diagnosis of catheter related sepsis. J Clin Pathol 1997; blood cultures to screen for catheter-related bloodstream infections, leaving differ- 50:278–282. ential quantitative blood cultures as a confirmatory and more specific technique.

Purpose of review Introduction To review recent evidence supporting the guidelines for Infections associated with the use of intravascular preventing catheter-related and catheter-associated catheters or devices represent 10–20% of all nosocomial infections. infections. They may complicate the stays of up to 10% of Recent findings intensive care unit (ICU) patients. Almost all patients A series of studies has confirmed, over the past few years, staying in an ICU require at least one intravascular device that education-based preventive programmes can reduce for fluid/drug administration and approximately half are these infections by one half to two thirds. The evidence central venous catheters (CVCs) [1]. According to data supporting some specific measures has increased for the from the National Nosocomial Infections Surveillance optimal timing for set replacement, for catheter-site system, it is estimated that at least 48 600 ICU patients dressing with chlorhexidine-impregnated devices, and for develop a CVC-related bloodstream infection every year the use of some coated or impregnated intravascular in US ICUs (approximately five episodes per 1000 devices. catheter-days). These infections, mostly caused by Summary coagulase-negative staphylococci, Staphylococcus aureus, Catheter-related and associated infections are largely Enterococcus species and Candida species, are associated preventable and should not be viewed as an unaffordable with considerable morbidity (prolonged length of stay tribute to technical medicine. Improvements in existing and increased costs) and mortality [2]. Although debated techniques and new technologies should all be integrated by experts with regard to magnitude, the attributable into a structured process of continuous improvement in the mortality of these infections may correspond to 5000– quality of care. 15 000 deaths directly caused by catheter-related infections; the benchmarking of rates is currently included in Keywords the assessment of quality of care in many institutions [3]. catheter, catheter-related infection, infection control, nosocomial infection, prevention As for other nosocomial infections, the cornerstone of prevention of intravascular device-associated or related Curr Opin Infect Dis 20:360–369. ß 2007 Lippincott Williams & Wilkins. infections relies first on a strict observation of the basic rules of hygiene, of which hand hygiene remains the first Department of Adult Intensive Care Medicine and Burn Centre, Centre Hospitalier and most important procedure [4]. More specific Universitaire Vaudois (CHUV), Lausanne, Switzerland measures, including the use of maximal sterile barriers Correspondence to Philippe Eggimann, MD, Department of Adult Intensive Care Medicine and Burn Centre, Centre Hospitalier Universitaire Vaudois (CHUV), for insertion, the optimal insertion site, skin preparation, BH 08-609, Bugnon 46, CH-1011 Lausanne, Switzerland detailed guidelines for catheter care and replacement, Tel: +41 21 314 2923; fax: +41 21 314 3045; e-mail: [email protected] and defining particular situations in which the use of antiseptic/antibiotic-coated devices may be used, have Current Opinion in Infectious Diseases 2007, 20:360–369 been addressed in many clinical studies, and are exten- Abbreviations sively discussed in regularly updated evidence-based CDC Centers for Disease Control and Prevention guidelines and recommendations [5–7]. A series of CHSS chlorhexidine–silver sulfadiazine CVC central venous catheter studies has confirmed, over the past few years, that ICU intensive care unit education-based preventive programmes can reduce the incidence of these infections by more than two thirds. ß 2007 Lippincott Williams & Wilkins 0951-7375 Accordingly, infections associated with or related to intravascular devices are now considered among the most preventable nosocomial infections, and have become a focus for the 100 000 Live Campaign of the Institute for Healthcare Improvement [8].

Prevention of vascular access-associated and related infections Recommendations and guidelines for the prevention of catheter-associated and related infections consisted for

360

decades of exhaustive catalogues of many specific between 2000–2006 is discussed. The interventions are measures previously demonstrated to be efficient [5]. catergorized as follows: Specific measures include the use of maximal sterile barriers during insertion, optimal insertion site prep- Sherertz et al. [10]: Education consisted of one day course aration, detailed guidelines for catheter insertion and including 1-h stations in which participants received replacement, and defining particular situations in which training in: (1) blood draws through vascular lines; the use of antiseptic/antibiotic-coated devices may be (2) arterial puncture for obtaining an arterial blood gas; useful. Data from surveillance programmes repetitively (3) insertion of arterial catheters and central venous showed that they are generally not used or are insuffi- catheters (CVCs); (4) urinary catheter insertion; (5) lum- ciently applied. bar puncture; (6) peripheral venous catheter insertion; (7) phlebotomy. As with any nosocomial infections, however, their prevention should rely first on general measures, including Eggimann et al. [11]: Multiple-approach intervention on a strict observation of the basic rules of hygiene, of targeted at the reduction of vascular-access infections which hand hygiene represent the first and most import- including a 30-min slide show and practical demon- ant procedure [4]. They should be continuously empha- strations completed by individual in-service training. sized and reinforced before considering any specific The programme included detailed written information measures [9]. on clinical pathways on hand hygiene procedures, for vascular access insertion, and device maintenance and use. Early in the 2000s four cohort studies showed that multimodal educational programmes were able to reduce Yoo et al. [12]: The intervention included: (1) distribution dramatically the rate of catheter-associated and related of new guidelines for the care of catheters (skin and hand infections by more than two thirds [10–13]. These pro- disinfection, occlusive gauze dressing) with insertion sets grammes all consisted of the systematic education of the that included a sterile gown and gloves, a mask, and a personnel in charge of the insertion and care of intravas- large sterile drape; (2) daily surveillance in the ICU to cular access in ICUs. Emphasizing general measures first, monitor all catheters by a checklist; (3) recommendation including a strict observation of the basic rules of hygiene, for systematic blood cultures and removal of the catheter these interventions produced a standardization of care at if the patient develops fever of unknown origin or any the bedside. Elaborated by personnel on the ward in close symptoms or signs of CR-BSI. collaboration with infection control specialists, and implemented using communication and education tools, Coopersmith et al. [13]: Based on audits to determined active participation and positive feedback, they all differences between the Centers for Disease Control and resulted in important behavioural changes. Prevention (CDC) guidelines and current practice, a task force built an education intervention to improve practices The concept of preventing catheter-related infections related to CVC insertion and care: (1) 10-Page self-study has evolved accordingly, and education-based program- module with pre and post-question exam including the mes were recommended as a first-line target in the 2002 following topics: (a) epidemiology and scope of the renewed guidelines for the prevention of intravascular problem; (b) risk factors; (c) aetiology; (d) definition; device-related infections [7]. and (e) methods to decrease risk. Specific risk-reduction strategies addressed included: (i) handwashing and asep- The objective of this paper is to review further impor- tic techniques; (ii) methods for detecting potential tant evidence on a series of topics that has appeared clinical signs and symptoms of local infection; (iii) tech- in the literature since the publication of these guide- niques for sending catheter-tip culture; (iv) routine lines. catheter site care; (v) replacing administration sets and fluids; (vi) cleaning and changing injection ports and Education-based programmes luer-lock caps; (vii) how to handle parenteral fluids and Increasing amounts of evidence come from at least 17 multidose vials; and (viii) procedure for drawing blood additional cohort studies, confirming that when included cultures. (2) Guidelines for catheter maintenance in education-based programmes, combined specific included changing injection caps and intravenous tubing measures can reduce intravascular device-associated and for fluids and medications every 72 h (or immediately if related infections by one half to two thirds (Table 1) blood accumulated in or around the cap or its integrity [14–25,26–30]. was compromised). In addition, transparent line dressings were replaced every 7 days, whereas gauze dressings In Table 1 the impact of multimodal educational pro- (which were used solely when there was bleeding or grammes that target the prevention of intravascular oozing at the insertion site) were changed every 48 h. device-related infections in adult intensive care units, The guidelines also recommended that dressings that

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 362 Nosocomial and hospital-related infections 74% 89.6% 1.18 96% 1573000 – 1.47 2006 – 5.0/1000 2.8/1000 0.001) < 12.9/1000 P 0.001) and stopcock < P 24%; – 34.0/1000 patient-days fi cant reduction, but improvements 2.9/1000 device-days) and 3.4/1000 device-days) and 21%; fi rst postgraduate year – students and physicians completing their (6.3 clinical sepsis (18.3 device-days). Overall nosocomial infections decreased by 65% (52.4 control programme study period were chlorhexidinesilver and sulfadiazine-bonded to that used by the NNIS system (6.3 with intravascular device siteGauze care: on device site 53 in documenting the dressing(11 date use (70 Date on IV administration set 0.6 infection (21.1 catheter-days) hygiene, use of maximalbarrier sterile precaution and skin preparation clinical sepsis (18.3 device-days) 34 months: US$103 600 Good gauze condition 48.7 pneumonia US$1 900 000 per year No impact on ventilator-associated Cost savings estimates of US$815 309 Presented as an active surveillance and All catheters inserted during the entire Primarily directed towards nurses Primarily directed towards nurses Increase in compliance with hand Other type of ICUs: 4.72 Signi fi cant increase in compliance Non-signi Signi fi cant decrease in urinary tract Estimated cost saving per year: Estimated cost savings over Non-teaching hospitals Methodology for surveillance comparable Prevention of eight deaths Intervention targeted at third-year medical Medical/surgical ICUs: 3.64 BSI including both primary bacteraemia BSI including both primary bacteraemia Effect (% reduction) Comments 55 75 75 ed infections in adult intensive care units, 2000 3.5 CR-BSI 100 Infection rate (/1000 device-days) pre-intervention 4.2 CR-BSI 69 4.2 CR-BSI 50 10.8 CR-BSI 66 2.1 Primary BSI 24 20.0 CR-BSI7.0 CR-BSI 45 46 45.9 CR-BSI 78 3.4 CR-BSI 18 11.3 CR-BSI 100 3.3 CR-BSI 27 24.6 BSI 4.3 BSI 24.6 BSI tudy tudy module 9.4 CR-BSI 49 components , ] 30 – 25,26 – self-study module self-study module German hospital infection surveillance system (KISS) module, behavioural intervention sequential interventions introduced sequentially over 2 years) Type of intervention [10 Education, multimodal Voluntary adherence to the Education, multimodal, Education, multimodal s targeted at the prevention of intravascular device-relat ng ICUs 1 Step-down unit 2 Coronary 1997 6 ICUs 2000 2 Mixed ICUs Education, multimodal, 1997 1 Medical ICU Education, multimodal 2000 1 Surgical ICU Education, multimodal 1998 1 Mixed ICU Education, multimodal 2001 84 ICUs 2001 2 Mixed ICUs 2002 1 Surgical ICU Education, multimodal, self-s 2005 69 ICUs 2002 1 Medical ICU Education, multimodal 2002 1 Medical ICU Education, multimodal 2004 1 Medical ICU Education, multimodal 2002 1 Surgical ICU Education, multimodal (5 2003 1 Medical ICU Education, multimodal, self-s 2000 1 Mixed ICU Infection control programme – – – – – – – – – – – – – – – 2001 1995 [13], 2002 1998 [18], 2004 2001 [19], 2004 1998 [15], 2003 1997 [11], 2000 1995 [24] . [16], 2003 1999 [10], 2000 1995 [14], 2003 1998 [20], 2004 2000 et al. et al. [17], 2004 1995 [21], 2005 2001 et al. et al. [22], 2005 2002 et al. et al et al. [12], 2001 1997 et al. et al. et al. et al. et al. et al. et al. Table 1 Impact of multimodal educational programme Study/year of publication Study period Setti Sherertz Eggimann Coopersmith Zuschneid Rosenthal Warren Yoo Coopersmith CDC [23], 2005 Eggimann Lobo Berenholtz Misset Wall Warren

were either soiled or no longer occlusive be replaced immediately. 9.4% 0.88) 1.42) Warren et al. [14]: Identical to the interventions in – –

34.4% Coopersmith et al. [13]. Zuschneid et al. [15]: The Hospital Infection Surveillance 0.5) 1 309 000 – – System (Krankenhaus Infektions Surveillance System; KISS) uses CDC deﬁnitions for reporting in ICUs and

48.5% per focuses on primary bloodstream infections, urinary tract infections, pneumonia, and bronchitis. The participating Cost savings estimate per year pneumonia: 54% (RR 0.71; 95% CI 0.61 (RR 1.29; 95% CI 1.17 Reduction in ventilator-associated US$350 000 Reduction in length of stay: 18% US$220 150 Femoral site insertion: 12.9 32.8 Dated dressings 26.6 100 discharges (RR 0.68; 95% CI 0.31 hospitals receive their rates of infection and device use Cost-saving estimate per year: See text for comments Improvement in compliance: Reduction of crude unadjusted mortality: Nurse-initiated intervention twice yearly so as to provide feedback and encourage infection control activities in the participating ICUs.

Rosenthal et al. [16]: The intervention included three Effect (% reduction) Comments 68 75 steps: (1) active surveillance for intravascular device-

ICU, intensive care unit; NNIS, National Nosocomial Infections associated infections and compliance with intravascular device site care; (2) education and training of all healthcare workers for catheter care on the basis of the CDC guidelines; (3) monthly performance feedback documenting rates of compliance with handwashing, gauze on CVC insertion sites, dates on intravenous adminis- 12.8 CR-BSI 77 11.3 CR-BSI 77 2.7 CR-BSI 100 Infection rate (/1000 device-days) pre-intervention 11.2 CR-BSI 21 46.3 BSI 4.0 BSI tration sets, and maintaining the condition of catheter ].

gauze dressings. 30 –

Misset et al. [17]: Implementation of infection control practices produced by the ICU staff together with the 25,26

– infection control unit according to the CDC recommendations including general measures such as hand hygiene ]

and antibiotic control, and speciﬁc guidelines targeted at text [10 30

– the prevention of ventilator-associated pneumonia and of intravascular device-related infections. 25,26

– Coopersmith et al. [18]: Identical to Coopersmith 2002 [13] self-study module Education, multimodal Type of intervention [10 Education, multimodal, Education, multimodal with an additional multifactorial behavioural intervention

ﬁ dence interval; CR-BSI, catheter-related bloodstream infection; targeted at improving compliance deﬁciencies noted in audits in intravascular device insertion and maintenance. CI, con Berenholtz et al. [19]: Five components sequentially introduced from February 1999 to November 2001: (1) educational intervention to increase provider awareness 1 BMT unit 1 Neurosurgical ICU of evidence-based infection control practices; (2) creation of a central catheter insertion cart; (3) asking providers 2003 1 Mixed ICU Education, multimodal 2005 103 ICUs 2002 1 Mixed ICU Education, multimodal 2003 12 ICUs 2004 1 Medical ICU Education, multimodal 2004 1 Mixed ICU – – – – – – daily whether catheters can be removed; (4) implementation of a checklist to be completed by the bedside nurse;

The type of intervention referred to is discussed in(5) the empowerment of nurses to stop procedures if guide- a lines are not followed. ], 2006 2003 ] 2000

) Warren et al. [20]: The intervention included: (1) review ], 2006 2002 ], 2006 2001 ], 2006 2003

and update of hospital or unit policies and procedures [30 [27 [25], 2005 2003 [28 concerning the insertion and the use of devices by infec- [26 [29 et al.

continued tion control team and ICU staff and members; (2) staff et al. ( et al. et al. et al.

et al. education by three methods: (a) didactic lectures and presentations; (b) self-study module with pre and post- Tsuchida Warren Table 1 BMT, Bone marrowSurveillance; transplant; RR, BSI, relative bloodstream risk. infection; Study/year of publication Study period Setting Pronovost Higuera Hatler Young test; (c) fact sheets and posters; (3) the primary messages

were: (i) hand hygiene procedures; (ii) avoidance of sterile drape (4100 by 5500 with 400 fenestration) and 2% femoral site; (iii) maximal sterile barrier precautions for chlorhexidine gluconate in 70% isopropyl alcohol for skin insertion; (iv) precise guidelines for catheter handling antisepsis. and dressing. Tsuchida et al. [27]: The intervention consisted of: Lobo et al. [21]: The intervention consisted of: (1) obser- (1) enhanced skin preparation by scrubbing with regular vation and 10 pretest questions about hand hygiene and bathing soap and tap water; (2) a new method for stabil- catheter insertion and handling; (2) education classes and ization of the catheter inserted into the internal jugular poster for hand hygiene and catheter insertion (skin vein, in which an additional dressing was placed over the preparation) and handling (dressing replacement). sterilized dressing; (3) education of the staff for maximal sterile precautions by teaching staff members and dis- Wall et al. [22]: The intervention consisted of: (1) edu- playing posters; (4) use of a check-list and observation of cation: provider, trainee supervision, insertion site, hand catheter insertion by link nurses to monitor compliance; hygiene procedures, skin antisepsis, and use of maximal (5) selection of a disinfectant that requires shorter contact sterile barriers; (2) standardized checklist for nurse time and has longer residual effect (from 10% povidone– including: hand hygiene, trainee supervision, maximal iodine to 0.5% chlorhexidine–78% ethanol). sterile barriers, skin preparation with chlorhexidine (2% solution), circumstance of catheter insertion; (3) audits Warren et al. [28]: Identical to the interventions in and monthly feedback. Warren et al. [20].

CDC [23]: The intervention consisted of: (1) education Hatler et al. [29]: The intervention consisted of: (1) staff for catheter insertion practices (maximum sterile barrier members received a one-page document detailing precautions; chlorhexidine solution for skin disinfection; new strategies to address the prevention of ventilator- avoidance of the femoral insertion site; guidelines for associated pneumonia and CR-BSI, thus increasing their dressing; removal of catheters no longer indicated); (2) abilities to control and perform the desired behaviours; promotion of educational module on device-associated (2) chart of expected activities was posted in each bloodstream infections and strategies for their preven- patient’s room; (3) daily rounds, led by the unit’s nursing tion; (3) promotion of standardized tools for recording supervisor, helped establish adherence to the targeted adherence to recommendations; (4) promotion of a stan- procedures as ‘normal’ behaviour for staff members; dardized list of contents for catheter insertion kits; (5) (4) daily goals were posted on white boards in patients’ measurement of CVC-associated bloodstream infection rooms; (5) regular feedback about the rates; (6) use of a rates, with quarterly reports allowing anonymous bench- rapid-cycle approach to increase the ﬂexibility of imple- marking between participating ICUs. menting the change measured for 2–4 weeks.

Eggimann et al. [24]: Identical to the interventions in Pronovost et al. [30]: the intervention included ﬁve of Eggimann et al. [11]. the highly ranked (CDC category IA) evidence-based recommendations identiﬁed as having the greatest effect Higuera et al. [25]: The intervention consisted of: (1) on the rate of catheter-related bloodstream infection and education: 1 h classes on epidemiology of nosocomial the lowest barriers to implementation [7]. The recom- infections, on hand hygiene, disinfection, on prevention mended procedures were: (1) hand washing; (2) using of intravascular device-associated bloodstream infections, full-barrier precautions during the insertion of CVCs; (3) on prevention of ventilator-associated pneumonia, and on cleaning the skin with chlorhexidine; (4) a subclavian prevention of catheter-associated urinary tract infections; site is preferred and the femoral site is avoided if possible; (2) performance feedback: monthly chart (hand hygiene (5) removing unnecessary catheters. They were pro- compliance, gauze on CVC insertion sites, dates on moted by local physicians and nurses team leaders as intravenous administration sets, maintenance of gauze follows: (a) staff were educated about practices to control dressings on catheter sites) posted on the walls of the infection and harm resulting from catheter-related blood- ICUs; (3) the infection control guidelines included stream infections; (b) central-line cart with necessary the introduction of the use alcohol hand-rubs or hand supplies was promoted or created; (c) a checklist was washing with povidone–iodine soap. used to ensure adherence to infection-control practices; (d) providers were stopped (in non-emergency situations) Young et al. [26]: The intervention consisted of: (1) if these practices were not being followed; (e) the removal educational sessions that emphasized the use of maximal of catheters was discussed at daily rounds; (f) the teams sterile barrier precautions and the use of chlorhexidine received feedback regarding the number and rates of for skin antisepsis during insertion of CVCs; (2) intro- catheter-related bloodstream infections at monthly and duction of a special customized CVC kit including a large quarterly meetings, respectively.

The successful prevention of infections has not only been recommended. In addition, subgroup analysis found demonstrated in cases of high initial rates, but also in no differences between CVCs and peripheral catheters, institutions with considerable experience in the preven- nor between participants who did and did not receive tion of nosocomial infections and initially low rates of parenteral nutrition, or between children and adults. infections [10,15,17,18,30]. A majority of these programmes has been shown to be largely cost-effective. Needleless catheter access systems Moreover, such programmes may also work when Designed to reduce the incidence of sharp injuries to impregnated catheters were already in use before the healthcare workers, needleless infusion systems have intervention [14]. been developed by several manufacturers. They have not been proved to be able to prevent catheter-related The most recent report concerned an interventional infections, and were scored as an unresolved issue in the cohort study involving 103 ICUs in 67 hospitals in 2002 guidelines [7]. Michigan with more than 375 000 catheter-days of observation [30]. The intervention, detailed in Table 1, was Niel-Weisse et al. [32] recently reviewed five random- remarkable in several points. The programme was pro- ized trials evaluating the effect of vascular access needle- moted by local physicians and nurses organized by team less closed systems on catheter-related infections among leaders. It was supported by the introduction of daily goal hospitalized patients. The quality of these trials and the sheets to improve clinician-to-clinician communication way they were reported were generally unsatisfactory, within the ICUs. Furthermore, it was integrated in a and their heterogeneity did not allow a meta-analysis to comprehensive unit-based safety programme to improve be performed. Nevertheless, there was a trend towards the safety culture, which also included an intervention to the needleless closed devices decreasing catheter-related reduce the incidence of ventilator-associated pneumonia. bloodstream infections. Catheter tip and hub colonization Over the 18 months after the implementation of the were significantly reduced in one out of three and two out intervention, the median rate of catheter-related blood- of three studies, respectively. From their detailed review, stream infections was reduced by 66%, from 2.7 (mean the authors specified that in contrast to several previous 7.7) to 0 (mean 1.4) per 1000 catheter-days. reports, they did not find a trend in needleless closed devices towards an increased risk of infection [33,34]. The series of mono and multicentre studies, which They concluded that there is currently insufficient includes a large variety of ICUs all around the world, evidence to recommend needleless closed vascular strongly suggests that the process of training is progress- devices, and that new studies are necessary to confirm ively, and hopefully definitely, moving from the that these systems reduce catheter-related infections. traditional widespread approach ‘see one, do one, teach one’ to an integrated and structured process of continuous Catheter site dressing with chlorhexidine-impregnated improvement of the quality of care. devices Gluconate of chlorhexidine is a powerful antimicrobial Optimal timing for intravenous administration agent widely used for surgical scrub and for hand hygiene, set replacement with a good safety profile. When used as a skin disin- The current guidelines recommend, scored with the fectant, it is more effective than povidine–iodine highest level of evidence, to replace administration sets, solutions in reducing catheter-related bloodstream infec- including secondary sets and add-on devices, no more tions [35]. Devices impregnated with chlorhexidine glu- frequently than at 72-h intervals, unless a catheter- conate (Biopatch; Ethicon Inc., Somerville, New Jersey, related infection is suspected or documented. It is recom- USA) are able to release it up to 10 days over the skin mended, with a slightly less high level of evidence, to surface when placed over the epidural catheter insertion decrease this delay to 24-h intervals for the adminis- site [36]. Whether these devices could be routinely used tration of blood products or lipid emulsions [7]. in the care of short-term intravascular accesses, however, remained unresolved in the 2002 guidelines [7]. Gilles et al. [31] from the Cochrane Anesthesia Group performed a systematic review to identify the optimal Ho and Litton [37] found 10 prospective, randomized, interval for the routine replacement of intravenous admin- controlled clinical trials comparing chlorhexidine- istration sets. From their analysis of the data extracted from impregnated dressings with placebo or povidine–iodine 13 out of 23 studies, the authors concluded that adminis- dressing, and included eight of them in a meta-analysis. tration sets that do not contain lipids, blood or blood The chlorhexidine-impregnated dressing reduced the products may be left in place for intervals of up to 96 h risk of intravascular catheter or exit-site colonization without an increase in the risk of infection. There was no [14.8 versus 26.9%; odds ratio (OR) 0.47; 95% confidence evidence to suggest that administration sets containing interval (CI) 0.34–0.65; P < 0.00001]. After excluding lipids should not be changed every 24 h as currently the study that compared chlorhexidine-impregnated

dressings with povidine–iodine dressings, they were CHSS-impregnated catheters (on their internal surface associated with a significant reduction in catheter-related only) and the effect on catheter-related infection. The bloodstream infections when compared with placebo pooled data did not show a significant reduction of (OR 0.40; 95% CI 0.21–0.75; P ¼ 0.004). Local cutaneous catheter-related bloodstream infections (OR 0.69; 95% reactions to chlorhexidine-impregnated dressings were CI 0.46–1.03). Statistical significance was achieved only reported in 5.6% of patients in three studies (OR 8.17; when both studies with a more appropriate type of 95% CI 1.19–56.14; P ¼ 0.04), and 96% of these reactions definition were separated out (OR 0.58; 95% CI 0.46– occurred in neonatal patients. The number needed to 0.96), or when only studies with a short average duration prevent one episode of bloodstream infection was 142 for of catheterization (maximum 8 days) were considered an average period of catheterization of 10 days and a (OR 0.34; 95% CI 0.15–0.75). The authors highlighted change of dressings every 5 days. The cost of preventing the fact that all methods of diagnosing catheter coloniza- one catheter-related bloodstream infection was estimated tion used in the studies were evaluated with uncoated to be US$532.5. The authors concluded that the use of catheters. They suggested that failure to use neutralizers chlorhexidine-impregnated dressings is safe and may be could lead to an underestimation of the rate of catheter- cost-effective in adult patients with intravascular devices. related bloodstream infections; conversely, the absence This should now be confirmed in a large prospective, of inhibitory agents may lead to an overestimation of the randomized controlled trial. antibacterial efficacy of coated catheters.

Coated or impregnated intravascular Falagas et al. [40] performed a meta-analysis of eight devices prospective randomized clinical trials including a total of The use of antibiotic or antiseptic-impregnated catheters 1715 rifampicin-based impregnated CVCs, 956 uncoated reduces device-related infections but remains controver- catheters, 451 catheters coated on their internal surface sial. In the last guidelines, experts recommended con- by CHSS, and 237 silver–platinum–carbon-impregnated sidering the use of such devices if the rate of catheter- catheters. Overall, the rifampicin/minocycline-impreg- related bloodstream infections remains higher than 3.3 nated catheters significantly reduced bacterial coloniza- episodes per 1000 CVC-days, after having enhanced tion (OR 0.46; 95% CI 0.31–0.69) and catheter-related other preventive measures, including multimodal edu- bloodstream infections (OR 0.23; 95% CI 0.14–0.40). On cative programmes [7]. the basis of the available data, no clear conclusions could be drawn regarding the impact of the use of rifampicin/ Several meta-analyses that included studies published minocycline-impregnated CVCs on the development of after the release of those guidelines appeared in the antimicrobial resistance. The authors concluded that literature. these catheters are safe and effective in reducing the rate of catheter colonization and related infections. Walder et al. [38] reviewed the impact of coated devices Regarding the development of resistance, reviewing in 23 trials, including chlorhexidine–silver sulfadiazine the effectiveness and safety of rifampicin-based regimens (CHSS) internal coating in 12, silver-based coating or in eradicating S. aureus carriage, the same authors cuffing in seven, and various antibiotic coatings in five. recently concluded that the available evidence suggested The major finding of that meta-analysis was that in that oral rifampicin is effective in its eradication [41]. five trials on CHSS coating (1269 CVCs), in which the They also specified that the development of resistance average insertion time ranged from 5.2 to 7.5 days occurs in a considerable proportion of patients. (median 6 days), the risk of bloodstream infections significantly decreased from 4.1% in controls to 1.9% The potential greater efficacy of catheters coated with with anti-infective catheters (OR 0.48; 95% CI 0.25– minocycline/rifampicin compared with those coated with 0.91). In five additional trials on CHSS coating (1544 CHSS in preventing infections has largely been attrib- CVCs), in which the average insertion time ranged uted to the fact that CHSS coating concerned only the from 7.8 to 20 days (median 12 days), the risk of blood- internal lumen of the device [42]. Since then, a second stream infections was 4.5% in controls and 4.2% with generation of CHSS catheters with coating of both the anti-infective catheters (OR 0.94; 95% CI 0.58–1.54). internal and the external surface was produced by the The authors concluded that antibiotic and CHSS coatings manufacturer. At least three studies [43–45], including are anti-infective for short insertion times (less than one two large multicentre ICU studies, have failed to demon- week). For longer insertion times (more than one week), strate a significant reduction in catheter-related blood- there were no data on antibiotic coating, and some stream infections. All showed a significant reduction in evidence of a lack of effect for CHSS coating. bacterial colonization. The overall low rate of infection in the placebo arms could, however, argue in favour of Geffers et al. [39] assessed the methodological quality of insufficient power. By pooling the data, Gastmeier and 11 randomized controlled studies (3131 catheters) with Geffers [46] demonstrated a significant reduction in the

rate of catheter-related bloodstream infections [relative lished meta-analyses [53,54]. These techniques risk (RR) 0.40; 95% CI 0.18–0.90]. should, however, be restricted to this setting when infection with coagulase-negative staphylococci is documen- New composite catheters, consisting of polyurethane ted but without systemic signs of sepsis or secondary combined with silver, carbon, and platinum, have been dissemination [55]. Accordingly, as a result of the risk of introduced onto the market. They were demonstrated to promoting the spread of resistance, and the lack of data in be able to release ions of silver with prolonged anti- other types of settings, they should continue not to be microbial activity (oligon) [47–51]. Those studies used routinely. showed a significant reduction in bacterial colonization. Even by pooling the data (822 impregnated versus 833 The 2002 guidelines recommend the use of a 2% solution non-impregnated catheters), Gastmeier and Geffers [46] of chlorhexidine for skin antisepsis before intravascular did not find a significant reduction in the rate of catheter- device insertion and further maintenance care. The related bloodstream infections (RR 0.81; 0.46–1.45). majority of the commercially available chlorhexidine- based solutions for use with intravascular devices consist To summarize the information of the many randomized of 2% chlorhexidine gluconate in 70% isopropyl alcohol. studies, the impact of these devices is in the same range Medical-grade ethanol has already been in use for many as those of the educational programmes. Nevertheless, years for the management or prevention of the occlusion they should never replace them. It should be highlighted of intravascular devices used for parenteral nutrition that in at least one of the studies demonstrating a sig- [56,57]. A series of reports has suggested that 25–70% nificant reduction in infections after the introduction of a of ethanol used as a lock solution may be of value both as multimodule educational programme, coated catheters an adjunct to the treatment of intravascular device- were used during the entire study period [14]. related bloodstream infections and for the prevention of infection with the use of long-term intravascular Borschel et al. [52] recently reported the impact in a devices [58,59,60]. Crnich and colleagues [61] demon- real-world setting after the introduction of CHSS CVCs strated that a 70% ethanol lock solution has a negligible (second generation with internal and external coating) in impact on the mechanical properties of polyetherur- all consecutive patients requiring central venous access in ethane and silicone catheters, despite continuous six adult ICUs at a large, tertiary care teaching hospital. exposure times as long as 10 weeks. These findings The intervention significantly increased the rate of should prompt further studies of ethanol as an anti- reduction of the incidence of catheter-related blood- infective lock solution for the prevention and treatment stream infections by approximately 4% per month, cor- of intravascular device-related bloodstream infections in responding to an overall 35% relative risk reduction over a other settings, including shorter catheterization times. 16-month period (from approximately nine to less than four episodes per 1000 device-days). With 41 infections Conclusion avoided for approximately 1647 coated catheters inserted, Catheter-related and associated infections are largely the authors estimated that the intervention saved preventable and should not be viewed as an unaffordable US$266 325 over one year. Assuming that not all of the tribute to technical medicine. Their prevention is a very observed effect was caused by the coated catheters, the active and stimulating field for clinical research. Improve- intervention would remain cost-effective even if only 13% ments in many specific technical measures, such as new of the observed decrease in infection rate was attributable catheter securement or fixation devices, will probably to it. Furthermore, the intervention was associated with a continue to be studied. New technologies are stimulating significant reduction in the rate of vancomycin use. the design of new types of impregnated devices. After being shown to be useful, all these improvements should If such a positive and cost-effective impact of antiseptic- then be integrated into structured multimodal edu- coated devices in a real-world setting could be confirmed cational programmes. Moreover, these interventions in large multicentre studies, it may contribute to increase should all be integrated in a structured process of con- the level of evidence to include them in all strategies tinuous improvement of the quality of care. targeted at the reduction of catheter-related infections. References and recommended reading New areas of development Papers of particular interest, published within the annual period of review, have been highlighted as: Antibiotic-lock or flush solutions are not routinely recom- of special interest mended for the prevention of device-related infections of outstanding interest Additional references related to this topic can also be found in the Current by the 2002 guidelines [7]. They were confirmed to be World Literature section in this issue (p. 436). able to reduce significantly the rate of catheter-related 1 National Nosocomial Infections Surveillance (NNIS). System report, data infections and be useful in the treatment of persistent summary from January 1992 through June 2004, issued October 2004. bacteraemia in long-term catheters by two recently pub- Am J Infect Control 2004; 32:470–485.

2 Warren DK, Quadir WW, Hollenbeak CS, et al. Attributable cost of catheter- 21 Lobo RD, Levin AS, Gomes LM, et al. Impact of an educational program associated bloodstream infections among intensive care patients in a non- and policy changes on decreasing catheter-associated bloodstream infec- teaching hospital. Crit Care Med 2006; 34:2084–2089. tions in a medical intensive care unit in Brazil. Am J Infect Control 2005; After adjusting for potential confounders in a cohort of 41 catheter-associated 33:83–87. bloodstream infections, these infections resulted in an attributable cost of 22 Wall RJ, Ely EW, Elasy TA, et al. Using real time process measurements to US$11 971, ICU length of stay of 2.41 days, and hospital length of stay of reduce catheter related bloodstream infections in the intensive care unit. Qual 7.54 days. Safety Healthcare 2005; 14:295–302. 3 Wenzel RP, Edmond MB. Team-based prevention of catheter-related infec- 23 Centers for Disease Control and Prevention. Reduction in central line- tions. N Engl J Med 2006; 355:2781–2783. associated bloodstream infections among patients in intensive care units – In a well-documented editorial, after new estimations of the epidemiology of Pennsylvania, April 2001–March 2005. MMWR Morb Mortal Wkly Rep catheter-related infection in the United States, the authors recommended to 2005; 54:1013–1016. the medical community to adhere to such concepts of caring behaviour that consistently lead to safety and comfort for patients. 24 Eggimann P, Hugonnet S, Sax H, et al. Long-term reduction of vascular access-associated bloodstream infection. Ann Intern Med 2005; 142:875– 4 Boyce JM, Pittet D. Guideline for hand hygiene in health-care settings. 876. Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. 25 Higuera F, Rosenthal VD, Duarte P, et al. The effect of process control on the Society for Healthcare Epidemiology of America/Association for Profes- incidence of central venous catheter-associated bloodstream infections sionals in Infection Control/Infectious Diseases Society of America. MMWR and mortality in intensive care units in Mexico. Crit Care Med 2005; 33: Morb Mortal Wkly Rep 2002; 51:1–45. 2022–2027. 5 Pearson ML. Guideline for prevention of intravascular device-related infec- 26 Young EM, Commiskey ML, Wilson SJ. Translating evidence into tions. Hospital Infection Control Practices Advisory Committee. Infect Control practice to prevent central venous catheter-associated bloodstream Hosp Epidemiol 1996; 17:438–473. infections: a systems-based intervention. Am J Infect Control 2006; 34:503–506. 6 Mermel LA, Farr BM, Sherertz RJ, et al. Guidelines for the management of An infection control intervention designed to facilitate the use of maximal sterile intravascular catheter-related infections. Clin Infect Dis 2001; 32:1249– barrier precautions and the use of chlorhexidine for skin antisepsis during the 1272. insertion of CVCs. See Table 1 for summary. 7 O’Grady NP, Alexander M, Dellinger EP, et al. Guidelines for the prevention of 27 Tsuchida T, Makimoto K, Toki M, et al. The effectiveness of a nurse-initiated intravascular catheter-related infections. Centers for Disease Control and intervention to reduce catheter-associated bloodstream infections in an urban Prevention. Morb Mortal Wkly Rep 2002; 51:1–29. acute hospital: an intervention study with before and after comparison. Int J 8 Berwick DM, Calkins DR, McCannon CJ, Hackbarth AD. The 100 000 lives Nurs Stud 2006; 23 December [Epub ahead of print]. campaign: setting a goal and a deadline for improving healthcare quality. An infection nurse-initiated multimodal intervention designed to reduce catheter- JAMA 2006; 295:324–327. related bloodstream infections. See Table 1 for summary. 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An educational multicentre intervention to prevent catheter-associated bloodstream infections and ventilator-associated pneumonia. See Table 1 and text 12 Yoo S, Ha M, Choi D, Pai H. Effectiveness of surveillance of central catheter- for summary and comments. related bloodstream infection in an ICU in Korea. Infect Control Hosp Epidemiol 2001; 22:433–436. 31 Gillies D, O’Riordan L, Wallen M, et al. Optimal timing for intravenous administration set replacement. Cochrane Database Syst Rev 2005; 19: 13 Coopersmith CM, Rebmann TL, Zack JE, et al. Effect of an education program CD003588. on decreasing catheter-related bloodstream infections in the surgical intensive care unit. Crit Care Med 2002; 30:59–64. 32 Niel-Weise BS, Daha TJ, Van den Broek PJ. Is there evidence for recommending needleless closed catheter access systems in guidelines? A systematic 14 Warren DK, Zack JE, Cox MJ, et al. An educational intervention to prevent review of randomized controlled trials. J Hosp Infect 2006; 62:406–413. catheter-associated bloodstream infections in a nonteaching, community A systematic review of five randomized trials evaluating the effect of vascular medical center. Crit Care Med 2003; 31:1959–1963. access needleless closed systems on catheter-related infections among hospi- 15 Zuschneid I, Schwab F, Geffers C, et al. Reducing central venous catheter- talized patients. See text for summary and comments. associated primary bloodstream infections in intensive care units is possible: 33 Danzig LE, Short LJ, Collins K, et al. Bloodstream infections associated with a data from the German nosocomial infection surveillance system. Infect needleless intravenous infusion system in patients receiving home infusion Control Hosp Epidemiol 2003; 24:501–505. therapy. JAMA 1995; 273:1862–1864. 16 Rosenthal VD, Guzman S, Pezzotto SM, Crnich CJ. Effect of an infection 34 McDonald LC, Banerjee SN, Jarvis WR. Line-associated bloodstream infec- control program using education and performance feedback on rates of tions in pediatric intensive-care-unit patients associated with a needleless intravascular device-associated bloodstream infections in intensive care units device and intermittent intravenous therapy. Infect Control Hosp Epidemiol in Argentina. Am J Infect Control 2003; 31:405–409. 1998; 19:772–777. 17 Misset B, Timsit JF, Dumay MF, et al. A continuous quality-improvement 35 Chaiyakunapruk N, Veenstra DL, Lipsky BA, Saint S. Chlorhexidine compared program reduces nosocomial infection rates in the ICU. Intensive Care Med with povidone–iodine solution for vascular catheter-site care: a meta-analysis. 2004; 30:395–400. Ann Intern Med 2002; 136:792–801. 18 Coopersmith CM, Zack JE, Ward MR, et al. The impact of bedside behavior on 36 Shapiro JM, Bond EL, Garman JK. Use of a chlorhexidine dressing to reduce catheter-related bacteremia in the intensive care unit. Arch Surg 2004; 139: microbial colonization of epidural catheters. Anesthesiology 1990; 73:625– 131–136. 631. 19 Berenholtz SM, Pronovost PJ, Lipsett PA, et al. Eliminating catheter-related 37 Ho KM, Litton E. Use of chlorhexidine-impregnated dressing to prevent bloodstream infections in the intensive care unit. Crit Care Med 2004; 32: vascular and epidural catheter colonization and infection: a meta-analysis. 2014–2020. J Antimicrob Chemother 2006; 58:281–287. 20 Warren DK, Zack JE, Mayfield JL, et al. The effect of an education program on A systematic review of 10 prospective randomized controlled clinical trials the incidence of central venous catheter-associated bloodstream infection in comparing chlorhexidine-impregnated dressings with placebo or povidine–iodine a medical ICU. Chest 2004; 126:1612–1618. dressings. See text for summary and comments.

38 Walder B, Pittet D, Tramer M. Benefit of antiseptic and antimicrobial coating 52 Borschel DM, Chenoweth CE, Kaufman SR, et al. Are antiseptic-coated of central venous catheters: a systematic review. Infect Control Hosp central venous catheters effective in a real-world setting? Am J Infect Control Epidemiol 2002; 23:748–756. 2006; 34:388–393. A prospective assessment of the clinical and economic effectiveness of anti- 39 Geffers C, Zuschneid I, Eckmanns T, et al. The relationship between methodo- septic-coated catheters for critically ill patients after their introduction for all logical trial quality and the effects of impregnated central venous catheters. patients requiring central venous access in the ICU. See text for summary and Intensive Care Med 2003; 29:403–409. comments. 40 Falagas ME, Fragoulis K, Bliziotis IA, Chatzinikolaou I. Rifampicin-impregnated 53 Safdar N, Maki DG. Use of vancomycin-containing lock or flush solutions for central venous catheters: a meta-analysis of randomized controlled trials. prevention of bloodstream infection associated with central venous access J Antimicrob Chemother 2007; 59:359–369. devices: a meta-analysis of prospective, randomized trials. Clin Infect Dis A meta-analysis of eight prospective randomized clinical trials comparing 2006; 43:474–484. rifampicin- based impregnated central venous catheters with uncoated catheters, A meta-analysis of the data on the efficacy of vancomycin-containing lock or flush with catheters coated on their internal surface by CHSS and with silver–platinum– solutions collected from seven randomized, controlled trials involving a total of 463 carbon-impregnated catheters. See text for summary and comments. patients (five in ICUs). The use of these solutions reduced the risk of intravascular 41 Falagas ME, Bliziotis IA, Fragoulis KN. Oral rifampin for eradication of device-related infection. The authors concluded that the use of an anti-infective Staphylococcus aureus carriage from healthy and sick populations: a sys- lock solution warrants consideration for patients who require central access but tematic review of the evidence from comparative trials. Am J Infect Control who are at high risk of bloodstream infections, such as patients with malignancy or 2007; 35:106–114. low-birthweight neonates. A review of the effectiveness, safety and impact on the emergence of resistance of 54 Van de Wetering MD, Van Woensel JBM. Prophylactic antibiotics for pre- rifampicin-based regimens in eradicating S. aureus carriage. See text for summary venting early central venous catheter Gram positive infections in oncology and comments. patients. Cochrane Database Syst Rev 2007; CD003295.pub2 42 Darouiche RO, Raad II, Heard SO, et al. A comparison of two antimicro- A meta-analysis of the data from nine trials with a total of 588 patients. The overall bial-impregnated central venous catheters. N Engl J Med 1999; 340:1–8. effect of administering an antibiotic before the insertion of the catheter did not 43 Brun-Buisson C, Doyon F, Sollet JP, et al. Prevention of intravascular significantly decrease the number of Gram-positive long-term tunnel’ed CVC catheter-related infection with newer chlorhexidine–silver sulfadiazine- infections. Flushing them with antibiotics and heparin significantly reduces the coated catheters: a randomized controlled trial. Intensive Care Med infection rate. The authors concluded that flushing the catheter with a vanco/ 2004; 30:837–843. heparin lock solution had a positive overall effect. 44 Rupp ME, Lisco SJ, Lipsett PA, et al. Effect of a second-generation venous 55 Segarra-Newnham M, Martin-Cooper EM. Antibiotic lock technique: a review catheter impregnated with chlorhexidine and silver sulfadiazine on central of the literature. 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Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Neonatal immune responses to coagulase- negative staphylococci Tobias Strunka,e, Peter Richmonda, Karen Simmera,b, Andrew Curriec, Ofer Levyd and David Burgnera

Purpose of review Abbreviations Coagulase-negative staphylococci have emerged as the CoNS coagulase-negative staphylococci most common nosocomial pathogen in neonatal intensive TLR Toll-like receptor TNF-a tumour necrosis factor alpha care units worldwide. Our understanding of the interactions TPN total parenteral nutrition between coagulase-negative staphylococci and the immune system is incomplete, especially in the newborn. ß 2007 Lippincott Williams & Wilkins 0951-7375 This review summarizes current knowledge on the human immune response to coagulase-negative staphylococci, with particular emphasis on the neonatal innate immune Introduction system. The group of coagulase-negative staphylococci (CoNS; Recent findings e.g. Staphylococcus epidermidis, Staphylococcus haemolyticus, There are very limited data on innate immune responses to Staphylococcus saprophyticus, Staphylococcus lugdunensis, coagulase-negative staphylococci in neonates. Levels of Staphylococcus schleiferi, etc.) are primarily ubiquitous colon- serum proteins, including transplacental anti-coagulase- izers of healthy skin and mucosal surfaces, and generally negative staphylococci immunoglobulin and complement, have low pathogenic potential [1]. CoNS are now the most correlate with gestational age, and this relative deficiency in commonly isolated group of organisms in the microbiology preterm infants contributes to their suboptimal opsonization laboratory, and have become increasingly important over and impaired bacterial killing of coagulase-negative the past two decades as nosocomial pathogens causing staphylococci. In adults, coagulase-negative staphylococci significant morbidity, mortality and healthcare costs world- elicit significant cytokine responses in vitro, which are wide [2,3–7]. probably partly mediated by Toll-like receptors, including Toll-like receptor type 2, but these pathways have not been In adults, CoNS infections usually affect patients with characterized in the high-risk neonatal population. indwelling plastic devices and the immunocompromised, Summary such as those with advanced HIV/AIDS, malignant dis- The susceptibility of human preterm neonates to coagulase- eases or after immunosuppression [8]. Advances in neo- negative staphylococci relates partly to the immaturity of the natal intensive care have led to a growing population of neonatal immune response. Strategies to reduce the burden children surviving even extreme prematurity, and the most of coagulase-negative staphylococci infections require a vulnerable groups for CoNS infections are the increasing thorough understanding of host–pathogen interactions, numbers of significantly preterm infants (< 32 weeks’ particularly the engagement of coagulase-negative gestation) [9,10]. Of note is the fact that premature infants staphylococci by the neonatal innate immune system. account for more than 70% of consequent neonatal and infantile severe illness and death, much of which is related Keywords to infection and its sequelae [9,11]. A large number of coagulase-negative staphylococci, immune response, late- extremely low birthweight infants (< 1000 g) have at least onset sepsis, preterm infant one episode of invasive bacterial infection [12–14]. Despite the greatly increased susceptibility of preterm Curr Opin Infect Dis 20:370–375. ß 2007 Lippincott Williams & Wilkins. infants to CoNS, the underlying immunological mechanisms have received scant attention. aSchool of Paediatrics and Child Health, bSchool of Women’s and Infants’ Health, cSchool of Medicine and Pharmacology, University of Western Australia, Perth, Australia, dDepartment of Medicine, Division of Infectious Diseases, Children’s Although much of the scientific effort has focused on Hospital Boston and Harvard Medical School, Boston, Massachusetts, USA and eDepartment of Pediatrics, University of Lu¨beck Medical School, early-onset sepsis (occurring within 72 h after birth), in Lu¨beck, Germany particular group B streptococcus, late-onset sepsis (onset Correspondence to Tobias Strunk, MD, School of Paediatrics and Child Health, after 72 h) is approximately 10 times more common in University of Western Australia, Princess Margaret Hospital for Children, preterm infants [14–16]. CoNS have emerged as the most GPO Box D184, WA 6840 Perth, Australia Tel: +61 8 9340 8125; fax: +61 8 9388 2097; e-mail: [email protected] prevalent and important neonatal pathogens, responsible for approximately 50% of all episodes of late-onset sepsis Current Opinion in Infectious Diseases 2007, 20:370–375 in neonatal intensive care units around the world. CoNS

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infections affect up to one half of very low birthweight as well as the long-term administration of lipids with infants, whereas term infants are rarely affected [3, total parenteral nutrition (TPN) are associated with a 17–19]. significantly increased risk [25,33–36]. Some of these associations may not be causal, however, but rather are Despite the marked improvement in neonatal intensive markers for the smallest and sickest infants, or may care, there is considerable morbidity, mortality and pro- reflect the deleterious effects of CoNS sepsis itself. longed hospitalization attributable to CoNS infection Surprisingly, long-term TPN administration is the only [3,14]. CoNS sepsis has been estimated to prolong the risk factor that has been investigated in detail. In infants, length of hospital stay, depending on birthweight, by TPN impairs neutrophil phagocytosis and the killing of 4–19 days, causing significantly increased healthcare CoNS as well as CoNS-induced tumour necrosis factor-a costs [20,21]. Markedly increased risks for the develop- (TNF-a) production in in-vitro whole blood assay ment of bronchopulmonary dysplasia and cerebral palsy [37,38]. in preterm infants affected by CoNS sepsis have been reported from small studies [22,23], and clearly warrant Pathogenesis and virulence factors of further investigation. Furthermore, one study found an coagulase-negative staphylococci association of delta-toxin producing CoNS with necrotiz- The formation of biofilm, an extracellular matrix consisting enterocolitis [24]. Reducing the burden of CoNS ing mainly of polysaccharides and proteins, constitutes one would thus reduce not only morbidity and mortality of the essential virulence factors of CoNS as it is critical to but also significant secondary complications and health- the successful colonization of host tissues, indwelling care cost. artificial devices and immune evasion [39–41]. Biofilm formation occurs significantly more frequently with clini- Clinical features of coagulase-negative cally invasive strains in comparison with commensal strains staphylococcal infections in preterm infants [42]. The formation of biofilm consists of two phases, in In preterm infants, CoNS infections most commonly occur which CoNS first adhere unspecifically to a surface and in the third week of life and manifest with the typical, but then proliferate and produce various proteins and poly- non-specific signs of infections in neonates, of which the saccharides to form a complex extracellular matrix commonest are an increased frequency of apnoea, worsen- [1,39,43]. The numerous molecular mechanisms involved ing respiratory status, lethargy, temperature instability and in the biofilm formation of CoNS remain incompletely gastrointestinal disorders [4,25]. Almost all CoNS are iso- characterized [44]. lated from blood culture, whereas infections of the central nervous system, heart or other organs occur infrequently Biofilm provides several lines of effective protection from [3,24]. It has recently been suggested that erythema the host immune response and from antimicrobial agents. toxicum neonatorum, affecting 50–70% of newborns, It was recently shown that some components of the biofilm reflects an innate immune response to CoNS that have produced by CoNS interfere with several major innate penetrated via hair shafts into the dermis. The phenom- immune response mechanisms. The polysaccharide enon is probably mediated by IL-6, which unlike many intercellular adhesin, restricted to a subpopulation of other cytokines is produced by neonates in equivalent S. epidermidis, and poly-g-DL-glutamic acid, ubiquitous quantities to adults after stimulation with Toll-like among S. epidermidis strains, provide significant protection receptor (TLR) type 2 [26,27]. from chemotaxis, degranulation and phagoyctosis of polymorphonuclear leukocytes and also confer resistance Risk factors for coagulase-negative to host antimicrobial peptides, such as cathelicidin, human staphylococcal infection b-defensin 3 and dermcidin [45,46]. Within the first days of life all neonates become colonized with CoNS, and the number of bacteria on the skin Unlike Gram-negative bacteria or S. aureus, CoNS do not dramatically increases within a short period of time produce a large arsenal of potent toxins inducing fulmi- [28–30]. Body sites with abundant CoNS colonization nant clinical responses [39,44]. Lipoteichoic acids and include the ear, axilla, nares and faeces, whereas the skin peptidoglycans of Gram-positive bacteria are significantly on the forearm and leg, most common sites for intravas- less potent inflammatory stimulants, typically requiring cular catheters, generally have a smaller bacterial load [31]. concentrations at mg/ml, whereas lipopolysaccharide at approximately 1000-fold lower concentrations (ng/ml) Epidemiological analyses have identified several risk will elicit similar cytokine responses. In keeping with factors for CoNS infection, including low gestational an overall reduced immune response, infection with age, low birthweight, procedures disrupting skin or muco- biofilm-positive CoNS results in a 34% smaller rise in sal integrity such as intravascular catheters, enteral tubes C-reactive protein, compared with biofilm-negative and mechanical ventilation [3,14,32]. Furthermore, the isolates [47]. A complex of bacterial peptides, derived administration of both antenatal and postnatal steroids, from S. epidermidis, called phenol soluble modulins, have

considerable pro-inflammatory potential, mediated via show significant differences between neonates and adults TLR-2 [48–50]. [60–63]. There are comparatively few analogous data on CoNS.Inadditiontosignificanthypogammaglobulinaemia, CoNS are frequently resistant to common antimicrobial preterm infants also display significantly reduced comp- agents, such as penicillins and aminoglycosides [51]. In lement activity of both classic and alternative pathways general, biofilm-producing strains of CoNS are more resist- [56,64,65]. Both the lack of effective antibodies and ant to a broader spectrum of antibiotics than non-biofilm- decreased complement activity contribute to inefficient producing strains, probably because the biofilm poses a polymorphonuclear cell opsonophagocytosis and bacterial nearly impermeable barrier to many antibiotics [47,52]. killing of CoNS compared with adults and term neonates Furthermore, compared with other forms of growth, [56,66]. The deficient complement deposition on CoNS of bacteria within a biofilm are in a state of reduced metabolic premature sera is corrected in vitro by the use of intrave- activity making them less susceptible to antimicrobial nous immunoglobulin [67]. Furthermore, a significantly agents that rely on ongoing cell division (e.g. b-lactams) impaired capacity of preterm polymorphonuclear cells to [53,54]. upregulate oxidative burst in response to stimulation with CoNS has been described [68]. TLR-2 and TLR-4 have Innate immune responses emerged as important activators for bacterial phagocytosis, The ‘immaturity of host response mechanisms’ is often but data relating to their role in neonates are not available postulated to contribute to the greatly increased suscepti- [69]. bility to bacterial infections in preterm infants, but the mechanisms underlying such impairment are incomple- The effect of biofilm on the efficient phagocytosis of tely defined [55]. Compared with early-onset infections, CoNS is unclear and the effects in vivo are likely to differ there is a striking lack of data on the host immune from in vitro observations; the biofilm formation of CoNS responses to pathogens causing late-onset sepsis, particu- is significantly diminished in planktonic growth, the larly CoNS. These data are, however, of paramount usual form of CoNS preparations employed for in vitro importance to understand the pathogenesis and ultimately studies [39,66,68,70,71]. develop interventions to reduce the disease burden. Here, we review the published literature on various aspects of Cytokine production the immune response to CoNS infections in neonates. We Substantially different cytokine profiles in response to used the following method for reviewing the English various stimuli have been observed in term and preterm literature: an internet-based PubMed search from 1985 neonates compared with healthy adults, and this contrib- to 2007; the search terms included ‘neonate’, ‘preterm utes significantly to the deficient innate immune responses infant’, ‘sepsis’, ‘late-onset’, ‘nosocomial infection’, ‘coag- to pathogens in newborn and preterm infants in particular ulase-negative staphylococcus’, ‘S. epidermidis’, ‘immune [55]. Little is known about the neonatal cytokine response’, ‘cytokine’. responses to CoNS, and only a few studies have investigated CoNS-induced cytokine production in vitro and Transplacental antibodies these data are limited to adults. Bayston et al. [72] Placentally transmitted IgG plays an important role as a described the strong induction of TNF-a in a whole-blood first-line defence mechanism, and low IgG levels in assay stimulated with cell-free supernatant of CoNS, preterm infants are associated with susceptibility to whereas other groups used whole, heat-inactivated CoNS late-onset sepsis [16]. The opsonic activity against to induce TNF-a, IL-1b, IL-6 and IL-12 in human S. epidermidis correlates with antistaphylococcal pepti- mononuclear leukocytes [73–75]. There are no analogous doglycan IgG and with gestational age in preterm infants data from newborn infants, but these are clearly warranted. [56]. Preterm infants who subsequently acquired CoNS Studies of other neonatal pathogens suggest that cytokine sepsis are indistinguishable from uninfected infants on profiles of in vivo infections show some correlation with the basis of their anti-CoNS IgG levels at birth [57], outcome [76–78]. possibly because of the insufficient complement-independent opsonic activity of preterm immunoglobulin Phagocyte signalling and function [56]. In contrast, the level of maternal group B strepto- There are very limited data on the initial TLR signalling coccal antibodies (to both group B streptococcus types Ia pathways that orchestrate the innate and adaptive immune and III) predicts the risk of neonatal group B strepto- response to CoNS [79]. As CoNS are Gram positive, it is coccus early-onset sepsis [58,59]. likely that they signal at least partly through TLR-2, and possibly TLR-4 [69]. S. epidermidis, the most commonly Complement, opsonophagocytosis and isolated strain of CoNS, secretes the TLR-2 agonist bacterial killing phenol-soluble modulin [50]. The potential involvement Many aspects of the innate immune responses to various of either of these receptors, or other TLRs, in the recog- neonatal pathogens, particularly group B streptococcus, nition of CoNS by the innate immune system may have

serious implications for preterm infants. For example, the Conclusion expression of TLR-4, although similar on term neonatal Infections with CoNS constitute the most common late- and adult monocytes [80,81], is significantly reduced in onset sepsis in preterm neonates, adding significantly to preterm infants [82]. In addition, although the expression morbidity, mortality and increased healthcare costs. of TLR-2 on neonatal and preterm monocytes was Therefore, it is of great importance to characterize the comparable to that in healthy adults [80,83], reduced innate immune responses to CoNS in preterm infants, signalling in response to TLR-2 agonists was observed which will be essential for the development of future in preterm cells, possibly as a result of lowered MyD88 prophylactic and therapeutic interventions. Successful expression. Finally, neonatal cytokine responses to a broad strategies to reduce CoNS infections among very preterm panel of TLR agonists (such as bacterial lipopeptides, low birthweight infants would reduce mortality and mor- heat-killed Listeria monocytogenes, zymosan particles, etc.) bidity, shorten hospital stays, reduce antibiotic usage, and are polarized towards high IL-6, low TNF-a production thereby reduce the overall human and financial costs of [27]. 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Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Clostridium difﬁcile: changing epidemiology and new treatment options Ed J. Kuijpera, Jaap T. van Disselb and Mark H. Wilcoxc

Purpose of review Abbreviations The review summarizes changes in the epidemiology and CDAD Clostridrium difficile-associated diarrhoea treatment of Clostridium difficile-associated disease. NAP1 North American pulsed-field type 1 PCR polymerase chain reaction Recent findings PFGE pulsed-field gel electrophoresis Recent outbreaks of Clostridrium difficile-associated

diarrhoea with increased severity, high relapse rate and ß 2007 Lippincott Williams & Wilkins significant mortality, have been related to the emergence of 0951-7375 a new, hypervirulent C. difficile strain in north America, Japan and Europe. Definitions have been proposed by the European Centre for Disease Prevention and Control to Introduction identify severe cases of Clostridrium difficile-associated Since the recognition of Clostridium difficile as the causative diarrhoea and to differentiate community-acquired cases agent of pseudomembranous colitis in 1978, this anaerobic from nosocomial-acquired cases. The emerging strain is spore-forming bacterium has emerged as an important referred to as North American pulsed-field type 1 and enteropathogen. Pathogenic C. difficile organisms release polymerase chain reaction ribotype 027. The emerging toxins that ultimately mediate diarrhoea and colitis. Colo- strain has also been detected in calf diarrhoea and ground nic injury and inflammation result from the production of meat samples in Canada. Attempts to prevent outbreaks two protein toxins: enterotoxin A (TcdA; 308 000 Mr) and caused by type 027 should focus on controlling the overall cytotoxin B (TcdB; 270 000 Mr). For epidemiological pur- use of antibiotics, and high-risk antibiotics such as poses C. difficile can be divided into more than 150 poly- cephalosporins, clindamycin and fluoroquinolones. Several merase chain reaction (PCR) ribotypes and 25 toxinotypes. new antibiotic and non-antibiotic alternatives have become PCR ribotyping is based on the comparison of patterns of available; there is currently no place for probiotic PCR products of the 16S–23S ribosomal RNA intergenic treatments. Patients who suffer multiple relapses of C. spacer region, and toxinotyping involves the detection of difficile-associated diarrhoea present a major therapeutic polymorphisms in the tcdA and tcdB and surrounding challenge. regulatory genes. Summary The early recognition of Clostridrium difficile-associated The illness associated with C. difficile (C. difficile-associated diarrhoea caused by NAP1/027 is necessary to start rapid diarrhoea; CDAD) ranges from mild diarrhoea to life- treatment, to prevent complications, and to prevent further threatening colitis. Typical clinical features include diar- spread of the bacterium. rhoea, lower abdominal pain and systemic symptoms such as fever, anorexia, nausea and malaise. Fulminant colitis Keywords occurs in 1–3% of patients, and is characterized by severe Clostridium difficile, emerging outbreaks, polymerase toxicity with high fever and diffuse abdominal pain and chain reaction ribotype 027 distention. Pseudomembranous colitis represents an advanced stage of disease, and although considered

Curr Opin Infect Dis 20:376–383. ß 2007 Lippincott Williams & Wilkins. ‘non-specific’, it is highly suggestive of C. difficile infection. Severely ill patients may have little or no diarrhoea as a aDepartment of Medical Microbiology, Leiden University Medical Center, Leiden, result of toxic dilatation of the colon (toxic megacolon) and the Netherlands, bInfectious Diseases, Leiden University Medical Center, Leiden, the Netherlands and cDepartment of Medical Microbiology, Leeds Teaching paralytic ileus that may result from a loss of colonic Hospitals and University of Leeds, Leeds, UK muscular tone. Mortality associated with toxic megacolon Correspondence to Ed J. Kuijper, Department of Medical Microbiology, is high, ranging from 25 to 40%. Recurrence of diarrhoea E4-64, Leiden University Medical Center, PO Box 9600, 2300 RC, occurs in 10–40% of patients. CDAD has been considered Leiden, the Netherlands Tel: +31 71 5263574; fax: +31 71 5248148; to be an infection of adults, but little information is e-mail: [email protected] available on the significance of C. difficile in children with Current Opinion in Infectious Diseases 2007, 20:376–383 diarrhoea. Klein et al. [1] found an unexpectedly high rate of 6.7% CDAD in children with diarrhoea who presented to a paediatric emergency department. Overall, a bacterial pathogen other than C. difficile was recovered from stool samples obtained from 7.3% of all patients tested, with

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Shiga toxin-producing Escherichia coli and Salmonella of the rapidly growing literature on the subject, it is species being the most common bacterial pathogens. proposed that a severe case of CDAD is characterized by any of the following: admission to an intensive care Recent outbreaks unit for treatment of CDAD or its complication; surgery Recent outbreaks of CDAD with increased severity, high (colectomy) for toxic megacolon, perforation or refractory relapse rates and significant mortality have been related colitis; death within 30 days after diagnosis if CDAD is to the emergence of a new, hypervirulent C. difficile strain either the primary or a contributive cause [15,17]. in 2003 in Canada and the United States [2–4,5,6–8]. There is no agreement on the inclusion of admission to The predominant strain is referred to as North American a healthcare facility as a criterion for a severe case [15,17]. pulsed-field type 1 (NAP1), PCR ribotype 027, and group In clinical trials, patients can be stratified on laboratory BI by restriction endonuclease analysis. Strain NAP1 parameters such as peak leukocyte, hypoalbuminaemia contains an 18-base pair (bp) and a deletion at 117 of and raised creatinine levels. A high leukocyte count the tcdC gene [9]. This strain has been associated with the (20.0 109/l or greater) and an elevated creatinine level in-vitro production of toxins A and B in quantities 16 and (200 mmol/l or greater) are associated with adverse out- 23 times, respectively, greater than production by control comes and the recurrence of diarrhoea [4]. In a study strains [10]. In a gut model of C. difficile infection, this encompassing 463 patients with a first recurrence of strain produced toxin for significantly longer than another CDAD [18], independent predictors of a second recur- epidemic PCR ribotype (001) (M. Wilcox, in prepara- rence were age and duration of hospitalization after the first tion). In addition, NAP1 also produces a binary toxin, recurrence. Of 51 patients (11.0%) who developed at least encoded by the cdtA gene (the enzymic component) and one major complication (i.e. shock, need for colectomy, the cdtB gene (the binding component). The extent to megacolon, perforation, or death within 30 days) during the which this toxin contributes to the pathogenicity of first recurrence, older age, a high leukocyte count, and C. difficile, however, is unknown. Geric et al. [11] renal failure at first recurrence were strongly associated reported that TcdA and TcdB-negative, but binary with a complicated CDAD [18]. toxin-positive C. difficile strains caused fluid accumulation in the rabbit isolated ileal loop assay, but did not lead to Definitions for severity of diarrhoea diarrhoea or death in the widely used hamster model of So far, a severity grading system of CDAD based on the C. difficile infection. C. difficile PCR ribotype 027 (and 001) amount and consistency of stools is lacking. A grading sporulate more frequently than other strains, which may system based on the intestinal output of diarrhoea was contribute to survival and spread [12]. developed and tested in a group of 37 allogenic stem cell transplant patients [19]. Mild diarrhoea was defined as The clonality of C. difficile PCR 027 is currently a topic of grade 1 diarrhoea when the intestinal output was 500 ml or research. PCR ribotype 027 exhibits at least two pulsed- less per day, moderate diarrhoea was defined as grade 2 field gel electrophoresis (PFGE) patterns with 94% when the output was between 501 and 2000 ml, and severe similarity: north American PFGE types 1a and 1b (NAP1a diarrhoea was defined as grade 3 when more than 3000 ml and NAP1b). As already demonstrated for PCR ribotype was produced. The grading scale was applied within 24 h 001, other typing techniques (DNA fingerprinting, rapid after diagnosing CDAD. Not unexpectedly, patients with enzymatic assay, arbitrarily primed PCR) may reveal severe diarrhoea were more likely to be treated for CDAD additional subgroups. Two recent publications also dem- and had a significant decrease in survival time, probably onstrate that multilocus variable-number tandem-repeat reflecting their underlying medical condition. The small analysis can differentiate various subtypes of type 027 sample size, the highly immunocompromised patients and [13,14]. the application of a 24 h interval are, however, important limitations of that study. There are also considerable Soon after the finding of the hypervirulent type in practical difficulties in accurately recording the volume Canada, this PCR ribotype 027, NAP1 and REA BI type of diarrhoeal stools, not least in confused or incontinent was also isolated in the United States and Europe patients. Another severity grading system was presented at [15,16]. This review summarizes recent findings on Infectious Diseases Society of America 2006 [20]. In con- the changing epidemiological and clinical features of trast to the previous system, this grading system applies to CDAD, and some new treatment modalities. the disease and not only to the symptoms of diarrhoea. The CDAD severity score was calculated by a scoring system Definitions for severity of the disease for fever (temperature > 388C), ileus, systolic blood The recent increase in the incidence of cases and number pressure less than 100 mmHg, white blood cell count of of intensive care admissions, colectomies and fatalities 15 000 cells/ml or greater, and computed tomography find- has raised interest in an easily applicable severity score of ings of the colonic wall. In a retrospective survey among CDAD that could be used as a predictive tool and to 102 patients, a good correlation was found of the CDAD stratify patients in clinical trials. To facilitate comparison severity score with clinical response to metronidazole.

A validation is required in a prospective study. Another suffer severe underlying medical conditions. Severe useful laboratory parameter could be serum lactate con- disease was 2.3 times more frequent [95% confidence centrations. A retrospective observational cohort study interval (CI) 1.1–4.9 times more frequent] when both among 165 cases of severe CDAD revealed that colectomy binary toxin genes and a partial tcdC deletion were present. could be lifesaving in patients aged 65 years or more, in patients with a leukocytosis of more than 20 109/l or Clostridium difficile-associated diarrhoea lactate between 2.2 and 4.9 mmol/l [21]. caused by polymerase chain reaction ribotype 027 in Europe Changes in clinical and epidemiological Soon after the finding of PCR type 027 in north America, manifestations in humans reports confirmed the presence of this new emerging strain An increase in the frequency, severity and relapse rate of in the United States, England, Scotland, Ireland, Belgium, C. difficile-associated diarrhoea was noted in Quebec in France, Austria (imported patient), Luxembourg (unpub- 2002 [2–4]. A review of 1721 case patients from 1991 to lished), Poland (March 2007) and the Netherlands [15, 2003 showed that the incidence of C. difficile enteric disease 16,22]. Very recently, PCR type 027 has also been found in per 100 000 individuals increased fourfold for the entire Switzerland (March 2007) and Denmark (March 2007). region and 10-fold for individuals older than 65 years [4]. The strain was also found in Japan, but unexpectedly was Furthermore, the disease seemed to be more serious and susceptible to fluoroquinolones [23]. This strain revealed refractory to therapy, as indicated by increased rates of more similarities with the historical PCR ribotype 027 toxic megacolon, disease requiring colectomy, associated isolates than with the currently circulating strains [6]. shock or death. Patients who suffered these severe com- The Centers for Disease Control and Prevention (CDC) plications of CDAD were characterized by the following: reported the strain to be associated with high rates of age older than 65 years, acquisition of infection in a morbidity and mortality during outbreaks in hospitals in hospital, peripheral leukocyte count higher than 20 at least 11 US states (http://www.cdc.gov/ncidod/dhqp/ 109 cells/l, renal failure, and severe immunosuppression. index.html). An increase in deaths related to CDAD The proportion of patients who died within 30 days after (ICD10-A04.7) has been reported in the United States diagnosis increased from 4.7% (eight out of 169) in 1991– from 0.29 per 100 000 population in 1999 to 0.77 in 2002 1992 to 13.8% (54/390) in 2003 (P < 0.001). Each case of [24]. Similarly, the number of death certificates in England nosocomial CDAD resulted in an average 10.7 additional and Wales that mentioned C. difficile increased from 1214 in days in hospital. The cumulative mortality 12 months after 2001to3807in2005.Between2004and2005thenumberof diagnosis was significantly higher among patients with deaths involving C. difficile increased by 69% (http://www. CDAD (37.3%) than among control subjects (20.6%) [3]. statistics.gov.uk/cci/nugget.asp?id=1735). Most of the deaths were in the elderly population. The results of a A recent survey in Canada in 12 hospitals encompassing mandatory surveillance of CDAD in individuals over 1703 patients [2] revealed a lower than previously reported 65 years of age revealed 51 690 CDAD cases in 2005, a attributable orcontributablemortalityrate ofCDAD,i.e. of 17.2% increase over 2004 [25]. In contrast to previous years, 6.9%. This mortality rate is more in line with that in reports non-001 types predominated, specifically types 106 and from the Netherlands (7%). The apparently lower 027 (Health Protection Agency, 2006). Type 106 has not mortality rate observed recently may, among other factors, been recognized elsewhere, and data on its severity or be explained by improvements in diagnostic algorithms relapse rate are currently unknown. resulting in the early recognition and rapid treatment of patients with CDAD. Until recently, the association Clostridium difficile-associated diarrhoea between the severity of CDAD and strain NAP1 had been caused by polymerase chain reaction ribotype based on indirect observation of the historical evolution of 027 in the community and among animals mortality or on surveys reporting a limited number of cases. Reports to date have focused on nosocomial CDAD, and In a recent published prospective study performed in 2005 only unpublished observations have mentioned com- patients in 88 hospitals in Quebec, 478 nosocomial isolates munity-acquired CDAD caused by 027. A preliminary ofC.difficilewerecollectedandinvestigated[5].Themost report by the CDC of an increase in CDAD in populations common pulsotypes were NAP1 (PCR ribotype 027) in previously considered to be at low risk has not been 57% and North American pulsed-field type B (PCR ribo- confirmed in other surveys [26]. In addition, only two type 001) in 18%. An association was found between NAP1 isolates were recovered from the patients involved in the and an increased incidence and a more severe course of CDC report, and, although toxin variant, these two isolates disease.CDADwastheattributablecauseofdeathfor2.6% were not PCR ribotype 027/BI/NAP1. It is likely that ofthepatients,andcontributedtodeathinanother4.5%.As CDAD caused by type 027 also occurs in the community aresultofCDAD,3%ofpatientshadtobetransferredtothe outside healthcare facilities. C. difficile also appears to be an intensivecare unit, and1% underwenta colectomy;most of important cause of enteric disease in a variety of animal these cases occurred in the older age groups that often species, including horses, dogs, cats, birds, rodents, and

especially neonatal pigs, suggesting that animals may serve ofthefluoroquinolones comesfromthestudybyPepinetal. as a reservoir for human pathogens. A prospective case– [32]. The authors found an adjusted hazard risk (AHR) of control study, performed among calves from 102 dairy 3.44 (2.65–4.47) for the development of CDAD. The risk farms in Canada, resulted in 31 C. difficile isolates in was dependent on the duration of treatment. The AHR 278 calves [27]. PCR ribotyping of 31 isolates showed tended to be lower for levofloxacin (AHR 2.52; 95% CI eight distinct patterns, of which two have been associated 1.68–3.79) than for ciprofloxacin (AHR 3.74; 95% CI 2.81– with outbreaks of severe disease (PCR types 017 and 027). 4.97). Among patients who received newer respiratory The authors concluded that C. difficile may be associated fluoroquinolones, the risk seemed higher in those who with calf diarrhoea, andcattle may be reservoirs of C. difficile received gatifloxacin (AHR 6.10; 95% CI 2.22–16.74) than for humans. C. difficile has also been isolated from 12 (20%) in those given moxifloxacin (AHR 2.04; 95% CI 0.50–8.31). of 60 retail ground meat samples purchased over a Almost one quarter of inpatients received fluoroquino- 10-month period in 2005 in Canada [28]. Eleven isolates lones,anditwascalculatedthatthepopulation-attributable were toxigenic, and eight (67%) were classified as toxino- fraction of CDAD was 35.9%. The population-attributable type III. C. difficile is an important cause of diarrhoea in fractions of second-generation cephalosporins and clin- horses, but a recent study among isolates from 38 diarrhoeic damycin was considerably lower, at 10.7 and 1.5%, respect- horses in Canada did notrevealtype 027[29].Interestingly, ively. Loo et al. [2] found CDAD patients more likely than type 017 A()B(þ) was found in eight isolates (16.6%). It matched controls to have received fluoroquinolones (OR should be pointed out, however, that as C. difficile are spore 3.9; 95% CI 2.3–6.6) or cephalosporins (OR 3.8; 95% CI forming and therefore long-lasting micro-organisms that 2.2–6.6). The authors found, however, that CDAD pa- are found ubiquitously in the environment it is difficult to tients received antibiotics more frequently and almost 50% delineate true reservoirs and transmission routes. more antibiotics (1.9 versus 1.3) than did controls; antibiotic duration was not examined [33]. Kazakova et al. [8] Antibiotics as risk factor for new emerging concludedthat a significantincrease in fluoroquinoloneuse Clostridium difficile-associated diarrhoea in the hospital was associated with an outbreak of diarrhoea Several studies have suggested that exposure to fluoroqui- caused by the 027 strain. Cephalosporins (OR 5.19; nolones is a major risk factor for the developmentof CDAD P ¼ 0.006), fluoroquinolones (OR 3.22; P ¼ 0.04) and caused by type 027 strains. If so, this would represent a proton pump inhibitors (OR 5.02; P ¼ 0.02) were indepen- major change in the epidemiology of hospital-acquired dendent risk factors for CDAD. The results are similar to CDAD, which is historically associated with the use those from a case–control study during an outbreak of cephalosporins and clindamycin. Obviously, such asso- of type 027 in a medium-sized hospital in the Netherlands. ciations to a large extent reflect in-hospital antibiotic The odds ratio for CDAD was 7.8 (95% CI 2.9–20.9) for policy, and anychange in prescription behaviour will reveal the use of cephalosporins, 28.8 (95% CI 2.6–319.2) for the ability of a new antibiotic to induce CDAD. For fluoroquinolones, and 57.5 (95% CI 6.8–483.6) for the example, ertapenem, a long-acting carbapenem, may be combined use of cephalosporins and fluoroquinolones. associated with an increase in C. difficile infection [30]. One The fact that the risk in those subjects was much higher of the first studies demonstrating that an increased use of (OR 57.5) than could be explained by simply summing the fluoroquinolones is associated with CDAD goes back to risk for the separate antibiotics (7.8 and 28.8, respectively) 2000 to Pittsburgh [7]. A retrospective case–control study could suggest a synergistic effect of cephalosporins and of case patients with C. difficile infection from January 2000 fluoroquinolones in the aetiology of CDAD. The popu- to April 2001 revealed clindamycin [odds ratio (OR) 4.8; lation-attributable risk percentage, i.e. the proportion of 95% CI 1.9–12.0], ceftriaxone (OR 5.4; 95% CI 1.8–15.8), CDAD cases in the study population that was attributable and levofloxacin (OR 2.0; 95% CI 1.2–3.3) independently to the use of cephalosporin or quinolone therapy, was associated with infection [7]. In contrast to ciprofloxacin, calculated as 56 and 33%, respectively. other quinolones such as levofloxacin, trovafloxacin, spar- floxacin, gatifloxacin and moxifloxacin have good activity Outbreak control and antibiotic policy against anaerobic bacteria. Ciprofloxacin has also, however, Attempts to control outbreaks caused by type 027 should been associated with CDAD caused by type 027. It has focus on controlling the overall use of antibiotics, focussing been suggested that the recent acquisition of resistance to on high-risk antibiotics such as cephalosporins, clindamy- the newer fluoroquinolones by this strain was the major cin and fluoroquinolones. Notably, substituting one of the reason for its wide dissemination [6], although fluroquino- fluoroquinolones with another have been unsuccessful lone resistance was known to be present in earlier epidemic [34]. Aggressive antibiotic restriction of high-risk anti- C. difficile strains. A recent study from Quebec also demon- biotics, reducing polypharmacy, the prevention of long strated a difference of gatifloxacin resistance of strains durations of therapy and avoiding inappropriate prescrib- belonging to the most common PFGE types in comparison ing are the first steps to decrease high incidence rates of with strains from other types, but not for the other fluor- CDAD. Up until now, those studies only included oquinolones [31]. The most compelling evidence for a role matched non-CDAD control patients. We found during

a one-year surveillance in the Netherlands that patients found in the quinolone-resistance determining region of with CDAD caused by type 027 had been prescribed gyrB [39]. It remains possible that different mutations significantly more fluoroquinolones (OR 2.88, 95% CI affect specific PCR ribotypes, because high-level resist- 1.01–8.20) than patients with non-027 types. An expla- ance to moxifloxacin and gatifloxacin in PCR ribotype 017 nation for this difference in the propensity to induce isolates were associated with a novel transversion mutation CDAD may be the higher level of fluoroquinolone resist- in gyrB (Asp-426, Val) of TcdA,TcdBþ strains [40]. ance of type 027 in comparison with other types, although certain other types have also demonstrated a high level New treatment options fluoroquinolone resistance [6,31]. It should be realized Treatment of C. difficile-associated disease has traditionally though that associations between CDAD and the use of been to discontinue the antibiotic agent implicated, and particular antibiotics are bound to be confounded by for those patients who have at least moderately severe or inhospital hygiene and crucially the risk of exposure to persistent disease, to give oral metronidazole or oral C. difficile. Also, local antibiotic policy, including the diver- vancomycin. Metronidazole is favoured on the basis of sity of the drugs used, total antibiotic use and physician’s cost, the results of clinical trials showing therapeutic choice of antimicrobial treatment for specific diagnoses are equivalence with vancomycin, and the notion that metro- likely to be key drivers of CDAD risk. nidazole would be less likely than vancomycin to promote colonic colonization and facilitate the spread of vancomy- Proton pump inhibitors and Clostridium cin-resistant enterococci. Unfortunately, treatment is com- difficile-associated diarrhoea plicated by a recurrence after treatment in 20–25% of the The controversy over proton pump inhibitor use and the cases, usually 1–10 days after treatment is discontinued. risk of CDAD remains. Two retrospective studies by Dial andcolleagues [35,36]suggestedthatcommunity-acquired CDAD caused by the new emerging PCR ribotype 027 CDAD in England was associated with previous proton strain appears to be more refractory to therapy with pump use. A hospital based case–control study found that metronidazole, although adequately controlled studies CDAD was independently associated with antibiotic use are required to verify this point. Oral vancomycin is also (OR 13.1; 95% CI 6.6, 26.1), acid suppression therapy (OR the preferred drug for patients who are seriously ill (or 1.90; 95% CI 1.10, 3.29) and female sex (OR 1.79; 95% CI have one of the characteristics described above, e.g. 1.06, 3.04) [37]. Two other recent large studies have failed admission to the intensive care unit, a leukocytosis of to demonstrate such an association [32,38]. Clearly, this 20 000 or greater, elevated serum creatinine and hypoal- issue awaits a prospective study. buminaemia), because it reaches high levels in the colon, and resistance has not yet been reported. Suitable alter- Antimicrobial resistance of Clostridium difficile natives for metronidazole and vancomycin have been polymerase chain reaction ribotype 027 tested in several studies. Nitazoxanide is a nitrothiazolide C. difficile isolates from a 2004 outbreak in Quebec, marketed for the treatment of intestinal parasites, but Canada, were all found to be susceptible to metronidazole, also has activity against C. difficile, and reaches high vancomycin, rifampin and meropenem, but were resistant colonic levels with oral administration. In a prospective, to bacitracin, cefotaxime, ciprofloxacin and levofloxacin, randomized, double-blind study 34 patients were treated and most (>80%) were resistant to ceftriaxone, clarithro- with metronidazole 250 mg four times per day for 7 days, mycin, gatifloxacin and moxifloxacin. The predominant 40 patients with nitazoxanide 500 mg two times per day NAP1 isolates were susceptible to clindamycin, whereas for 7 days, and 36 patients with nitazoxanide 500 mg two the North American pulsed-field type 2 isolates were times per day for 10 days [41]. Low, but comparable resistant [31]. Such studies invariably use systemic break- responses were observed for metronidazole (57.6%) and points to define resistance, and the relevance of these nitazoxanide (65.8 and 74.3%, respectively). No signifi- values to the concentrations of antibiotics achieved in cant difference was seen in terms of recurrence rate. the gut is often not considered. This characteristic Eight patients with multiple CDAD recurrences were antimicrobial susceptibity pattern encompassing suscepti- given a 2-week course of rifaximin therapy when they bility to clindamycin but resistance to newer fluoroquino- were asymptomatic, immediately after completing their lones and erythromycin has been recognized previously last course of vancomycin therapy [42]. Seven of the eight and may be of benefit for microbiological laboratories to patients experienced no further diarrhoea recurrence. recognize the 027 strain [16]. The molecular mechanism of Controlled studies of rifaximin in CDAD are needed. high-level resistance to newer fluoroquinolones in type The results of a recent study indicated that there is no 027 strains has not yet been established, although a role for routine rifampicin as an adjunct to treatment with publication from Ireland showed that five PCR ribotype metronidazole for hospitalized patients with C. difficile- 027 isolates from one cluster had a single transition associated diarrhoea [43]. The study was, however, small, mutation (C to T), resulting in the amino acid substitution and of 279 CDAD patients, only 39 were included. Thr-82-Ile in gyrA [39]. No amino acid substitutions were In addition, the cure rate in both treatment groups was

unacceptably low. Fusidic acid has been considered as considered of prime importance to prevent disease. In line another therapeutic option for CDAD, but a prospective with this notion, vaccines containing formaldehyde-inac- randomized double-blind trial comparing 400 mg metro- tivated toxins A and B have been developed. In healthy nidazole three times a day (n ¼ 55) with 250 mg fusidic volunteers, this vaccine induced high levels of IgG immu- acid three times a day for 7 days (n ¼ 59) revealed the noglobulins and some promising initial experience has development of resistance in 55% patients who remained been gained in a few patients with recurrent CDAD culture positive [44]. [49]. Passive immunotherapy effectively prevents lethal disease in the hamster model of CDAD, raising hope that Tolevamer, a novel non-antibiotic polymer that binds and monoclonal antibodies to toxin A and toxin B could find neutralizes C. difficile toxins A and B, has been compared application as adjunct therapy (Medarex, Princeton, New with two different doses (3 and 6 g per day) in a three-arm Jersey, USA; phase II). A similar approach concerns the use study encompassing289patientswith vancomycin (500 mg of anti-C. difficile bovine immunoglobulin secretory IgA per day) for the treatment of mild to moderately severe concentrate prepared from the milk of cows immunized CDAD [45]. The multicentre study was randomized, against C. difficile and its toxins [50]. The immune whey double blinded and actively controlled. Tolevamer at a protein concentrate neutralized the cytotoxic effects of dosage of 6 g per day was found to be not inferior to purified toxin A/B on cultured human cells, protected vancomcyin treatment. Although underpowered for this hamsters against lethal C. difficile-induced colitis, and endpoint, a trend was observed towards a lower recurrence was safe to use in humans [51]. In an uncontrolled pro- rate of tolevamer (10%) than vancomycin (19%). Currently, spective cohort study of the immune whey in over a phase III trial with a tolevamer 9 g-per-day regimen is 100 patients with CDAD, the rate of relapse was reduced being performed in comparison with vancomycin and by approximately half as compared with that in contem- metronidazole.Unlikenitazoxanide,tolevamerisnotavail- porary controls (J.T. van Dissel, 2007, in preparation). able in the marketplace; therefore, clinicians will not have Clearly, the findings on active and passive immune therapy access to this drug until completion of the phase III trial. support the further clinical development of these Par-101 (Optimer Pharmaceuticals, San Diego, California, approaches that now await confirmatory data from phase USA) is another new agent that is currently under inves- III randomized controlled trials. tigation in a multinational, multicentre, double-blind, randomized study to compare the safety and efficacy of Conclusion the drug when taken with 125 mg vancomycin every 6 h for Outbreaks of CDAD with increased severity, high relapse 10 days. Par 101 is an 18-membered macrocycle with poor rates and significant mortality have been related to the oral absorption. It has moderate activity against certain emergence of a new, hypervirulent C. difficile strain Gram-positive cocci and excellent activity against C. referred to as NAP1 and PCR ribotype 027. Interim difficile. Many other drugs are still under development, definitions for surveillance studies have been proposed including ramoplanin (Oscient Pharmaceuticals, Waltham, by experts from Europe and the United States. The Massachusetts, USA; phase II completed) and rafalazil emerging strain has also been detected in calf diarrhoea (ActivBiotics, Lexington, Massachusetts, USA; phase II). and ground meat samples in Canada, suggesting that annimals may serve as a reservor for human disease. Probiotics and relapsing Clostridium difficile- Patients who suffer multiple relapses of C. difficile-associ- associated diarrhoea ated diarrhoea present a major therapeutic challenge, but Patients who suffer multiple relapses of C. difficile-associ- several new therapeutic apporaches have become avail- ated diarrhoea present a major therapeutic problem, able. Outbreaks caused by type 027/NAP1 are difficult to because repeated cycles of antibiotics may trigger more control and require early recognition, prompt treatment, relapses. In such cases, various therapies including choles- effective evironmental cleaning, and a restrictive use of tyramine, Lactobacillus GG, Saccharomyces boulardii and antibiotics. Several new antibiotic and non-antibiotic even replacement of the colonic flora by stools of healthy alternatives have become available as alternatives for relatives have been tried. Many of these alternative, vancomcyin and metronidazole, but there is currently no non-antibiotic treatments, however, were found to be only place for probiotic treatments. modestly effective [46]. In therapy-refractory patients, intravenous pooled human immunoglobulin has been References and recommended reading administered, with approximately two-thirds of patients Papers of particular interest, published within the annual period of review, have been highlighted as: responding in two small series [47,48]. of special interest of outstanding interest Immunotherapy for - Additional references related to this topic can also be found in the Current Clostridium difficile World Literature section in this issue (pp. 435–436). associated diarrhoea 1 Klein EJ, Boster DR, Stapp JR, et al. Diarrhea etiology in a Children’s Hospital After the acquisition of C. difficile and subsequent colon- Emergency Department: a prospective cohort study. Clin Infect Dis 2006; ization by the bacterium, the host immune response is 43:807–813.

2 Loo VG, Poirier L, Miller MA, et al. A predominantly clonal multiinstitutional 20 Belmares J. Parada JP. Miskevics SA, et al. A severity of C. difficile-associated outbreak of Clostridium difficile-associated diarrhea with high morbidity and disease (CDAD) index predicts treatment outcomes with metronidazole. mortality. N Engl J Med 2005; 353:2442–2449. Alexandria, VA, USA: Infectious Diseases Society of America 2006. 3 Pepin J, Valiquette L, Cossette B. Mortality attributable to nosocomial 21 Lamontagne F, Labbe AC, Haeck O, et al. Impact of emergency colectomy on Clostridium difficile-associated disease during an epidemic caused by a survival of patients with fulminant Clostridium difficile colitis during an hypervirulent strain in Quebec. Can Med Assoc J 2005; 173:1037–1042. epidemic caused by a hypervirulent strain. Ann Surg 2007; 245:267–272. 4 Pepin J, Valiquette L, Alary ME, et al. Clostridium difficile-associated diarrhea An important study, suggesting that the serum lactate concentration may be of in a region of Quebec from 1991 to 2003: a changing pattern of disease importance in the decision to perform an emergency colectomy. severity. Can Med Assoc J 2004; 171:466–472. 22 Indra A, Huhulescu S, Hasenberger P, et al. First isolation of Clostridium 5 Hubert B, Loo VG, Bourgault AM, et al. Portrait of the geographic dissemina- difficile PCR ribotype 027 in Austria. Euro Surveill 2006; 11: E060914.3. tion of the Clostridium difficile North American pulsed-field type 1 strain and 23 Kato H, Ito Y, Van den Berg RJ, et al. First isolation of Clostridium difficile 027 the epidemiology of C. difficile-associated disease in Quebec. Clin Infect Dis in Japan. Euro Surveill 2007; 12: E070111.3. 2007; 44:238–244. An excellent one-year prospective surveillance study in 88 Quebec hospitals. 24 Wysowsky D. Increase of deaths related to enterocolitis due to Clostridium Clinical patient data were compared with strain characteristics and provide the difficile in the United States, 1999–2002. Public Health Reports 2006; first evidence for a more severe disease of NAP1/027 in comparison with other 121:361–362. types. 25 Health Protection Agency. Mandatory surveillance of healthcare-associated 6 McDonald LC, Killgore GE, Thompson A, et al. An epidemic, toxin gene- infection report, 2006. London, UK: Health Protection Agency 27 November variant strain of Clostridium difficile. N Engl J Med 2005; 353:2433– 2006. 2441. 26 Chernakl E, Johnson CC, Weltman A, et al. Severe Clostridium difficile- 7 Muto CA, Pokrywka M, Shutt K, et al. A large outbreak of Clostridium difficile- associated disease in populations previously at low risk – four states. MMWR associated disease with an unexpected proportion of deaths and colectomies Morb Mortal Wkly Rep 2005; 54:1201–1205. at a teaching hospital following increased fluoroquinolone use. Infect Control 27 Rodriguez-Palacios A, Sta¨mpfli HR, Duffield T, et al. Clostridium difficile PCR Hosp Epidemiol 2005; 26:273–280. ribotypes in calves, Canada. Emerg Infect Dis 2006; 12:1730–1736. 8 Kazakova SV, Ware K, Baughman B, et al. A hospital outbreak of diarrhoea A case–control study of calves from 102 dairy farms. PCR ribotypes 027 and 017 due to an emerging epidemic strain of Clostridium difficile. Arch Intern Med were found among eight different ribotypes. 2006; 166:2518–2524. 28 Rodriguez-Palacios A, Staempfli HR, Duffield T, Weese JS. Clostridium 9 Maccannell DR, Louie TJ, Gregson DB, et al. Molecular analysis of Clostridium difficile in retail ground meat, Canada. Emerg Infect Dis March 2007; 13. difficile PCR ribotype 027 isolates from eastern and western Canada. J Clin Available from: http://www.cdc.gov/EID/content/13/3/485.htm Accessed: Microbiol 2006; 44:2147–2152. April 2007. 10 Warny M, Pepin J, Fang A, et al. Toxin production by an emerging strain of 29 Arroyo LG, Staempfli H, Weese JS. Molecular analysis of Clostridium difficile Clostridium difficile associated with outbreaks of severe disease in North isolates recovered from horses with diarrhea. Vet Microbiol 2007; 120:179– America and Europe. Lancet 2005; 366:1079–1084. 183. 11 Geric B, Carman RJ, Rupnik M, et al. Binary toxin-producing, large clostridial 30 Itani KM, Wilson SE, Awad SS, et al. Ertapenem versus cefotetan prophylaxis toxin-negative Clostridium difficile strains are enterotoxic but do not cause in elective colorectal surgery. N Engl J Med 2006; 355:2640–2651. disease in hamsters. J Infect Dis 2006; 193:1143–1151. 31 Bourgault AM, Lamothe F, Loo VG, Poirier L. CDAD–CSI Study Group. In vitro The results of this study showed that binary toxin causes fluid accumulation in an Clostridium difficile isolated ileal loop model. Unexpectedly, hamsters challenged with strains producing susceptibility of clinical isolates from a multiinstitutional binary toxin did not get diarrhoea or die. outbreak in Southern Quebec, Canada. Antimicrob Agents Chemother 2006; 50:3473–3475. 12 Fawley WN. Underwood S. Freeman J, et al. Effect of hospital cleaning agents 32 et al. and germicides on the survival of epidemic Clostridium difficile strains. Infect Pepin J, Saheb N, Coulombe MA, Emergence of fluoroquinolones as the Clostridium difficile- Control Hosp Epidemiol 2007; in press. predominant risk factor for associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis 2005; 41:1254–1260. 13 Marsh JW, O’Leary MM, Shutt KA, et al. Multilocus variable-number tandem- 33 Wilcox MH, Freeman J. Epidemic Clostridium difficile. 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Proton pump inhibitor use and In contrast with automated sequence detection as applied by Marsh et al., smaller risk of community-acquired Clostridium difficile associated disease defined short tandem repeats (2–9 bp) were used to facilitate faster multicolored capillary by prescription for oral vancomcyin therapy. Can Med Assoc J 2006; 175: electrophoresis for fragment analysis. Reference strains and clinical isolates were 745–830. tested. 36 Dial S, Delaney JA, Barkun AN, Suissa S. Use of gastric acid-suppressive 15 Kuijper EJ, Coignard B, Tu¨ll P. Emergence of CDAD in Northern America and agents and the risk of community-acquired Clostridium difficile-associated Europe. Clin Microbiol Infect 2006; 12 Suppl. 6: 2–18. Available from: disease. JAMA 2005; 21:2989–2995. http://www.ecdc.eu.int/Health_topics/Clostridium_difficile/Guidance.html 37 Yearsley KA, Gilby LJ, Ramadas AV, et al. Proton pump inhibitor therapy is a Accessed: April 2007. risk factor for Clostridium difficile-associated diarrhoea. Aliment Pharmacol An overview of the situation of C. difficile PCR ribotype 027 worldwide in 2006, Ther 2006; 24:613–619. with epidemiological and microbiological data. Interim definitions are provided for community-acquired CDAD and nosocomial-acquired CDAD, based on the 38 Lowe DO, Mamdani MM, Kopp A, et al. Proton pump inhibitors and hospi- source and onset of disease. talization for Clostridum difficile associated disease: a population-based study. Clin Infect Dis 2006; 43:1272–1276. 16 Kuijper EJ, Van den Berg RJ, Debast S, et al. Clostridium difficile PCR ribotype 027, toxinotype III in the Netherlands. Emerg Infect Dis 2006; 39 Drudy D, Kyne L, O’Mahony R, Fanning S. gyrA Mutations in fluoroquinolone- 12:827–830. resistant Clostridium difficile PCR-027. Emerg Infect Dis 3 March 2007; 13 Available at: http://www.cdc.gov/EID/content/13/3/504.html. 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43 Lagrotteria D, Holmes S, Smieja M, et al. Prospective, randomized inpatient 47 Wilcox MH. Descriptive study of intravenous immunoglobulin for the treatment study of oral metronidazole versus oral metronidazole and rifampin for treat- of recurrent Clostridium difficile diarrhoea. J Antimicrob Chemother 2004; ment of primary episode of Clostridium difficile-associated diarrhea. Clin 53:882–884. Infect Dis 2006; 43:547–552. 48 McPherson S, Rees CJ, Ellis R, et al. Intravenous immunoglobulin for 44 Noren T, Wullt M, Akerlund T, et al. Frequent emergence of resistance in the treatment of severe, refractory, and recurrent Clostridium difficile diarrhea. Clostridium difficile during treatment of C. difficile-associated diarrhea with Dis Colon Rectum 2006; 49:640–645. fusidic acid. Antimicrob Agents Chemother 2006; 50:3028–3032. 45 Louie TJ, Peppe J, Watt CK, et al., Tolevamer Study Investigator Group. 49 Sougioultzis S, Kyne L, Drudy D, et al. Clostridium difficile toxoid vaccine in Tolevamer, a novel nonantibiotic polymer, compared with vancomycin in recurrent C. difficile-associated diarrhea. Gastroenterology 2005; 128:764– the treatment of mild to moderately severe Clostridium difficile-associated 770. diarrhea. Clin Infect Dis 2006; 43:411–420. A three-arm, multicentre, randomized, double-blind phase II study with two different 50 Van Dissel JT, De Groot N, Hensgens CM, et al. Bovine antibody-enriched doses of tolevamer and vanomycin. A trend was observed towards a lower relapse whey to aid in the prevention of a relapse of Clostridium difficile-associated rate in tolevamer-treated patients. A new preparation of tolevamer has been diarrhoea: preclinical and preliminary clinical data. J Med Microbiol 2005; prepared to prevent hypokalaemia and a higher dosage will be used in a new study. 54:197–205. 46 McFarland LV. Meta-analysis of probiotics for the prevention of antibiotic 51 Young KW, Munro IC, Taylor SL, et al. The safety of whey protein concentrate associated diarrhea and the treatment of Clostridium difficile disease. Am J derived from the milk of cows immunized against Clostridium difficile. Regul Gastroenterol 2006; 101:812–822. Toxicol Pharmacol 2007; 47:317–326.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Glycopeptide-resistant enterococci: deciphering virulence, resistance and epidemicity Rob J.L. Willems and Marc J.M. Bonten

Purpose of review Abbreviations Since their first discovery, glycopeptide-resistant CC17 clonal complex 17 enterococci have emerged as important nosocomial GRE glycopeptide-resistant enterococci GREF glycopeptide-resistant Enterococcus faecium pathogens first in the US, followed by the rest of the HGT horizontal gene transfer world. In this review the most recent findings that HiRECC high-risk enterococcal clonal complex MLST multilocus sequence typing relate to enterococcal epidemiology, virulence and VRE vancomycin-resistant enterococci glycopeptide-resistance maintenance will be

discussed. ß 2007 Lippincott Williams & Wilkins Recent ﬁndings 0951-7375 Frequent horizontal gene transfer and recombination, resulting in high-level genome plasticity, facilitating rapid responsiveness of enterococci to changing Introduction environmental conditions may have contributed to the The ﬁrst reports on clinical vancomycin-resistant Enter- worldwide emergence. For Enterococcus faecium this ococcus faecium isolates in Europe in 1988 marked a has resulted in the development of a distinct genetic dramatic change in the perspective on enterococcal infec- subspecies, clonal complex 17, responsible for the majority tions. From that time on enterococci were no longer of glycopeptide-resistant enterococci-related hospital considered as mere colonizers of the gastrointestinal tract, burden. Preliminary data also suggest that such high-risk occasionally causing nosocomial infections, but as emer- enterococcal clonal complexes may exist within ging nosocomial pathogens. This change in perspective Enterococcus faecalis. The last 2 years have not only was nourished by the epidemic increase of glycopeptide- disclosed novel determinants implicated in enterococcal resistant enterococci (GRE) in hospitals in the US follow- pathogenesis, but also showed that enterococci are able to ing the initial discovery in Europe. By 1993, the prevalence sense their environment and regulate virulence gene of vancomycin-resistant enterococci (VRE) had already expression accordingly. Linkage of glycopeptide resistance increased 20-fold in the intensive care units of US hospi- in enterococci to plasmid maintenance systems holds a tals. More recent data suggest that VRE prevalence has doomed perspective for controlling antibiotic resistance started to level-off at around 30%, due to almost complete emergence. penetration of glycopeptide resistance (around 70–80%) Summary into the nosocomial E. faecium population, while in Recent developments have improved our understanding Enterococcus faecalis it has not increased above 10%. This of enterococcal population structure, pathogenesis demonstrates that glycopeptide resistance is primarily a and glycopeptide-resistance maintenance. This may concern in E. faecium. In this review we will integrate the contribute to the development of novel intervention most recent knowledge on the epidemiology, population strategies to prevent enterococcal infections and biology, antibiotic resistance and virulence of GRE. contain the spread of glycopeptide-resistant enterococci. Epidemiology of glycopeptide-resistant Keywords enterococci enterococcus, glycopeptide-resistance maintenance, While before the turn of the century the emergence of population structure, virulence glycopeptide-resistant E. faecium GREF among hospitalized patients is primarily seen in the US, in recent

Curr Opin Infect Dis 20:384–390. ß 2007 Lippincott Williams & Wilkins. years accumulating data indicate that this evolution has more and more a global dimension. According to Department of Medical Microbiology, University Medical Center Utrecht, European Antimicrobial Resistance Surveillance System Utrecht, The Netherlands data (www.earss.rivm.nl), the prevalence of GREF among Correspondence to Rob J.L. Willems, Department of Medical Microbiology, nosocomial enterococcal blood stream infections is already University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands above 5% in 10 countries, of which three countries Tel: +31 30 2507630; fax: +31 30 2541770; e-mail: [email protected] exhibited prevalence rates above 10% and ﬁve countries Current Opinion in Infectious Diseases 2007, 20:384–390 even above 25%. Five years ago, in 2002, only seven, four and zero countries suffered from GREF prevalence rates above 5, 10 and 25%, respectively, demonstrating that in

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several European countries the emergence of GREF in (CC17 and non-CC17) using a mixed whole-genome hospitals has followed that of the US, but with a 10- to microarray based on the total genomes of nine E. faecium 15-year delay. Also, GREF are encountered in other parts isolates (CC17 and non-CC17) revealed that hospital- of the world, including South America, Asia and Australia, associated CC17 isolates were, based on gene content, illustrating the pandemic spread of hospital-associated genetically highly related, and grouped together in a GREF [1,2,3,4,5,6,7]. At the same time, however, the hospital clade distinct from non-CC17 human community European animal reservoir of GREF, which was significant and animal isolates [25]. These microarray data not only during the 1980s and 1990s due to the use of avoparcin as confirmed distinct clustering of hospital-associated clinical an antimicrobial growth promoter, has decreased after the and outbreak isolates, but also identified more than European Union banned its use [8,9]. This suggests that 100 genes that were specifically enriched among CC17 hospital-associated GREF have their own transmission isolates [25]. These CC17 specific genes included not dynamics apparently not directly linked to the animal only antibiotic-resistance genes, but also putative viru- reservoir. In addition to these epidemiological data, studies lence genes, genes encoding metabolic pathways, phage on the population structure of E. faecium added to the genes, integrated plasmids and insertion sequence hypothesis that increased circulation of GREF in and elements. Due to the fact that the set of more than between hospitals is primarily due to enhanced prevalence 100 CC17-enriched genes are not clustered on one geno- of a particular hospital-adapted E. faecium clonal complex, mic region, acquisition of these genes most probably has CC17. CC17 seems to be distinct from animal-associated occurred multistep-wise and has been pivotal in the pro- E. faecium and has over time acquired glycopeptide gression of E. faecium from an enteric commensal towards a resistance through horizontal gene transfer (HGT). The hospital-adaptation pathogen. Previously, such a multistep emergence of CC17 E. faecium coincided with an increase evolutionary process has been referred to as genetic capit- in the number of E. faecium nosocomial infections rela- alism or the Mathew effect [26,27]. tive to infections caused by E. faecalis [10,11]. Based on the large number and broad range of genes Population structure and genetic evolution of acquired by CC17, all likely to contribute to the selective Enterococcus faecium and Enterococcus advantage of CC17 in the hospital environment, we have faecalis classified CC17 as an example of a high-risk enterococcal First insights into the population structure of E. faecium clonal complex (HiRECC). Within healthcare settings a were obtained from molecular-typing data [12]. These data HiRECC can act as donor of resistance genes to other pointed towards host specificity of E. faecium and the nosocomial pathogens, like Staphylococcus aureus, which existence of a distinct subpopulation, designated CC17 may lead to the emergence of vancomycin-resistant based on multilocus sequence typing (MLST). CC17 S. aureus. A GREF is thought to be the donor of vanco- appeared responsible for the vast majority of hospital mycin-resistance genes in at least one of the five reported associated clinical infections and outbreaks. Interestingly, vancomycin-resistant S. aureus cases [28]. this CC17 was not confined to US hospitals, the country with presumably the highest number of enterococcal infec- A MLST scheme was developed for E. faecalis in 2006 tions, but was found to be globally dispersed [12]. Soon [29]. Among the initially identified 55 sequence types and after the initial publications describing CC17, formally four major clonal complexes, two, CC2 and CC9, may known as lineage C1, additional publications confirmed represent an E. faecalis HiRECC. These two clonal com- the emergence of this nosocomial E. faecium subpopulation plexes, previously designated the BE complex, and the in Brazil, Germany, Italy, Korea, The Netherlands, Singa- ACB clone exclusively contained isolates from hospital- pore and Sweden [1,2,5,13,14–16,17,18,19]. ized patients, and included invasive as well as hospital outbreak isolates [29,30]. Furthermore, a study of almost The ecological success of CC17 in the hospital environ- 300 E. faecalis isolates from Polish hospitals identified two ment is, most likely, partly related to increased antibiotic additional HiRECC, i.e. ST40 and CC87 [31]. Vancomy- resistance. Both ampicillin and quinolone resistance are cin resistance, a relatively rare feature in E. faecalis,was specifically associated with this clonal complex [12,20–22]. found relatively frequently in these E. faecalis HiRECCs. Increased antibiotic resistance within this clonal complex Hospital adaptation of particular E. faecalis clones was also is, however, not the whole story. Already at the moment of concluded from a study on E. faecalis isolates from healthy identification, the enterococcal surface protein gene, esp,a volunteers, intensive care unit patients and blood isolates virulence gene contained on a putative pathogenicity from a single institute [32]. island, was found to be specifically linked to CC17 or the C1 lineage [12,20,21,23,24]. A recent comprehensive From the MLST-based studies of E. faecium and E. comparative genomic analysis of 97 E. faecium isolates from faecalis it became also apparent that frequent HGT different ecological (humans and animals; hospital and and recombination shaped the population structure of community associated) and evolutionary backgrounds both enterococcal species. Nonclonality in E. faecalis and

E. faecium was demonstrated among others by the lack of persist in the absence of glycopeptide antibiotics. A congruence between the topologies of MLST gene trees, possible explanation for glycopeptide-resistance persist- representing housekeeping genes. Comparative genomic ence is that vanA elements on mobile genetic elements are hybridization of nine E. faecalis isolates on an E. faecalis linked with postsegregational killing ‘plasmid addiction’ V583-based microarray showed considerably diversity in systems [40,42,43,44]. These systems encode an anti- gene content. Compared to E. faecalis V583, up to 23% of toxin that neutralizes a toxin encoded by the same plasmid. genes were divergent, mainly found in the exogenously If during replication the plasmid is lost, the antitoxin, acquired accessory genome, and included antibiotic which is unstable, is degraded. This results in toxin- resistance, virulence and integrated plasmid genes mediated killing of plasmid-free daughter cells. If [33]. Comparative genomic hybridization analysis of vancomycin resistance is linked to such a plasmid, this 30 E. faecalis clinical and food strains on 202 E. faecalis mechanism creates a replication advantage for plasmid- genes identified 78 (39%) variable genes, of which containing (i.e. VRE) bacteria. There are different classes 51 were equally detected in clinical and food strains, of postsegregational killing systems, of which four were and 15 enriched in clinical strains, while 10 genes were found ubiquitously and often physically linked to vanA absent in all but V583 strains [34]. elements: mazEF, axe–txe, relBE and v–e–z [42]. Of these systems, axe–txe was previously found in a clinical Both MLST as well as the microarray data of E. faecium and E. faecium isolate on a multidrug-resistance plasmid pRUM E. faecalis clearly demonstrate enormous genome plasticity [45]. The v–e–z system was found in VRE recovered from and diversity, which may facilitate survival and persistence poultry in Norway, 3–8 years after the ban on avoparcin, of these microorganisms in environments that require and appeared to reside on the same plasmid as the vanA continuous adaptation to changing conditions, such as a element [40,43,44,46]. hospital. Virulence High rates of recombination imply high rates of genetic Research on Enterococcus virulence or virulence determi- exchange – a phenomena well known in enterococci. In nants has concentrated on adhesion/biofilm formation and both E. faecalis and E. faecium, numerous publications regulation of expression. Adhesion to extracellular matrix describe plasmid-based transfer of antibiotic resistance proteins and biofilm formation on indwelling medical genes, of which some recent examples will be highlighted devices, like catheters and stents, are considered important below. Whether recombination of chromosomal DNA properties of enterococci that contribute to their patho- fragments that include the MLST housekeeping genes genic behavior [47,48]. It becomes more and more is mediated by these mobile genetic elements or includes apparent that adhesion and biofilm formation is a complex additional mechanisms like transduction and natural process involving multiple bacterial factors, either acting transformation remains to be investigated. in concert or solitary, depending on the environmental conditions and type of biofilm assay. This hampers the Horizontal gene transfer and maintenance of interpretation of studies focusing on the role of particular glycopeptide resistance individual determinants in biofilm formation. One of Clonal expansion of glycopeptide-resistant HiRECC is the disputed virulence determinants is the enterococcal not the only way that vancomycin resistance is spread. surface protein Esp encoded by the esp gene found on a This became apparent in the 1980s when vancomycin- pathogenicity island in both E. faecalis and E. faecium resistance genes were found to be located on plasmids [23,49]. No correlation was found between the presence [35]. Also, recent literature documents transmission of or absence of the esp gene in clinical isolates and biofilm vancomycin resistance through clonal spread as well as formation in some studies [50–53], while Esp-mediated HGT [36–38]. HGT of vancomycin resistance most biofilm formation was demonstrated by others [54,55,56]. likely occurs in the gastrointestinal tract, which implies In addition to Esp, other enterococcal loci have been cocolonization, at least transiently, of a VRE donor and a implicated in biofilm formation. Extended work on the vancomycin-susceptible acceptor strain. In-vivo transfer previously identified bop locus in E. faecalis involved in of vanA and vanB genes has been documented in the biofilm formation linked a biofilm-positive phenotype intestine of mice [39,40] and in the intestines of human with levels of gut colonization in a mouse model [57]. volunteers [41]. It was even demonstrated that in-vivo Yet another novel transferable locus in E. faecalis, desig- vancomycin-resistance genes were transferred at higher nated bee, which conferred a high biofilm phenotype, was rates than in vitro, supporting the notion that in-vitro found on a large conjugative plasmid [58]. Alanine esters models may underestimate transfer efficiencies [40]. of enterococcal lipoteichoic acid and two sagA-like genes, salA and salB,ofE. faecalis, also appeared to be involved in Despite the fact that after the European-wide ban on the biofilm formation [59,60]. Both studies also confirmed use of avoparcin in animal husbandry in 1997 prevalence that cell surface charge and hydrophobicity are two import- rates of VRE significantly decreased, VRE are able to ant parameters that influence biofilm formation. In fact,

van Merode et al. [61] claimed that heterogeneity in cell E. faecalis [30,70,71]. HGT of an approximately 27-kbp surface charge enhances initial adherence to abiotic sur- internal fragment of the E. faecalis pathogenicity island, faces, irrespective of the presence of other strain-specific including the cytolysin gene cluster and the esp gene, traits like esp or GelE. The discovery of pleomorphic could be transferred at high frequencies during transient surface pili encoded by the ebp locus that are antigenic colonization of the mouse gastrointestinal tract [70]. Due in humans during endocarditis and involved in biofilm to HGT this pathogenicity island is not confined to a single production of E. faecalis was exciting [62]. Insertional clonal complex in E. faecalis, but is found in enterococci inactivation of acm in a clinical E. faecium isolate using a belonging to different genetic lineages in animals and novel temperature-sensitive vector completely abolished humans [30,71]. adherence to collagen I and IV [63]. This is the first report in which a putative virulence gene is inactivated in a Resistance to new antibiotics clinically relevant E. faecium strain through allelic In recent years three new antibiotics have been introduced exchange. Until now, studies on the role of particular for the treatment of patients with GRE: the glycylcycline virulence genes in clinical E. faecium isolates have been tigecycline, the lipopeptide daptomycin and the oxazoli- hampered by the lack of proper molecular tools to con- dinone linezolid. Both daptomycin and linezolid have struct insertional deletion mutants. This improved vector been used, after Food and Drug Administration approval will be helpful in the successful construction of other in September 2003 and March 2000, respectively. Resist- isogenic mutants in E. faecium. ance to linezolid was documented shortly after its introduction. Rapid emergence of linezolid resistance was In addition to the fact that biofilm formation is a multi- already observed after 12 days of linezolid therapy [72] factorial process, variation in gene expression may com- and nosocomial spread of linezolid-resistant GREF was plicate linkage of particular genes to this process. We have described in a tertiary care academic medical center in the shown that Esp expression in E. faecium is variable [56]. US [36]. Daptomycin has low potential for the develop- Some esp gene-positive strains hardly produce Esp and ment of resistance in vitro. So far resistance has been biofilm, while other strains have high levels of Esp expres- reported in five patients (four E. faecium and one E. faecalis) sion and biofilm formation [56]. Esp expression in that suffered enterococcal bacteremia [73]. Clinical experi- E. faecium was regulated by growth conditions, with ence with tigecycline, approved for use in the US since elevated expression at 37 compared to 218C, and when June 2005, is still limited. Two recent studies report on the grown under anaerobic conditions compared to aerobic potent in-vitro activity of tigecycline against E. faecalis conditions [56]. Growth-condition- and growth-phase- and E. faecium including 60 clinical GREF isolates with dependent expression of virulence genes was also shown minimum inhibitory concentrations required to inhibit in E. faecalis [64], and transcription of the E. faecalis ace the growth of 50% of organisms of 0.047–0.06 mg/l and gene was upregulated after growth in the presence of minimum inhibitory concentrations required to inhibit the collagen type I and type IV [65]. The exact regulatory growth of 90% of organisms of 0.094–0.12 mg/l [74,75]. pathways that control these effects are not known. It is possible that the fsr locus is involved. It was already known Conclusion that the fsr locus controlled expression of gelatinase and Boosted by the rapid emergence of VRE in the US in the protease, but last year this locus appeared as an important 1990s and the increase in Europe after the turn of the global regulator controlling expression of numerous genes century, several novel findings and new insights mark [66]. In addition, croRS, a regulator implicated in the enterococcal research over the last 2 years. Epidemiologi- environmental stress response [67], or the prfA-like cal and population biological studies showed the existence regulator [68] might be involved in growth condition of distinct enterococcal subpopulations, like E. faecium regulation of virulence gene expression. CC17 and putative E. faecalis HiRECC, specifically adapted to the hospital environment, representing clear The Esp enterococcal virulence determinant has not only examples of adaptive diversification splitting enterococcal been shown to be involved in adhesion and biofilm lineages into derived groups due to frequency-dependent formation to abiotic surfaces, but also in increased conju- ecological interactions. Enterococcal genomics provided a gation frequencies [69]. This suggests that either Esp plays more detailed insight in the genetic diversity and genome a direct role in cell–cell interaction or that Esp may serve as plasticity of E. faecalis and E. faecium. These studies also a marker for strains with enhanced potential to acquire new revealed the enormous difference in gene contents genetic elements. between CC17 isolates and other E. faecium isolates, which might argue in favor of calling CC17 a distinct E. faecium Not only resistance genes (see above), but also virulence subspecies. Regarding enterococcal pathogenesis, it genes may disseminate through HGT. In the last 2 years becomes more and more apparent that adhesion to extra- three publications have reported on the horizontal transfer cellular matrix proteins and biofilm formation are import- of the pathogenicity island on which the esp gene resides in ant features. New molecular tools became available to

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Lett Appl Microbiol resistance is still considered the most significant enter- 2006; 43:274–279. ococcal resistance trait, also because of its transmissibility 17 Mascini EM, Troelstra A, Beitsma M, et al. Genotyping and preemptive isolation to control an outbreak of vancomycin-resistant Enterococcus faecium. Clin to S. aureus. Exciting new knowledge on linkage of Infect Dis 2006; 42:739–746. This study shows how a combination of molecular typing to discriminate E. faecium vancomycin-resistance to plasmid maintenance systems CC17 from non-CC17 in combination with preemptive isolation controlled a predicts that reduction in human vancomycin use may hospital outbreak with VRE. not in parallel result in reduced numbers of VRE – a 18 Peta M, Carretto E, Barbarini D, et al. Outbreak of vancomycin-resistant Enterococcus spp. in an Italian general intensive care unit. 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67 Muller C, Le Breton Y, Morin T, et al. The response regulator CroR modulates 71 Shankar N, Baghdayan AS, Willems R, et al. Presence of pathogenicity island expression of the secreted stress-induced SalB protein in Enterococcus genes in Enterococcus faecalis isolates from pigs in Denmark. J Clin Microbiol faecalis. J Bacteriol 2006; 188:2636–2645. 2006; 44:4200–4203. 72 Seedat J, Zick G, Klare I, et al. Rapid emergence of resistance to linezolid 68 Giard JC, Riboulet E, Verneuil N, et al. Characterization of Ers, a PrfA-like during linezolid therapy of an Enterococcus faecium infection. Antimicrob regulator of Enterococcus faecalis. FEMS Immunol Med Microbiol 2006; Agents Chemother 2006; 50:4217–4219. 46:410–418. This study describes that resistance to linezolid, one of the few novel and promising antibiotics, in E. faecium can occur rapidly during therapy. 69 Lund B, Billstrom H, Edlund C. Increased conjugation frequencies in clinical 73 Wiedemann B. Test results: characterising the antimicrobial activity of dap- Enterococcus faecium strains harbouring the enterococcal surface protein tomycin. Clin Microbiol Infect 2006; 12 (Suppl 8):9–14. gene esp. Clin Microbiol Infect 2006; 12:588–591. 74 Abb J. In vitro activities of tigecycline, daptomycin, linezolid and quinupristin/ 70 Coburn PS, Baghdayan AS, Dolan GT, Shankar N. Horizontal transfer of dalfopristin against glycopeptide-resistant Enterococcus faecium. Int J virulence genes encoded on the Enterococcus faecalis pathogenicity island. Antimicrob Agents 2007; 29:358–360. Mol Microbiol 2007; 63:530–544. 75 Waites KB, Duffy LB, Dowzicky MJ. Antimicrobial susceptibility among This study describes HGT of the E. faecalis pathogenicity island or parts of it pathogens collected from hospitalized patients in the United States and in encoding virulence genes at high frequency during transient colonization of the vitro activity of tigecycline, a new glycylcycline antimicrobial. Antimicrob intestinal tract of mice. This has important implications not only for genome evolution Agents Chemother 2006; 50:3479–3484. and diversity, but also for transmission of virulence determinants that could render A US nation-wide overview of the activity of tigecycline against different pathogens commensal E. faecalis strains into pathogenic strains. including enterococci.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Measuring the impact of multidrug resistance in nosocomial infection Stijn Blota,b, Pieter Depuydta, Koenraad Vandewoudea,b and Dirk De Bacquerc

Purpose of review Introduction The review examines potential confounders hampering Nosocomial infection is the most frequent complication measurement of the impact of multidrug resistance in associated with hospitalization and has adverse clinical and nosocomial infections. Methodological techniques dealing economic outcomes [1]. Multidrug resistance (MDR) in with the problem of confounding are discussed and current pathogens causing nosocomial infection is ever-increasing findings in how multidrug resistance affects outcome in and is now recognized as a major health problem. From the patients with nosocomial infection are highlighted. very beginning of the history of antimicrobial chemother- Recent findings apy, microbial pathogens have acquired various resistance Outcome comparisons between patients infected with mechanisms to counter the effect of antibiotics, keeping multidrug-resistant pathogens and patients infected with pace with the development of new classes of antimicrobial susceptible microorganisms are hampered by confounders drugs. MDR has been recognized to contribute to such as differences in disease severity, prolonged unfavourable clinical outcome and increased resource hospitalization prior to onset of infection (exposure time), the utilization. Apart from increased risk of dying in the causative pathogen, the type of infection, and the rate of individual patient affected by MDR infection, the weight appropriate empirical antimicrobial therapy. The confounding of resistance is cumbersome as it boosts the health- effect can be countered by means of either multivariable economic burden of infection through prolonged hospital regression techniques or matched cohort studies, or a stay, the prescription of newer, broad-spectrum and more combination of both. Recent literature on the impact of expensive antimicrobial drugs and the urgency to take multidrug resistance (methicillin-resistant Staphylococcus costly infection control measures [2–5]. Accurately esti- aureus, vancomycin-resistant enterococci, extended- mating the impact of MDR is difficult due to the presence spectrum b-lactamase producing Enterobacteriaceae, etc) is of multiple patient characteristics that may confound conflicting and highly dependable on the way disturbing uniform analysis. The purpose of this review is to sum- variables are accounted for. marize the methodological problems encountered when Summary measuring this impact of MDR and to discuss to what Recent data underscore that the impact of multidrug extent it affects patients’ outcome. resistance on the outcome of nosocomial infection might differ depending on the study population, type of infection, type of pathogen and appropriateness of therapy, and hence, The problem of confounding that any conclusion drawn prior to accurate accounting for Measurements of the impact of MDR on patients’ and imbalanced confounders is premature and potentially false. health-economical outcome parameters are essentially derived from observational, epidemiological studies; due Keywords to obvious ethical constraints, experimental designs such antibiotic resistance, infection, mortality, nosocomial, as randomized controlled trials cannot be conducted to outcome investigate this question in human subjects. The main methodological issue in these epidemiological studies is to

Curr Opin Infect Dis 20:391–396. ß 2007 Lippincott Williams & Wilkins. provide adequate correction for confounding. Confound- ing refers to the presence of factors that are associated with aIntensive Care Department, Ghent University Hospital, bUniversity College Ghent, the acquisition of nosocomial infection with MDR strains, Department of Healthcare and cDepartment of Public Health, Ghent University, Ghent, Belgium that are independent predictors of outcome, and that are not instrumental in the biological pathway leading from Correspondence to S. Blot, PhD, Ghent University Hospital, Intensive Care Department, De Pintelaan 185, 9000 Ghent, Belgium exposure (infection) to outcome [6]. The impact of MDR Tel: +32 9 240 62 16; fax: +32 9 240 49 95; e-mail: [email protected] should preferably be measured in a design to compare Current Opinion in Infectious Diseases 2007, 20:391–396 patients infected with MDR pathogens and patients infected by antimicrobial-susceptible microorganisms. Abbreviations Such an approach does not, however, eliminate confound- MDR multi-drug resistance or multidrug resistant MRSA methicillin-resistant Staphylococcus aureus ing. Several factors interfering with a correct outcome comparison have been identiﬁed (Table 1). First, patients ß 2007 Lippincott Williams & Wilkins infected with MDR pathogens have more detrimental 0951-7375 underlying conditions and are usually sicker than patients

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Table 1 Major confounders in studies aiming to assess the impact of multidrug resistance Confounder Comment Disease severity Patients infected with multidrug-resistant pathogens generally have more comorbidities (chronic renal insufficiency, chronic obstructive pulmonary disease, chronic heart failure, diabetes etc) and are more prone to develop acute organ failure necessitating intensive care unit admission. Exposure time Patients infected with multidrug-resistant pathogens generally experienced a hospitalization prior to the onset of infection. This implies imbalance in the risk for infection and suggests a more complicated hospitalization course. Causative microorganism Different microorganisms have different virulence attributes and invasive potential. Additionally, the drugs of choice might differ in efficacy (glycopeptides vs. carbapenems). Type of infection Stratification for type of infection is desirable because sicker patients (often colonized with multidrug-resistant pathogens) are more likely to develop more severe infections such as pneumonia or intra-abdominal infections. Bloodstream infection is the best choice for assessing the impact of resistance, but controlling for source of infection remains necessary. Antimicrobial Antimicrobials used to treat multidrug-resistant pathogens might be less effective, in terms of tissue and bacterial cell-wall penetration, than those used to treat susceptible microorganisms (e.g. glycopeptides vs. penicillinase-resistant penicillins). Inappropriate therapy Inappropriate empirical antimicrobial therapy is more frequently observed among patients infected with multidrug-resistant pathogens. Adjustment for appropriateness of therapy is necessary given its adverse impact on the outcome of severe infection.

with infections caused by antimicrobial-susceptible micro- Apart from confounding, MDR in nosocomial infection organisms. In other words, the a priori probability of death may have an unfavourable effect through different mech- in patients affected by MDR pathogens is likely to be anisms, whether or not related to treatment effects. Argu- higher. Hence, accounting for disease severity and acute ably the most important of these mechanisms is an illness will be an essential element in the study design. increased risk for initial inappropriate therapy. Several Secondly, patients infected with MDR microorganisms studies have found important detrimental effects of generally experience a longer hospitalization prior to the delayed appropriate antimicrobial therapy on survival in infection. Prolonged hospitalization might reflect severe nosocomial infection. Furthermore, MDR aetiology unfavourable aspects that influence both the risk of being has been identified as probably the most important infected by MDR pathogens, and the risk of death. independent risk factor for inappropriate empirical anti- Although it is still a matter of some discussion whether biotic therapy. Apart from this, and as mentioned above, or not the length of stay prior to infection adversely affects suboptimal pharmacodynamic and pharmacokinetic patients’ outcome [7,8,9], accounting for exposure time characteristics of antibiotics used to treat MDR may con- (as number of days at risk of infection) is generally recom- tribute to decreased infection clearance and unfavourable mended when addressing the issue of impact [10,11]. outcome, such as glycopeptides in MRSA infection [14]. As several strategies have been described to increase appro- Another potential confounder is the causative pathogen priateness of antimicrobial therapy in MDR nosocomial under study. Different types of microorganisms probably infection, such as de-escalation and surveillance-guided have different virulence factors and pathogenic potential. therapy [15,16,17,18], it is increasingly desirable to deter- The impact of methicillin resistance in Staphylococcus mine how much of the added cost of MDR infection is aureus may differ from that observed in coagulase-negative ‘preventable’ by optimal antibiotic therapy. As such, it staphylococci. In addition, the potential of the antimicro- could be argued that measuring the impact of MDR should bial armamentarium left to treat MDR pathogens, in terms be done accounting for the effect of therapy, although this of tissue and microbial cell-wall penetration, might vary is not a confounder in a strict sense. Finally, it should be considerably (e.g. glycopeptides vs. carbapenems). It mentioned that many of these potential confounders are therefore appears methodologically most correct to assess clustered, thereby providing an additional problem of the impact of MDR stratified for type of pathogen collinearity (see below). (S. aureus, enterococci, Enterobacteriaceae, etc). Measuring the impact of multidrug resistance: In addition, the type of infection should be equal or addressing confounding balanced between groups under comparison. High-risk Observational studies may be prospective or retrospec- sources such as intra-abdominal infections or pneumonia tive and may compare patients infected with MDR are associated with a particularly grim prognosis [5,12,13]. strains with controls infected with the corresponding Microbiologically documented bacteraemia represents non-MDR strain. To separate the effect of MDR from the most optimal in vivo model to investigate the patho- that of potential confounders on the outcome parameter genic significance of resistance. Even then, however, the under study, one has to rely on specific study designs or primary source of infection should be taken into account. statistical techniques; the most widely used are matching

procedures and multivariable logistic regression analysis. confoundingvariables toa single covariatethat summarizes These techniques can be used separately or can be all of the relevant information about the confounders combined within the same study. (the propensity score) [22,23]. In the context of MDR infection, the propensity score is the probability of devel- Unmatched cohort studies using multivariable oping the event of nosocomial MDR infection for a patient regression analysis conditional on the patient’s observed pre-event covariates. In these studies, a dataset of nosocomial infections is To estimate the propensity score, a first model is fitted to analysed in totality as they contain a prospectively col- predicttheeventofdevelopingnosocomialMDR infection lected, consecutive series of patients [19]. By including all incorporating a number of patient characteristics as pre- cases, selection bias will be limited, but on the other hand, dictors or covariates, such as comorbidity and exposure case-mix may profoundly confound the results. As men- time. The summary of a subject’s covariates in this model is tioned earlier, these cohorts are frequently ill-balanced for the expected probability of developing the event of noso- disease severity and exposure time. Advanced statistical comial MDR infection or the propensity score. Thereafter, techniques based on multivariable modelling (logistic this propensity score can be introduced as a covariate in a regression analysis, Cox regression analysis) are needed second multivariable regression analysis evaluating the to correct for this imbalance. Such analysis allows for association between the event of nosocomial MDR infec- determining whether there exists an independent associ- tion and outcome. ation between MDR and a binary outcome parameter such as mortality. This association may be quantified by means Matched cohort studies of an adjusted odds ratio. To quantify attributable cost, In a matched cohort study, outcome is compared between such as length of stay or other healthcare-related costs, this a group of subjects with MDR nosocomial infection and a kind of analysis is less useful. group without, but with a similar distribution of patient characteristics influencing risk for infection and outcome Ideally, all potential confounders should be entered in the [24,25]. As potential confounders are equally present and multivariate model as predictors or covariates, but due to distributed in both groups, it is assumed that the effect of statistical limitations, this goal can seldom be reached. As confounding will be minimized. The quality of a matched not all potential risk factors can be included, as in the cohort analysis will depend on the quality of the matching analysis, covariates have to be carefully selected on the procedure, allowing for comparison between cases and basis of clinical experience or a review of prior literature. controls that have similar a priori outcome expectancies. Moreover, to avoid collinearity, one has to carefully avoid As with cohort studies, propensity scores may be used to including sets of predictors that have strong associations match patient groups. As far as we know, however, this between themselves, as this can provide largely overlap- design has not yet been used to measure the impact of ping information, which multivariable analysis cannot MDR in nosocomial infection. As it is important to match resolve. Collinearity has to be expected when regression as closely as possible, some cases may be lost for the models are unstable, and when confidence intervals of analysis, leading to reduced sample size (and power to adjusted odds ratios are excessively wide. In addition, detect differences), or to the introduction of possible associations may be unreliable if the model includes too selection bias. many independent variables and too few outcome events. Although controversial, analysis with fewer than ten out- Case–control studies come events per explanatory variable may lead to unstable In case–control studies, patients are grouped by outcome andincorrectresults[20].Ontheotherhand,bylimitingthe (survivors vs. nonsurvivors) after which the prevalence of number of predictors, the analysis may be biased by uncon- an infection caused by MDR pathogens is compared retro- trolled confounding. Due to these statistical limitations, spectively between both groups. Many methodological when differences in severity of underlying diseases and issues may influence the validity of the results. In particu- acute illness are multiple and immense, regression models lar, the selection of controls can seriously affect the study’s may failtoadjust adequately. Insuch circumstances,cohort vulnerabilitytobias[26].Additionally,case–controlstudies studiestendtomagnifytherelationshipbetweenresistance are inefficient when the incidence of outcome (death) is and adverse outcome [21]. Often, information about the higherthantheprevalenceofexposure(bacteraemiacaused patient’s characteristics and disease severity is summarized by MDR pathogen). As this is generally the case in high- using severity scores, such as the Charlson’s index for risk groups used for this study purpose (e.g. critically-ill comorbidity or the APACHE II-score. As an alternative patients), this design cannot be recommended and tool to control for confounding, the propensity score has (matched) cohort studies are to be preferred. been proposed. Propensity score analysis represents a versatile method of controlling for confounding, especially Nosocomial multidrug-resistant pathogens when the number of potential confounders is relatively Some examples of nosocomial multidrug-resistant patho- large. The basic premise is to reduce the large number of gens are given below.

Methicillin-resistant Staphylococcus aureus (MRSA) source of confounding. Studies that successfully included Methicillin resistance in S. aureus remains the biggest accurate adjustment for appropriate therapy generally concern in nosocomial infections [27]. In a hospital-wide showed no significant excess mortality [12,38,39]. study comparing 96 patients with MRSA bacteraemia Additionally, in infections caused by MDR Pseudomonas with 252 patients with bacteraemia caused by methicil- aeruginosa, no excess mortality is found after controlling for lin-susceptible S. aureus (MSSA), published in 2006, inappropriate therapy [40,41]. The impact of MDR in methicillin resistance was associated with a significant Acinetobacter baumannii infections is hard to assess due to its excess in length of hospitalization and hospital charges intrinsic resistance to multiple antimicrobials, and hence, [28]. Hospital mortality, however, was not different (23% the lack of a control group infected with susceptible vs. 20%), which is in contrast with a previous meta-analysis isolates. In a matched cohort study comparing patients by the same investigators, and an earlier matched cohort with A. baumannii bacteraemia with uninfected controls, study by another research group conducted in a subset of no significant attributable mortality could be demon- critically-ill patients [5,29]. In another recent study, Das strated [42]. In this specific cohort, the rate of appropriate et al. [30] found significantly higher attributable mortality therapy was high (85%). This study indirectly suggests rates in patients with MRSA bacteraemia, but no inde- that the clinical impact of MDR in A. baumannii infections pendent association with mortality was found after adjust- is of minor importance if adequate therapy is initiated ment for age, respiratory source of the bacteraemia, and sufficiently early [43]. inappropriate therapy. An increased economic cost associated with methicillin resistance has been observed in Candida nonalbicans spp. patients with nosocomial pneumonia as well as in patients Regarding Candida infections, antifungal resistance usu- with bacteraemia [31], and similarly, an adverse impact ally is defined as resistance to fluconazole [44]. Flucona- in terms of survival has remained a point of discussion zole resistance, however, remains rare [45], so most [16,32,33]. Zahar et al. [32] found higher crude mortality epidemiological studies have focused on the clinical rates among patients with MRSA ventilator-associated impact of Candida nonalbicans species. Some of these pneumonia (59% vs. 40%; P ¼ 0.024), but this difference species are likely to be dose-dependent-susceptible disappeared after controlling for time in the intensive care (C. glabrata) or alternatively have intrinsic resistance to unit before ventilator-associated pneumonia and imbal- fluconazole (C. krusei). Prior exposure to fluconazole in anced parameters at time of admission. Strangely enough, patients who develop Candida infection is a risk factor for adjustment for appropriateness of therapy did not alter the Candida nonalbicans involvement [46,47,48]. Again, impact of methicillin resistance. In contrast to this, other such an observation implies that patients at risk for investigators demonstrated a pivotal role of appropriate Candida nonalbicans represent a more severely-ill popu- therapy in MRSA infections [34]. lation. Accordingly, after adjustment for confounding covariates, no excess mortality could be demonstrated Antibiotic resistance in enterococci among patients infected by nonalbicans spp. [49,50]. Enterococci are increasingly important nosocomial pathogens. Their resistance patterns show a strong geographic Conclusion variation. Resistance to ampicillin and gentamycin is Recent data underscore that the impact of MDR might associated with worse outcomes, but again, the link with differ depending on the study population, type of infection substantial delays in appropriate therapy is obvious. This and appropriateness of therapy, and hence, that any con- also seems to be the key issue in studies regarding vanco- clusion drawn prior to accurate accounting for imbalanced mycin-resistant enterococci. The relationship between confounders is premature and potentially deceptive. vancomycin resistance and high mortality is strong [35]. Adjustment for appropriate therapy is very difficult, how- References and recommended reading Papers of particular interest, published within the annual period of review, have ever, as the imbalance is extreme: inappropriate empirical been highlighted as: treatment in cases of vancomycin-susceptible enterococci of special interest is rare, whereas dramatically high rates (even 100%) have of outstanding interest Additional references related to this topic can also be found in the Current been described in series caused by vancomycin-resistant World Literature section in this issue (pp. 436–437). strains [36,37]. 1 Cosgrove SE, Carmeli Y. 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Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Improving the tools in the ﬁght against cytomegalovirus or strengthening David to defeat Goliath Jose´ G. Montoya

Division of Infectious Diseases and Geographic Medicine, Department of Medicine, reported to correlate with long-term graft dysfunction in Stanford University School of Medicine, Stanford, California, USA heart transplant recipients [4]. Correspondence to J.G. Montoya, Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Room S-169, M/C 5107, Stanford, CA 94305, USA CMV is not only the most common viral infection cur- Tel: +1 650 853 4824; fax: +1 650 329 9853; e-mail: [email protected] rently affecting immunocompromised patients, but also Current Opinion in Infectious Diseases 2007, 20:397–398 carries the highest burden of morbidity and mortality of all opportunistic agents (see the paper by Bob Rubin Abbreviation in this issue of Current Opinion in Infectious Diseases CMV cytomegalovirus pp. 399–407). The proportion of specific CMV syndromes may significantly vary among immunocompro- ß 2007 Lippincott Williams & Wilkins mised patients; retinitis is by far the most common 0951-7375 syndrome observed in AIDS patients, but it is extremely rare in transplanted patients [5]. The allograft appears to Of the herpes viruses capable of causing disease in be the most common site of infection for patients with humans, cytomegalovirus (CMV) is the largest solid organ transplants and disseminated disease for those (230 kbp) in the family (Table 1). The role of each gene with hematopoietic cell transplants. Gastrointestinal dis- continues to be elucidated by several groups of investi- ease seems to be unusually high for all groups. A clear gators [1]. In the herpes family, CMV also has the understanding of the tropism of CMV for different organs unwelcome distinction of being responsible for the and its capacity to express a different profile of disease widest spectrum of disease in immunocompromised according to the host is crucial in our attempts to decrease patients (Table 1). Of note, it is clear that the role in its pathological burden in immunocompromised patients. human disease of other members of the herpesviridae family has been underrecognized and the full etiologic Despite all the tools available for CMV to invade and potential for all herpes viruses is yet to be established [2]. produce disease in humans, significant progress continues to be made in our understanding, diagnostic capabilities CMV is ubiquitous and easily transmissible through respir- and treatment options in the prevention and treatment of atory secretions and other body fluids. Excretion rates in CMV infection and disease. child-care centers can be as high as 70% in children 1–3 years of age. The age-adjusted CMV seroprevalence for In this issue of Current Opinion in Infectious Diseases, individuals 6 years old and older in the US has been the papers by Rubin pp. 399–407 and by Potena and reported to be 58.9% [3]. As for all the members of the Valantine pp. 425–431 elegantly emphasize the indirect herpesviridae family, once CMV infects a host, it estab- and long-term effects of CMV subclinical reactivation lishes life-long latency. It is important to distinguish and disease. According to Potena and Valantine pp. 425– between CMV infection (virus is dormant in a latent state 431, it appears that significant loss of graft function and and not causing disease), CMV disease (virus is actively viability can occur in patients with detectable CMV DNA replicating and being responsible for symptoms and signs) in polymorphonuclear leukocytes even though replica- and CMV subclinical viremia (virus is detected in serum, tion of CMV is not demonstrated by sensitive assays such whole blood or peripheral blood polymorphonuclear as polymerase chain reaction performed in serum and in leukocytes, but it is not causing apparent symptoms or the absence of recognizable CMV disease. This new signs). The presence of CMV-specific IgG antibodies in information may have major implications for the ongoing the absence of CMV IgM specific antibodies best estab- controversy regarding the selection of the best pro- lishes latency. The presence of disease can be best deter- phylactic strategy to decrease CMV-associated morbidity mined in symptomatic patients by the demonstration of and mortality. As per the thorough review by Puius and viral DNA (polymerase chain reaction, in-situ hybridiz- Snydman in this issue (pp. 419–424), only a randomized ation), viral particles or antigens (pp65, immunohisto- clinical trial would be able to establish whether the chemistry, shell vial assay) or the whole infectious virion benefits of universal prophylaxis are different from those (culture) in body fluids and/or tissues. Subclinical viremia, of a preemptive intervention. Should the findings of the established by the presence of CMV DNA in peripheral Stanford investigators be confirmed [4] by other groups blood polymorphonuclear leukocytes, has been recently and in other solid organ transplant settings, however, the

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Table 1 Nomenclature, classiﬁcation, molecular weight for and diseases caused by members of the human herpesviridae (HHV) family Serial Arabic Genome Examples of diseases caused by herpes viruses HHV subfamilies number designation length (kb) in immunocompromised patients

a Human herpes simplex-1 HHV-1 152 Disseminated disease, encephalitis, pneumonia, retinitis Human herpes simplex-2 HHV-2 152 Disseminated disease, pneumonia, encephalitis Varicella zoster virus (VZV) HHV-3 125 Disseminated disease, pneumonia, encephalitis, retinitis b Cytomegalovirus HHV-5 230 Disseminated disease, retinitis, pneumonia encephalitis, gastrointestinal disease, liver and biliary disease Human herpesvirus-6 HHV-6 160 Disseminated, encephalitis Human herpesvirus-7 HHV-7 145 Disseminated disease? g Epstein–Barr virus HHV-4 172 Posttransplant lymphoproliferative disease, retinitis Kaposi’s sarcoma-associated herpesvirus HHV-8 165 Kaposi’s sarcoma, Castleman’s disease

method used to trigger an intervention in the preemptive to clinicians. We are certainly far from defeating this giant approach may have to be reevaluated. Further diagnostic virus belonging to the b herpes family, but we are now tools with the same potential and with therapeutic one step closer towards that goal. implications are assays aimed at the detection of CMV- specific T lymphocytes. Drew (pp. 408–411) presents an excellent summary of the progress made in the diagnostic References front in our fight against CMV. 1 AuCoin DP, Smith GB, Meiering CD, Mocarski ES. Betaherpesvirus- conserved cytomegalovirus tegument protein ppUL32 (pp150) controls The therapeutic armamentarium against CMV needs to cytoplasmic events during virion maturation. J Virol 2006; 80:8199– be further expanded. The drug of choice for prophylaxis 8210. 2 and treatment of CMV disease is still intravenous or oral Krueger G, Ablashi D. Human herpesvirus-6. 2nd ed. Oxford: Elsevier; 2006. 3 Staras SA, Dollard SC, Radford KW, et al. Seroprevalence of cytomegalovirus ganciclovir or valganciclovir, but bone marrow toxicity infection in the United States, 1988–1994. Clin Infect Dis 2006; 43:1143– may be a limiting factor for the administration (see the 1151. paper by Bob Rubin in this issue pp. 399–407) of the drug. 4 Potena L, Holweg CT, Vana ML, et al. Frequent occult infection with cytomegalovirus despite antiviral prophylaxis in cardiac transplant recipients. Intravenous foscarnet or cidofovir can be used alternatively J Clin Microbiol 2007 [Epub ahead of print.] when ganciclovir results in toxicity, intolerance or viral 5 Montoya JG, Giraldo LF, Efron B, et al. Infectious complications among 620 resistance, but renal toxicity is one of the major barriers consecutive heart transplant patients at Stanford University Medical Center. Clin Infect Dis 2001; 33:629–640. to their use. The combination of ganciclovir plus foscarnet 6 Mylonakis E, Kallas WM, Fishman JA. Combination antiviral therapy for [6] or leflunomide [7] may be useful in the setting of ganciclovir-resistant cytomegalovirus infection in solid-organ transplant reci- ganciclovir-resistant CMV disease. Maribavir, a member pients. Clin Infect Dis 2002; 34:1337–1341. of the benzimidazole chemical class, has in-vitro greater 7 John GT, Manivannan J, Chandy S, et al. A prospective evaluation of leflunomide therapy for cytomegalovirus disease in renal transplant recipients. efficacy than ganciclovir against CMV, including CMV Transplant Proc 2005; 37:4303–4305. strains resistant to ganciclovir [8]. Maribavir has been 8 Drew WL, Miner RC, Marousek GI, Chou S. Maribavir sensitivity of cytome- reported to reduce the incidence of CMV infection com- galovirus isolates resistant to ganciclovir, cidofovir or foscarnet. J Clin Virol 2006; 37:124–127. pared with placebo in allogeneic stem cell transplants [9]. 9 Winston D, Van Burik J, Pullarkat et al. Prophylaxis against Cytomegalovirus infections with oral Maribavir in allogenic stem cell transplant recipient: results of a randomized, double-blind, placebo-controlled trial. 48th Newer diagnostic and therapeutic tools in the fight American Society of Hematology Annual Meeting, Orlando, FL, USA, against CMV disease have been recently made available 2006: 593.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. The pathogenesis and clinical management of cytomegalovirus infection in the organ transplant recipient: the end of the ‘silo hypothesis’ Robert H. Rubin

Purpose of review Introduction Cytomegalovirus infection is initiated when tumor necrosis The interaction of replicating, invasive microorgan- factor binds to the cytomegalovirus receptors of latently isms with the human host is a complex one, involving infected cells, resulting in the reactivation of the virus and numerous cascades and pathways (e.g. sepsis and acute the production of clinical disease of two types: direct respiratory distress syndrome, both the classical and infection causing pneumonia, mononucleosis, colitis and alternative complement cascades, and clotting and clot other viral-related syndromes, and indirect infection in which lysis), the elaboration of toxins (e.g. clostridial and an array of cytokines are released by the host that produce streptococcal toxins) and the mediators of immunologic much the same effect as does the rejection process. injury (both native and specific), and acute and chronic Recent findings inflammation. In the transplant recipient the effects of These effects fall into three categories: allograft injury, an immunosuppressive therapy will modify the clinical increase in superinfection with opportunistic pathogens effects of these processes while increasing the pace of and an increase in the incidence of B cell the illness and the subsequent mortality, while attenu- lymphoproliferative disease. Other factors that modulate the ating the symptom complex produced by the infection. clinical impact of reactivated cytomegalovirus in the An interesting paradox develops in these patients: while transplant patient include the past experience of the host the microbial burden, a major indicator of the gravity of with the virus (primary infection, donor seropositive and a particular illness, is significantly increased, the clinical recipient seronegative), the degree of major course is greatly modified – a minimum of symptoms for histocompatibility complex mismatch, the viral burden and a significant period of time, followed by a rapid deteriora- the amount of calcineurin inhibitor the patient receives. tion and a high mortality rate [1]. Summary Optimal therapy for diagnosing, treating and preventing The range of disease observed, the possibility of person- these indirect effects still remains to be defined; the direct to-person spread and the requirement for extended effects, in contrast, are well managed with valganciclovir. therapy are directly related to this paradox. For virtually all infections occurring in the transplant patient, the key Keywords to survival is early recognition and aggressive therapy. As allograft, cytokine, cytomegalovirus, ganciclovir clinical presentation of infection is often occult, a more ‘invasive’ approach to diagnosis is often required, i.e. Curr Opin Infect Dis 20:399–407. ß 2007 Lippincott Williams & Wilkins. innocuous appearing skin lesions must be biopsied and high-definition chest computed tomography scans, rather Harvard Medical School, Center for Experimental Pharmacology and Therapeutics, than conventional chest X-rays, are essential. There is Harvard-MIT Division of Health Sciences and Technology and Division of Infectious Disease, Brigham and Women’s Hospital, Boston, Massachusetts, USA also a time pressure in approaching these patients, Correspondence to Robert H. Rubin, MD, FACP, FCCP, Division of Infectious whereas in the normal host it may be acceptable to delay Disease, Brigham and Women’s Hospital, 25 Francis Street, Boston, aggressive biopsy procedures while cultures are pending MA 02115, USA Tel: +1 617 525 7868; e-mail: [email protected] and empiric therapy is being tried. In the transplant patient, the aim should be specific diagnosis within Current Opinion in Infectious Diseases 2007, 20:399–407 24 h of presentation. Prevention of infection, not surpris- Abbreviations ingly, becomes an important goal in the management of EBV Epstein–Barr virus transplant patients (Fig. 1) [1]. CMV cytomegalovirus MHC major histocompatibility complex PCR polymerase chain reaction effect Cytomegalovirus (CMV) adds further complexity to this PTLD posttransplant lymphoproliferative disease TNF tumor necrosis factor system:

ß 2007 Lippincott Williams & Wilkins (1) CMV is the single most important infection occurring 0951-7375 in transplant patients, causing a broad range of clinical syndromes and requiring prolonged courses of therapy. Its systemic, multiorgan effects are

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Figure 1 Temporal aspects of cytomegalovirus infection in the hepatitis C virus and CMV have bidirectional effects, organ transplant recipient with CMV increasing the viral burden of hepatitis C virus and vice versa [1,5,6]. (b) Oncogenesis. Certain forms of malignancy are promoted by CMV, with the (a) clearest evidence for this being EBV-related B cell Transplant patient lymphoproliferative disease, whose incidence is Microbial increased 7- to 10-fold by CMV infection [1,5,6]. burden Normal host (c) Allograft injury. CMV appears to promote classical rejection, as well as a vasculopathy of the allograft, both of which appear to play a role in determining the longevity of the allograft [1,3,4,5,6–8,9,10].

The purpose of this chapter will be to define the inci- Time dence, pathogenesis and management of both the indirect and direct effects of CMV in the solid organ transplant (b) patient. Prevention, as well as management of sympto- Normal matic disease, will be emphasized. Symptom severity Immunocompromised host Epidemiology and consequences of cytomegalovirus The unique clinical behavior of CMV is directly related to its properties as a herpes virus, with latency, cell association and oncogenicity being characteristic of this b herpes virus. In this context, the term latency is utilized to Time describe the phenomenon of life-long infection – after acute lytic infection, the virus remains dormant in a variety (a) Microbial burden, a major determinant of the severity of the infection, is significantly increased in the immunocompromised transplant recipi- of tissues, but capable of being reactivated at any time post ent. (b) Owing to the antiinflammatory effects of the antirejection therapy, initial infection. The presence of specific antibody to CMV symptoms of infections are greatly muted and the presentation occult, in the absence of replicating virus is a marker for dormant until the infection is far advanced, with a poor prognosis. CMV infection. The term cell association connotes the requirement for intimate contact between cells to transmit infection to spread within the host to produce systemic directly related to its vasculitic impact and its poten- infection, and to render humoral immunity inefficient. tial for causing chronic and/or recurrent disease. More The key host defense against CMV is mediated by major than 50% of transplant patients, by the end of the first histocompatibility complex (MHC)-restricted, virus- year posttransplant, have, or did have, laboratory and/ specific, cytotoxic T cells – just that portion of host or clinical evidence of CMV replication. defense most affected by present-day immunosuppressive (2) CMV should be regarded not only as an important therapy. The importance of this last is the demonstration pathogen by itself, but also as a model for the con- that the greater the MHC mismatch between donor and sequences of viral infection. Thus, such infections as recipient, the greater the impact of the infection. Acqui- those caused by other herpes group viruses [human sition of CMV infection can be accomplished by intimate herpes virus-6, -7 and -8, and Epstein–Barr virus contact in the community, transfusion or transplantation of (EBV)] and the hepatitis viruses are likely to have latently infected cells from a seropositive donor. Person- similar effects to those produced by CMV [2,3,4,5,6]. to-person transmission is uncommon in most situations, (3) The effects of CMV on the transplant recipient can including adult dialysis and transplant units; nurses and be divided into two general categories: the direct other pediatric personnel can acquire CMV from children causation of clinical infectious disease syndromes with congenital CMV. The difference between children (e.g. fever, mononucleosis, pneumonia, colitis, etc.) and adults in this category of acquisition is related to and the indirect effects of the virus that are due to personal hygiene as well as a higher titer of virus in the mediators produced by the host in response to the urine and secretions. viral invasion. These indirect effects are best considered as being of three types [1,5,6]. (a) Immuno- There are three forms of CMV, as determined by prior modulation. CMV contributes to the net state of and current experience with the virus (Table 1) [1,5,6]: immunosuppression, thus predisposing to opportunistic superinfection with bacterial, fungal and even (1) Primary CMV disease, which occurs when an allo- viral agents. In the case of virus–virus interactions, graft (or a transfusion product) is obtained from a

Table 1 Clinical disease due to cytomegalovirus in organ transplant patient recovery different form of immunosuppression Serologic results Donor Recipient Nature of immunosuppression Incidence of symptomatic disease

þ Conventional Alab-1 50% with either form of immunosuppression þþ Conventional 10–25% þþ Conventional þ Alab-1 30–50% þ Conventional 10% Conventional þ Alab-1 30% Conventional immunosuppression: tacrolimus/prednisone/mycophenolate. Conventional immunosuppression þ Alab, tacrolimus/prednisone/cyco- phenolate; Alab-1, antithymocyte globulin (or OKT3) þ conventional immunosuppression; Alab-2, Anti-interleukin-2 (no cytokine storm).

seropositive donor and is transplanted into a serone- Pathogenesis of cytomegalovirus infection in gative recipient. With no antiviral protocol utilized, organ transplant recipients approximately 60% of these DþR– patients will The key step in the pathogenesis of CMV infection is become clinically ill, usually at around 4 weeks reactivation from latency. This critically important repli- posttransplant. Although clinical disease tends to cative function has three distinct stages, which together be more severe when acquired from an allograft, as take around 24 h: (a) an immediate early phase which opposed to a transfusion, it is deemed appropriate produces proteins that regulate the as-yet unexpressed to utilize ‘pedigreed’ blood products free of trans- genes and which is the critical point in this process, (b) an mission risk, both to prevent the direct manifes- early phase that results in the synthesis of viral DNA tations of the infection and to decrease the risk of polymerase and other proteins, and (c) a late phase in the indirect effects. Although laboratory markers of which the major structural proteins are synthesized, and the risk of transmission with an allograft are not the completed virus is assembled and released [1,3,4]. currently available, an important observation has Initiation of this process is accomplished by the presence been when both kidneys are transplanted from a of tumor necrosis factor (TNF), released in response to an particular cadaveric donor into seronegative recipi- inflammatory process, which binds to the TNF receptor ents – either both recipients become symptomatic or on latently infected cells, which is then responsible for neither do [1,11–14]. a signaling process that involves activation of protein (2) Reactivation disease. This occurs when a seropositive kinase C and nuclear factor-kB. The resulting activated individual reactivates endogenous virus which then p65/p50 nuclear factor-kB heterodimer translocates into has the potential for producing clinical disease. the nucleus and binds to the CMV immediate early Although an incidence of direct manifestations of enhancer region, which initiates viral replication. There 10–30% is often quoted in patients who are CMV are two other pathways that can accomplish the task of seropositive prior to transplant, the nature of the viral reactivation. The first of these pathways is activated immunosuppression required will have a major effect by the stress catecholamines, epinephrine and norepi- on these numbers. Of particular interest is the role of nephrine. This results in increasing concentrations of asymptomatic, reactivation infection in the causation cyclic AMP, which, in turn, stimulates the reactivation of the indirect effects of the virus [1,11,12,15–18]. process. A variation of this theme involves proinflamma- (3) Superinfection. This occurs when an allograft from a tory prostaglandins, which stimulate the cyclic AMP seropositive donor is transplanted into a seropositive mechanism. Through these mechanisms, inflammation, recipient and the virus that is reactivated is of donor infection and stress are responsible for CMV reactivation origin. The evidence that is currently available sup- [1,19–26]. ports the hypothesis that superinfection carries a greater risk for both the direct and indirect effects Any process that results in TNF release will promote the of CMV. There is considerable heterogeneity geneti- reactivation of CMV, including such diverse conditions cally and antigenically among different isolates of as liver failure, significant infection, allograft rejection, human CMV. The transplantation of an allograft from EBV-related B cell lymphoproliferative disease (particu- a seropositive donor into a seropositive recipient larly when associated with fever), amphotericin infusion, (a DþRþ donor and recipient combination) appears allergic reactions and antilymphocyte antibody therapy. to have a less happy outcome than the transplantation It is useful to think of the clinical effects of CMV of an organ with a D–Rþ phenotype. There appears reactivation as having ‘two waves’. A CMV seropositive to be a higher incidence of clinical disease with individual presenting with pneumococcal or Gram- DþRþ transplants, as opposed to D–Rþ pheno- negative sepsis can make a full recovery from their types. Less well defined are the effects of these phe- primary infection and then 3–4 weeks after their notypes on the indirect effects of the virus [1,11,12]. ‘cytokine storm’ (in which TNF plays a prominent role)

replicating CMV can be easily demonstrated. Whether or vascular injury; in the heart allograft intracoronary ultra- not clinical illness results from these events is determined sound is the diagnostic procedure of choice. Rupture of primarily by the patient’s net state of immunosuppres- CMV-infected endothelial cells appears to be a common sion [1,19–26]. event in viremic individuals, with viral debris then pha- gocytosed by ‘professional’ phagocytes, particularly poly- There has been considerable interest in the cause(s) of morphonuclear leukocytes. This series of events is the ‘late CMV’, i.e. the occurrence of CMV infection not 4–6 basis for the pp65 antigenemia assay, in which these weeks posttransplant as expected, but weeks to months debris-laden leukocytes can be identified by immuno- later. The two most common causes of this phenomenon fluorescence and provide a semiquantitative measure- are (a) an incomplete protective effect of an antiviral ment of the viral burden. The ability of CMV to cause prophylactic regimen, which serves to prolong the incu- a viral vasculitis is an important factor in the multiorgan bation period without changing the incidence, and (b) the system dysfunction that can occur [1]. second-wave phenomenon, where TNF release triggers the replicative process of this mediator [1,19–24,27]. The direct effects of CMV are similar in all the forms of organ transplantation, with one notable exception – the The next steps in the pathogenesis of CMV infections organ transplanted is far more affected than a native are amplification and dissemination, which are largely organ. Thus, it is recognized that hepatitis is an important determined by the microbial burden and the net state finding in a liver transplant recipient, but is relatively of immunosuppression. The particular host defense of trivial in the native liver; similarly, the attack rate and importance is the MHC-restricted, virus-specific, cyto- severity of CMV pneumonia are greater in lung and heart/ toxic T cell response and, as will be discussed below, it lung recipients than in other organ transplant recipients, is the integration of this function with the viral load that and CMV myocarditis is a clinical entity seen almost determines prognosis. The nature of the immunosuppres- exclusively in the heart transplant recipient. Similarly, sive program is a major determinant of these factors. pancreatitis (occasionally to an extent that true pancreatic Table 1 summarizes the available data in this area abscesses are formed) is observed in pancreatic trans- [1,28–31]. plant recipients. There are a variety of explanations for these observations: there could be a synergy between the The immunosuppressive program currently in use can be effects of the virus and the effects of rejection; the thought of as being of two types in terms of the effects on greatest viral burden is in the allograft; and, perhaps most CMV. (a) Antithymocyte globulin, cytotoxic drugs like interesting, the allograft can be regarded as a privileged cyclophosphamide and azathioprine, and high-dose site for viral replication because the MHC-restricted, steroids for rejection are powerful agents in terms of virus-specific, cytotoxic T cells have a decreased ability reactivating latent virus. (b) In particular, the calcineurin to eliminate virally infected cells in the presence of MHC inhibitors do not reactivate virus, nor does mycophenolate mismatch between donor and recipient [1,5,6,33]. or moderate doses of steroid. Cyclosporine and tacrolimus do not reactivate virus; however, once replicating virus is For a long time, the diagnosis of tissue infection due to present, these drugs are potent stimuli for amplification CMV caused some difficulty: typically, the microscopic and dissemination of the virus [what we have termed as examination revealed only a few cells that had one of having an in-vivo polymerase chain reaction (PCR) the three classical findings caused by lytic infection: cyto- effect]. There are now a variety of antithymocyte globu- megaly,nuclearand/orcytoplasmicinclusionbodiestypical lins (both monoclonal and polyclonal) that are used in for CMV, and cells that stain positively for the virus on transplantation. Those in whom no fever is induced by immunofluorescent staining. In addition, an intense, acti- their antibody and cause the release of a minimum of vated population of inflammatory cells is present in this TNF (i.e. the interleukin-2 inhibitors) cause rather small tissue that can play a role in the clinical course of the amounts of CMV to be reactivated. Antithymocyte glo- patient. This inflammatory infiltrate in the absence of bulin and the monoclonal antibody OKT3 are T cell overwhelming infection in the past led to the concept that depleting, and their use is associated with a cytokine CMV is a ‘scavenger virus’, invading and extending the storm, followed by an increase in replicating virus [1,32]. pathology that is presently invading and damaging. As this Systemic spread of CMV is highly cell associated, with hypothesis gained credence, an alteration in the pathology lytic virus the most visible cells present. It is likely that of vascular injury was brought to the forefront. Those few the vascular endothelial cell is a prime target for CMV cells with lytic viral infection produced prodigious infection, thus accounting for a number of effects of the amounts of interleukin-1 that, in turn, was responsible virus: diffuse vascular injury, producing a characteristic for the activated inflammatory cell population [1,11,12]. vasculopathy that is responsible for vascular injury developing over months to years. Conventional angiography The earliest stages of CMV clinical disease are similar has proven to be an insensitive method for studying to any systemic viral infection: unexplained fever and

constitutional symptoms, with a general condition con- antigenemia or PCR assay has been a major advance. sistent with mononucleosis (including a 5–10% atypical Unfortunately, extensive disease of the gut is frequently lymphocytosis). Respiratory symptoms are the rule associated with negative viremia assays. The preferred (dry cough, mild tachypnea and mild/moderate dyspnea). approach to diagnosis is by endoscopy and biopsy, with In addition to invasive infection due to this virus, com- the not infrequent need for follow-up endoscopy to plement activation is common, with impairment of provide ‘proof of cure.’ respiratory function the result [1]. The indirect effects of cytomegalovirus on the By far the most common radiologic abnormality is a organ transplant recipient: the infectious bilateral, symmetrical peribronchovascular (‘interstitial’) disease implications and alveolar infiltrate predominantly affecting the lower CMV is an important contributor to the net state of lobes. Uncommonly, this can proceed to a diffuse, multi- immunosuppression, which potentiates superinfection lobar pneumonia with respiratory failure (particularly in with a variety of pathogens. CMV-induced leukopenia lung transplant patients). Focal disease and even nodules is a marker of a significantly increased risk of serious can be caused by CMV. The X-ray picture found in the superinfection. For example, CMV infection is associated great majority of these patients is, however, one that with an increased incidence of pulmonary infection with closely resembles that produced by Pneumocystis. Dual Pneumocystis, Aspergillus and a variety of Gram-negative infection with Pneumocystis and CMV is frequent, especi- pathogens. As part of these events, Gram-negative bacil- ally when anti-Pneumocystis prophylaxis is not being pro- lary colonization of the respiratory tract is increased, vided. CMV in the lung and/or systemically is sometimes serving as a reservoir from which pulmonary invasion associated with superinfection with invasive opportunis- can occur. Septicemia with a variety of agents, particu- tic infection due to such organisms as Aspergillus, Crypto- larly Listeria, Candida and Gram-negative organisms, is coccus or Nocardia. Indeed, an atypical X-ray can be due to likewise increased by CMV infection. With regard to CMV, but an intensive search for other pathogens is posttransplant infections, especially opportunistic patho- necessary. Around 10–20% of patients with significant gens, 90% or more occur in the setting of CMV-induced CMV infection in the lung will have dual or sequential immunosuppression. Indeed, exceptions to this associ- infection. Another clue to the presence of an additional ation usually connote an excessive environmental hazard pathogen is a pulmonary process that is progressing that requires immediate attention to forestall an rapidly, rather than over several days [1]. epidemic.

The gastrointestinal tract is a major target for CMV The host defense defects that develop in the CMV- disease. The most important site of invasion is the gut infected patient include the following: a decreased from mouth to anus. The result of CMV invasion is number and, probably, function of polymorphonuclear dramatic: diffuse inﬂammation, hemorrhage, frank leukocytes, and impaired cell-mediated immunity in the ulceration, perforation and, possibly, pneumatosis intes- face of relatively intact humoral immunity. The extent of tinalis. The stomach is the most common site of CMV the cell-mediated immune abnormalities include depres- invasion, with resultant structural and functional abnorm- sed responsiveness to recall skin test antigens as well as alities: subjective complaints of nausea, abdominal full- to in-vitro testing. Clinical recovery from CMV in the ness, emesis and/or dysphagia. These symptoms have absence of antiviral therapy is largely dependent on the been correlated with gastritis and poor gastric emptying presence or development of MHC-restricted, CMV- due to the invading virus. Essentially any site in the speciﬁc, cytotoxic T cell and natural killer cell response. gut can be involved, with esophagitis and duodenitis The greater the MHC mismatch between donor and ( ulceration) not uncommon. ‘Pseudolymphoma’ of recipient, the more impaired this response will be. There the duodenum responsive to antiviral therapy is well is a dynamic relationship between viral burden and recognized. Invasion of the colon, particularly the right the lymphocyte responses. Natural killer cells appear colon with hemorrhage, is common. Unusual entities to be important in controlling the virus during the early include pseudomembranous colitis, localized disease phases of replicating virus. From that point on, the mimicking a neoplasm or ischemic colitis. Functional cytotoxic T cell response is the key response. Active gut disturbance is often observed, such as acute colonic CMV infection suppresses both natural killer cell and pseudoobstruction (Ogilvie’s syndrome), which is com- monocyte function, with these monocytes inhibiting, in parable to poor gastric emptying. Primarily curiosities, turn, the T cell response. CMV infection causes a down- CMV hemorrhoiditis and cholecystitis can be demon- regulation of class I MHC antigens, which is thought to strated [1]. inhibit the host response to the virus. In addition, exhaus- tion of antiviral cytotoxic T cells may also play a role. As will be discussed below, our ability to diagnose Other infections ‘promoted’ by CMV include human and quantitate the microbial burden in the blood by herpes virus-6, hepatitis C virus and toxoplasmosis [1].

An important marker of disordered cell-mediated immu- in patients with symptomatic and asymptomatic CMV nity induced by CMV is based on the ability to define the infection. An entity termed cardiac allograft vasculopathy nature of the lymphocyte cell surface markers by flow is highly associated with CMV. It has been shown that cytometry and labeled monoclonal antibodies directed CMV infection of both vascular smooth muscle and the against lymphocyte cell surface markers. Normally, the endothelium occurs regularly, and can be the foundation ratio of CD4 (helper/inducer cells) to CD8 (cytotoxic/ of chronic allograft injury. Chronic vascular injury of this suppressor cells) cells is around 1.5–2.0. In the face of a type has been reported in kidney and liver transplant variety of viral infections, most notably CMV and EBV, recipients as well. Bronchiolitis obliterans in lung and there is a significant change (to 0.1–0.5), with clinical heart/lung recipients is highly associated with CMV recovery being associated with the normalization of this infection, particularly when there is relapsing CMV ratio. The great majority of opportunistic infections occur disease. in the subset of patients with the viral-induced changes in the lymphocyte subsets. The occurrence of opportunistic What has been reported in all forms of organ transplan- infections with normal circulating lymphocytes can be a tation is that all of the inflammatory changes reported clue to the presence of a significant environmental can occur in the absence of CMV, but are far more hazard. Indeed, transplant patients have been described common in the presence of this, and possibly other, as ‘sentinel chickens’, placed in the hospital and com- viruses. Other factors that can increase the effects of this munity environment to monitor any evidence of excess process include ischemia reperfusion injury and, perhaps, trafficking in microbes, which then triggers an aggressive which CMV genotype is involved. In particular, reports search and correction effort when this problem has been from the University of Oregon have noted that genotype identified. gB1 is associated with an increased occurrence of acute rejection episodes. In sum, the clinical data fits well The effects of cytomegalovirus on the the final common pathway hypothesis [1,34–38]. allograft In 1970, it was hypothesized that CMV infection could Adding significantly to this hypothesis are animal studies. lead to clinically important allograft injury. One measure In a rat aortic allograft model early rat CMV infection was of the controversy that was triggered was a question that associated with inflammation of the endothelium, smooth was frequently asked, ‘Which comes first, rejection or muscle cell proliferation and intimal atherosclerotic virus?’. The answer is now clear: it is YES, i.e. there is a changes. Bronchiolitis obliterans that appears to be iden- bidirectional trafficking in mediators that are important in tical can be produced by either rejection and virus, and the pathogenesis of both processes. Years ago, the so- be significantly increased by the two together. Kidney called ‘silo hypothesis’ of pathogenesis was defined. rejection and ischemia can produce progressive, chronic According to this hypothesis, ligand–receptor interaction nephropathy, as can CMV, with synergy between these was the first step, followed by the triggering of a pathway processes being demonstrable. Ganciclovir, a potent within the silo that, after completion of the pathway, led antiviral compound, can block the CMV component in to a unique histopathology that provided important these experiments, as can antirejection therapy. insights into the ligand, the receptor and the pathway that were involved in this process. A different molecule Perhaps the most compelling information to emerge is a would bind to a different class of receptors, travel a series of studies of antiviral prophylaxis, in which antiviral different intrasilo pathway and produce a different his- administration that is successful provides significant topathology. Although such a mechanism may be at work protection against CMV-associated allograft injury with some processes, Nature tends to be more conserva- [1,39–41]. tive than that: a pathway that works will be employed for a variety of effects. The norm will not be multiple silos, Cytomegalovirus and the pathogenesis of but rather a final common pathway into which a number cancer in transplant patients of pathways reach. This makes it possible for simul- The clearest relationship between CMV and malignancy taneous processes to produce a similar effect or even is with EBV-associated posttransplant lymphoprolifera- effects that are additive or synergistic. tive disease (PTLD). The incidence of PTLD is increased 7- to 10-fold in patients with symptomatic Extensive clinical studies in renal, liver, lung and heart CMV disease. Conversely, PTLD is almost uniformly transplant recipients have reported a link between CMV associated with CMV reactivation. Whether PTLD and allograft injury, particularly in the presence of six- occurs is related to the specific immune response that antigen MHC mismatch. Perhaps the most convincing can be generated. We would suggest that there is a clinical data come from studies in heart transplant reci- bidirectional relationship between these two processes, pients, where a number of studies have reported both with the link being chemokines and growth factors pro- acute rejection and accelerated coronary atherosclerosis duced by the host in response to CMV invasion [1,11,12].

Prevention and treatment of cytomegalovirus problems. The valine esters of acyclovir and ganci- disease in organ transplant recipients clovir provide excellent bioavailability (indeed, they Attempts to prevent CMV infection and its consequences can duplicate what is accomplished with intravenous can be divided into three major strategies, which are not therapy). necessarily mutually exclusive. (a) Protective matching (3) Histocompatibility match. Six-antigen mismatch of donor and recipient so that an organ from a CMV between donor and recipient is associated with per- seropositive donor is not transplanted into a seronegative sistent and/or relapsing CMV infection, as might be recipient, in order to avoid primary infection; avoiding-six expected given the MHC restriction on the key host MHC mismatches would also fit into this category. defense – the cytotoxic T cell response. It is already (b) Immunologic interventions – active immunization clear that more intensive and sustained antiviral with a CMV vaccine and passive immunization with a therapy is needed for these patients. There is pre- variety of intravenous immunoglobulin preparations. The liminary information that there is a ‘dose–response’ possibility that a live, attenuated vaccine can be admi- aspect to this as well, i.e. a five-antigen mismatch nistered while future kidney transplant recipients are requires more therapy than a four-antigen mismatch, still on dialysis has been demonstrated. Unfortunately, etc. Optimization of the management of such the potency of this vaccine in individuals receiving patients requires further study. immunosuppressive therapy was thought suspect and (4) Immunosuppression management is an important this strategy is currently not being pursued, with the aspect of CMV strategies. A truism of treatment exception of early efforts to develop a subunit vaccine. and prevention protocols aimed at CMV is that Intravenous immunoglobulin preparations, particularly exogenous immunosuppression should be signifi- hyperimmune anti-CMV preparations, are moderately cantly decreased in the face of infection. In addition, effective as a preventive strategy, although clearly not the following details of the immunosuppression pro- as potent as antivirals. Still under consideration is gram must be observed and accounted for: when combination or sequential therapy with hyperimmune antilymphocyte categories of drug are utilized, globulin and an antiviral, particularly in the treatment of particularly those like OKT3 and antithymocyte glo- patients with high burdens of the virus and serious bulin which produce a cytokine storm (TNF, etc.), clinical disease. (c) Antiviral therapy used to prevent preemptive and prolonged antiviral therapy is significant CMV disease [1,42–45]. needed; pulse dose steroids, high-dose cyclosporine or tacrolimus likewise require extended therapy. There are three different modes in which antimicrobial Perhaps most important is the recognition that ‘one therapy can be administered: prophylactic (the adminis- size does not fit all’, i.e. the duration of therapy tration of drug to an entire population before an event to cannot be determined on first encounter with the prevent clinically important disease), therapeutic (the patient. Although a common response to the question treatment of clinically important infection) and preemp- ‘how long to treat’ is 2–3 weeks, this is likely to be tive (the administration of a drug to a subpopulation of wrong: microbial burden, the need for acute rejection individuals at particular risk for clinically important dis- therapy, the gravity of the illness and the other factors ease as shown by a particular laboratory or clinical/ listed will modify the course of therapy required. epidemiologic finding that defines the risk). There are several variables that affect the efficacy and choice of a particular antiviral compound: Antiviral strategies in the transplant recipient: treatment of symptomatic cytomegalovirus (1) Potency of a particular compound. At present the disease drugs that are in common use are ganciclovir (and its The treatment of symptomatic CMV disease is with various forms, valganciclovir, oral ganciclovir and ganciclovir, with initiation of therapy with intravenous intravenous ganciclovir), acyclovir (and its various ganciclovir, followed by a prolonged course of valganci- forms, valacyclovir and intravenous acyclovir) and clovir. Therapy is continued until all evidence of the foscarnet (a powerful anti-CMV drug with significant virus, clinically and microbiologically, is eliminated; at renal toxicity, including the occurrence of severe that point we add a ‘buffer’ to protect against relapse. A potassium and calcium losses). typical course of treatment is 3–4 weeks of acute therapy (2) Bioavailability of the drug of interest is of great followed by 2–4 months of buffer therapy. Primary drug importance, as the choice of drug should be in part resistance (in the absence of previous drug exposure) determined by its pharmacokinetic properties. All of is very unusual in the organ transplant recipient. The the drugs listed in the above have poor bioavailability development of resistance is observed primarily in the when administered orally (below 10%). The intrave- face of inadequate dose and/or duration of therapy, and in nous formulations do provide adequate blood levels, lung, liver and heart/lung recipients with a particularly but only through central lines that carry their own high viral burden. If resistance is encountered, then

foscarnet becomes the therapy of choice, with there being References and recommended reading some evidence that foscarnet plus ganciclovir offers at Papers of particular interest, published within the annual period of review, have been highlighted as: least additive therapy. Relapsing infection merits more of special interest intense and prolonged therapy. This usually occurs in of outstanding interest Additional references related to this topic can also be found in the Current patients at risk for primary infection (DþR–). One issue World Literature section in this issue (pp. 445–446). that requires further study is the management of asymp- 1 Rubin RH. Infection in the organ transplant recipient. In: Rubin RH, Young LS, tomatic infection. Is any viral replication dangerous, editors. Infection in the organ transplant recipient, 4th ed. New York: Kluwer/ particularly in terms of the indirect effects [1,11,12]? Plenum; 2002. pp. 573–680. 2 Humar A, Malkan G, Moussa G, et al. Human herpesvirus-6 is associated with cytomegalovirus reactivation in liver transplant recipients. J Infect Dis 2000; Prevention of cytomegalovirus disease and 181:1450–1453. infection 3 Pereyra F, Rubin RH. Prevention and treatment of cytomegalovirus infection in The availability of relatively rapid viral diagnostic tests solid organ transplant recipients. Curr Opin Infect Dis 2004; 17:357–361. has advanced the management of CMV considerably. 4 Nebbia G, Mattes FM, et al. Exploring the bidirectional interactions between human cytomegalovirus and hepatitis C virus replication after liver transplan- Two types of assays provide comparable information: the tation. Liver Transpl 2007; 13:130–135. pp65 antigen assay prepared on a blood sample and a Informative review on CMV. PCR assay on blood provide accurate information on the 5 Carstens J, Andersen HK, et al. Cytomegalovirus infection in renal transplant presence or absence of viremia. With both assays, asymp- recipients. Transpl Infect Dis 2006; 8:203–212. 6 Lemstrom KB, Bruning JH, Bruggeman CA, et al. Cytomegalovirus infection- tomatic viremia can be demonstrated in many patients enhanced allograft arteriosclerosis is prevented by DHPG prophylaxis in the 2–5 days before symptoms occur, leading to the idea of rat. Circulation 1994; 90:1969–1978. preemptive therapy based on ‘viremia monitoring’. 7 Humar A, Gillingham KJ, et al. Association between cytomegalovirus disease and chronic rejection in kidney transplant recipients. Transplantation 1999; Logistically difficult, this approach has some merit, 68:1879–1883. reserving therapy for those who need it most. The 8 Hussain T, Burch M, et al. Positive pretransplantation cytomegalovirus ser- viremia assays are usually negative in patients with ology is a risk factor for cardiac allograft vasculopathy in children. Circulation 2007; 115:1798–1805. colitis or pneumonia. Perhaps the biggest question, 9 Li L, Chaudhuri A, et al. Subclinical cytomegalovirus and Epstein–Barr virus however, is whether or not preemptive therapy is useful viremia are associated with adverse outcomes in pediatric renal transplantation. Pediatr Transplant 2007; 11:187–195. in the management of the indirect effects of the virus Interesting review on determining the longevity of the allograft. [1,46,47]. 10 Stoica SC, Cafferty F, et al. The cumulative effect of acute rejection on development of cardiac allograft vasculopathy. J Heart Lung Transplant 2006; Prophylaxis with valganciclovir or valacyclovir is effective 25:420–425. 11 Rubin RH. Infectious disease complications of renal transplantation [clinical in preventing CMV disease – of both the direct and conference]. Kidney Int 1993; 44:221–236. indirect types. One would have hypothesized that a 12 Soulillou JP, Cantarovich D, Le Mauff B, et al. Randomized controlled trial of a ganciclovir preparation would be most effective. Trials monoclonal antibody against the interleukin-2 receptor (33B3.1) as compared with rabbit antithymocyte globulin for prophylaxis against rejection of carried out to date show around 90% efficacy, with equal renal allografts. N Engl J Med 1990; 322:1175–1182. results with oral ganciclovir (despite poor bioavailability), 13 Chou S. Newer methods for diagnosis of cytomegalovirus infection. Rev Infect oral valganciclovir and valacyclovir after 4–6 months of Dis 1990; 12 (Suppl 7):S727–S736. 14 Chou SW. Neutralizing antibody responses to reinfecting strains of cytome- prophylaxis. In those patients failing prophylaxis, late, galovirus in transplant recipients. J Infect Dis 1989; 160:16–21. symptomatic CMV (6–12 months posttransplant) is not 15 Emery VC, Sabin CA, Cope AV, et al. Application of viral-load kinetics to uncommon. We have hypothesized that the explanation identify patients who develop cytomegalovirus disease after transplantation. for this result is a sluggish cytotoxic T cell response. In Lancet 2000; 355:2032–2036. 16 Sia IG, Wilson JA, Groettum CM, et al. Cytomegalovirus (CMV) DNA load patients receiving prolonged valganciclovir therapy, the predicts relapsing CMV infection after solid organ transplantation. J Infect Dis T cell response is immature since the antiviral regimen is 2000; 181:717–720. so potent; with the other lesser regimens, ‘escape’ from 17 Emery V, Sabin C, Cope A, et al. Application of viral-load kinetics to identify patients who develop cytomegalovirus disease after transplantation. Lancet the antiviral leads to a potent cytotoxic T cell response. 2000; 355:2032–2036. This hypothesis, which we term endogenous vaccination, 18 Griffiths P, Cope A, Hassan-Walker A, Emery V. Diagnostic approach to merits testing. cytomegalovirus infection in bone marrow and organ transplantation. Transpl Infect Dis 1999; 1:179–186. 19 Fietze E, Prosch S, Reinke P, et al. Cytomegalovirus infection in transplant Conclusion recipients. The role of tumor necrosis factor. Transplantation 1994; 58:675– CMV remains an important pathogen in organ transplan- 680. 20 Stein J, Volk HD, Liebenthal C, et al. Tumour necrosis factor alpha stimulates tation, particularly in the production of indirect effects the activity of the human cytomegalovirus major immediate early enhancer/ that are caused by CMV-induced cytokine release. The promoter in immature monocytic cells. J Gen Virol 1993; 74:2333–2338. pathogenesis of CMV disease is initiated by TNF, which 21 Prosch S, Staak K, Stein J, et al. Stimulation of the human cytomegalovirus IE enhancer/promoter in HL-60 cells by TNF-alpha is mediated via induction of reactivates latent infection in seropositive tissues. NFkB. Virology 1995; 208:107–116. Optimal therapy for diagnosing, treating and preventing 22 Docke WD, Prosch S, Fietze E, et al. Cytomegalovirus reactivation and tumour these indirect effects still remains to be defined; the necrosis factor. Lancet 1994; 343:268–269. 23 Mutimer D, Mirza D, Shaw J, et al. Enhanced (cytomegalovirus) viral replication direct effects, in contrast, are well managed with valgan- associated with septic bacterial complications in liver transplant recipients. ciclovir. Transplantation 1997; 63:1411–1415.

24 Mutimer DJ, Shaw J, O’Donnell K, Elias E. Enhanced (cytomegalovirus) viral 36 Grundy JE, Super M, Lui S, et al. The source of cytomegalovirus infection in replication after transplantation for fulminant hepatic failure. Liver Transpl Surg seropositive renal allograft recipients is frequently the donor kidney. Trans- 1997; 3:506–512. plant Proc 1987; 19:2126–2128. 25 Kutza AS, Muhl E, Hackstein H, et al. High incidence of active cytomegalovirus 37 Grundy JE, Lui SF, Super M, et al. Symptomatic cytomegalovirus infection in infection among septic patients. Clin Infect Dis 1998; 26:1076–1082. seropositive kidney recipients: reinfection with donor virus rather than reactivation of recipient virus. Lancet 1988; 2:132–135. 26 Ho M. Cytomegalovirus infection in patients with bacterial sepsis [editorial; comment]. Clin Infect Dis 1998; 26:1083–1084. 38 Nordoy I, Muller F, et al. The role of the tumor necrosis factor system and interleukin-10 during cytomegalovirus infection in renal transplant recipients. 27 Reinke P, Prosch S, Kern F, Volk H. Mechanisms of human cytomegalovirus J Infect Dis 2006; 181:51–57. (HCMV) (re)activation and its impact on organ transplant patients. Trans Infect Dis 1999; 1:157–164. 39 Lowance D, Neumayer HH, Legendre CM, et al. Valacyclovir for the prevention of cytomegalovirus disease after renal transplantation. International 28 Quinnan GV Jr, Kirmani N, Rook AH, et al. Cytotoxic t cells in cytomegalovirus Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group. infection: HLA-restricted T-lymphocyte and non-T-lymphocyte cytotoxic N Engl J Med 1999; 340:1462–1470. responses correlate with recovery from cytomegalovirus infection in bone- marrow-transplant recipients. N Engl J Med 1982; 307:7–13. 40 Valantine HA, Gao SZ, Menon SG, et al. Impact of prophylactic immediate posttransplant ganciclovir on development of transplant atherosclerosis: 29 Rook AH, Quinnan GV Jr, Frederick WJ, et al. Importance of cytotoxic a post hoc analysis of a randomized, placebo-controlled study. Circulation lymphocytes during cytomegalovirus infection in renal transplant recipients. 1999; 100:61–66. Am J Med 1984; 76:385–392. 41 Valantine H. Role of CMV in transplant coronary artery disease and survival 30 Reusser P, Riddell SR, Meyers JD, Greenberg PD. Cytotoxic T-lymphocyte after heart transplantation. Transpl Infect Dis 1999; 1:25–30. response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and 42 Simmons R, Weil R, Tallent M, et al. Do mild infections trigger the rejection of disease. Blood 1991; 78:1373–1380. renal allografts? Transplant Proc 1970; 2:419–423. 31 Biron CA, Byron KS, Sullivan JL. Severe herpesvirus infections in an adoles- 43 Rubin RH. Cytomegalovirus disease and allograft loss after organ transplan- cent without natural killer cells. N Engl J Med 1989; 320:1731–1735. tation [editorial]. Clin Infect Dis 1998; 26:871–873. 32 Hibberd PL, Tolkoff-Rubin NE, Cosimi AB, et al. Symptomatic cytomegalo- 44 Plotkin SA, Starr SE, Friedman HM, et al. Vaccines for the prevention of virus disease in the cytomegalovirus antibody seropositive renal transplant human cytomegalovirus infection. Rev Infect Dis 1990; 12 (Suppl 7):S827– recipient treated with OKT3. Transplantation 1992; 53:68–72. S838. 33 Evans PC, Soin A, Wreghitt TG, et al. An association between cytomega- 45 Plotkin SA, Starr SE, Friedman HM, et al. Effect of Towne live virus vaccine on lovirus infection and chronic rejection after liver transplantation. Transplanta- cytomegalovirus disease after renal transplant. A controlled trial. Ann Intern tion 2000; 69:30–35. Med 1991; 114:525–531. 34 Rubin RH, Cosimi AB, Tolkoff-Rubin NE, et al. Infectious disease syndromes 46 Torres HA, Kontoyiannis DP, et al. Gastrointestinal cytomegalovirus disease in attributable to cytomegalovirus and their signiﬁcance among renal transplant patients with cancer: a two decade experience in a tertiary care cancer center. recipients. Transplantation 1977; 24:458–464. Eur J Cancer 2005; 41:2268–2279. 35 Chou SW. Acquisition of donor strains of cytomegalovirus by renal-transplant 47 Korkmaz M, Kunefeci G, et al. The role of early colonoscopy in CMV colitis of recipients. N Engl J Med 1986; 314:1418–1423. transplant recipients. Transplant Proc 2005; 37:3059–3060.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Laboratory diagnosis of cytomegalovirus infection and disease in immunocompromised patients W. Lawrence Drew

Purpose of review Introduction To review new developments in PCR technology as they In this review of cytomegalovirus (CMV) diagnostic apply to detecting cytomegalovirus viremia and pneumonia, testing, I have selected four areas of focus: detection recent advances in detecting CMV resistance to antivirals of CMV viremia, specifically CMV deoxyribonucleic and assays of specific CMV lymphocyte function. acidemia by PCR, as compared to antigenemia; the utility Recent findings of quantitative CMV PCR assay on bronchoalveolar This review summarizes the attempts to use real time PCR lavage (BAL) samples; detection of CMV resistance, for cytomegalovirus deoxyribonucleic acidemia and to especially direct assays of resistance mutations in clinical compare it to conventional PCR and antigenemia, it also specimens; and the utility of assays for CMV specific reviews the use of quantitative PCR on bronchoalveolar T cells in transplant recipients. lavage to assist in the diagnosis of CMV pneumonia. Phenotypic assays of susceptibility in tissue culture are I have restricted the review to papers published within much too slow to assist clinical decisions, taking weeks for the past 12 months that have provided important addi- completion. Genotypic assays may be performed directly tions to our knowledge. To my surprise there have been on clinical samples such as blood, and cerebrospinal fluid precious few such publications especially with respect to and can be done by sequencing in a very few days. detecting CMV viremia or most importantly, CMV dis- Finally, assays of lymphocytic functional responsiveness ease. Most of the papers on this subject report the use of to cytomegalovirus can be used to identify transplant PCR for detecting CMV DNA, both qualitatively and recipients at continuing risk for cytomegalovirus disease. quantitatively, including ‘real time’ assays and compare Summary these assays to CMV antigenemia detected by immuno- Assays for CMV DNA or antigen in blood are superior to fluorescence. Heretofore most reports on detecting CMV culture for documenting viremia and pneumonia. Genotypic DNA by PCR have utilized ’in-house’ assays with vari- assays have largely replaced phenotypic assays for CMV able primers, making it difficult to extrapolate results resistance to antivirals. Lymphocyte responses to CMV from one institution to another. The Cobas Amplicor antigen(s) may identify patients at risk for CMV disease. CMV Monitor (CACM; Roche, Brandenburg, New Jersey, USA) is commercially available and would allow Keywords laboratories to generate data with more widespread appli- antigenemia, antiviral resistance, CMV DNA, CMV cability. Piiparinen et al. [1] found the Cobas assay to be pneumonia, PCR comparable to the pp65 antigenemia assay in renal transplant recipients, being monitored for CMV viremia or Curr Opin Infect Dis 20:408–411. ß 2007 Lippincott Williams & Wilkins. treated with ganciclovir. In general, the various PCR assays agree with each other and with the antigenemia University of California, San Francisco, UCSF Medical Center at Mount Zion, assay (Table 1). Lengerke et al. [2] found 89–92% San Francisco, California, USA concordance between an in-house quantitative PCR on Correspondence to W. Lawrence Drew, University of California, San Francisco, UCSF Medical Center at Mount Zion, 1600 Divisadero Street, Box 1629, whole blood and the CACM on plasma. While the in- Room B 201, San Francisco, California 94115, USA house PCR amplified a portion of the immediate early Tel: +1 415 885 7315; fax: +1 415 353 9583; e-mail: [email protected] human cytomegalovirus (HCMV) gene, the CACM assay amplifies a 365 bp region of the HCMV DNA polymerase Current Opinion in Infectious Diseases 2007, 20:408–411 gene. Detection of HCMV infection was slightly slower Abbreviations (a mean of 35 days posttransplant until positivity) with BAL bronchoalveolar lavage the CACM assay than the in-house PCR (a mean of 30.5 CACM Cobas Amplicor CMV Monitor days). Eighteen percent of patients were positive only by CMV cytomegalovirus ELISA enzyme-linked immunosorbent assay the in-house assay but none of these nine developed HCMV human cytomegalovirus CMV disease suggesting an unnecessarily sensitive assay. PMN polymorphonuclear leukocyte Only two patients were in-house negative and CACM positive. ß 2007 Lippincott Williams & Wilkins 0951-7375 Allice et al. [3] found a 98% correlation between a Taqman real time PCR (Q-CMV Real Time System

408

Amplimedical, Buttiglera, Torino, Italy) amplifying the 0.33) 0.49) 92%) 0.72)

¼ immediate early gene and CACM, both assays using ¼ – r ¼ r

r peripheral blood leukocytes. These authors found good

1/200 000 correlation of quantitative CMV DNA by both PCR > assays when compared to pp65 antigen assays on samples containing at least one pp65 positive cell. The important pp65 1/200 000 ( ¼ > difference between the two PCR assays was the intra- in 100% of samples in 54% in plasma 46% in 83% of samples

ELISA PCR (corr 79%) assay and between assay variation. The coefficient of þ þ þ þ Real time PCR (corr 98%) pp65 HCMV PCR (corr 89 ¼ ¼ ¼ ¼ variation for the real time assay was below 28% versus a PBMN 35% PBL 54% 0.69) 0.76) 0.69) ¼ ¼ ¼ between runs coefficient of variation of 21.3–49.6 for r r r ( ( ( CACM. The real time assay copy numbers were signifi- LC PCR, EIA PCR, Results LC PCR corr with pp65 test ( EIA PCR corr with pp65 test ( pp65 Ag, CACM CACM Real time PCR cantly lower for patients with under 20 antigen positive cells versus over 20 positive cells while CACM copy numbers did not differ significantly for these two levels of antigenemia. The authors conclude that the real time PCR assay offers several advantages, including speed and precision versus the CACM. Other assay compared pp65 antigenemia DP PCR, pp65 antigenemia CACM pp65 antigenemia LC PCR immunosorbenty; assa HCMV, human cytomegalovirus; LC, light Pumannova et al. [4] also concluded that the light cycler real time quantitative PCR was superior to an enzyme- linked immunosorbent assay (ELISA) PCR, both of which amplified the same segment of the CMV genome, the UL 83 gene encoding pp65, and utilized the same buffy coat patient samples. The light cycler assay was superior in inter-assay and intra-assay variability but failed to detect CMV DNA in 17% of samples positive by the ELISA PCR. All light cycler negative, ELISA quantitative PCR whole blood

’ positive samples had low levels of CMV DNA. No sample was positive by light cycler and negative by the ELISA PCR PBN (leukocytes) (HCMV-PCR) PCR. Given the superior performance characteristics and PCR assay compared Real-time PCR ‘ In house PCR PBMN (mononuclears) rapidity, these authors felt that light cycler was more suitable for routine diagnosis.

Yaghobi et al. [5] performed a double primer assay to monitor 26 bone marrow transplant patients and evalu- Quantitative ated several types of blood samples. Two primers amplifying both the immediate early (IE) and late (LA) gene worked better than one increasing the number of posi- blood neutrophils.

, double primer; EIA, enzyme immunosorbent assay; ELISA, enzyme-linked tives by 11.6%. Plasma or peripheral blood leukocytes plasma (CACM) plasma (CACM) Light cycler PCR (LCPCR) ELISA PCR Assay tested Cobas Amplicor Quantitative Double primer PCR plasma (polymorphonuclear leukocytes; PMNs) were more often positive than peripheral blood monocytes and plasma was often positive 7–10 days earlier than PMN samples.

Martin-Davila et al. [6] attempted to determine a CMV DNA copy number equivalent to several levels of antigenemia. The study population was 164 samples from liver transplant recipients being monitored weekly 68 samples cell transplants transplants for preemptive therapy with ganciclovir. The Cobas Patients Amplicor quantitative PCR (CACM) on both plasma

] 14 stem cell transplants and leukocytes was compared to a quantitative anti- ] 72 allogenic stem [4 genemia assay, enumerating the number of CMV anti- [2 ] 32 liver transplant Cobas Amplicor

gen positive cells/200 000 PMNs. An antigenemia of 10 . [5] 26 bone marrow et al. [3

et al. positive cells equated with a plasma HCMV DNA of et al 1330 copies/ml or a PMN cutoff of 713/5 000 000 cells. et al. For an antigenemia level of 20 positive cells/200 000, Lengerke Table 1 Comparison of tests Author Yaghobi CACM, Cobas Amplicor CMV Monitor;cycler; Corr, PBMN, correlation; peripheral DP blood monocytes; PBN, peripheral Pumannova Allice the comparable copy numbers for plasma was again

1330 copies/ml but increased to 4755 copies/5 000 000 pneumonia than a blood viral load, since two of the five PMN cells. Use of DNA copy numbers to guide pre- CMV pneumonia patients had negative blood viral load. emptive therapy should decrease the amount of drug The number of patients with CMV pneumonia was small administration when a qualitative PCR is used for and the assay used was not PCR, so this study is not guidance. The authors found that the quantitative directly comparable to previous reports. PCR assays reached these cutoff values earlier posttransplant than antigenemia assays (33% earlier for cell PCR, Cytomegalovirus resistance to antivirals 16% for plasma PCR). Unfortunately the quantitative cell CMV resistance to antivirals was a substantial problem in PCR was, as expected, highly sensitive (100%) but poorly AIDS patients during the pre-highly active antiretroviral specific (54%) for the diagnosis of CMV disease while the therapy era and is now a growing problem in transplan- sensitivity and specificity of the plasma PCR using 1330 tation. It is particularly notable in patients undergoing lung copies/ml was no better than that of the antigenemia transplantation or other complex multiorgan transplants assay using 10 positive cells/200 000. and less a problem in, for example, kidney transplantation. It is also comparatively infrequent in recipients of hema- To summarize, new evidence suggests that real time PCR topoietic stem cell transplants but we have recently for CMV viremia is comparable to conventional PCR but encountered it in several of these patients [9]. Historically, that the diagnostic parameters of quantitative PCR antiviral resistance testing was performed in tissue culture are similar to the quantitative antigenemia assay. Dis- and required days to weeks for a result. Very recently the advantages of the antigenemia assay include the need gene mutations responsible for resistance have been ident- for sufficient leukocytes in peripheral blood (at least an ified and gene sequence mapping can rapidly detect absolute neutrophil count of 200), which may not occur in whether resistance is likely or not [10,11]. Sequence early, posthemopoietic stem cell transplant patients. Also analysis can be performed on CMV isolates from blood there is a requirement for immediate processing (within or other samples but can also be performed directly on the 6 h). On the other hand, in my analysis, quantitative PCR clinical specimens if sufficient viral DNA is detected. assays are more expensive than the antigenemia assays but Direct assays can be performed in 24–48 h while blood probably give more reproducible values. cultures followed by genotypic assay can take up to several weeks. Jabs et al. [12] verified that the results of direct PCR of bronchoalveolar lavage CMV DNA sequencing compared favorably to culturing The utility of CMV PCR on BAL fluid has been recently and then sequencing when analyzing UL 97, the gene studied in two patient groups: patients who have pneu- responsible for ganciclovir resistance. Using the same monia and hematologic malignancies, with or without stem patient sample, there was over 90% agreement between cell transplantation and patients after lung transplantation the UL 97 gene sequences from blood samples directly who have had routine or diagnostic BALs. In the hema- amplified by PCR and those from culture isolates. Plasma tologic patients with pneumonia, the qualitative CMV and leukocytes performed similarly. Presumably, similar PCR on BALs was not clinically useful. Of 18 positive results would be found when sequencing UL 54, the DNA CMV PCRs from 134 patients (13.4%), four were from polymerase gene, which contains mutations responsible patients with definite or probable CMV pneumonia but the for cidofovir and foscarnet resistance. other 14 were from patients who did not appear to have that diagnosis. In three of the four patients with CMV pneu- Monitoring human cytomegalovirus specific monia, the virus was identified by other methods [7]. immunity The most innovative studies in CMV infected, immuno- A quantitative CMV DNA assay, hybrid capture, of logically impaired patients involve assays of CMV BALs from lung transplant patients, was employed in specific lymphocytes. The number and functionality of the study by Chemaly et al. [8]. Prior reports on efforts to CMV specific CD4þ and CD8þ cells can be measured. use quantitative CMV PCR assays in this patient popu- In general peripheral blood lymphocytes are stimulated lation had been conflicting. In this study 43 consecutive by CMV antigens or by CMV infected cells or cell lysates. BAL samples from 27 lung transplant patients were Responsiveness to these challenges can be measured by analyzed. Of 15 patients who had positive BAL cultures, interferon production using cytokine flow cytometry and five had CMV pneumonitis and BAL viral loads over human leukocyte antigen tetramer staining or by the 500 000 copies/ml. The remaining 10 patients did not enzyme linked immunospot (ELISPOT) technique. have CMV pneumonia based on negative histology The number of responsive cells can be calculated as a including immunochemistry and a clinical course not percentage of the total CD4þ or CD8þ population, compatible with CMV pneumonia. In all 10 of these determined by direct immunofluorescence flow cyto- patients BAL viral load was under 500 000 copies/ml. metry. To facilitate quantitation of CMV specific The high BAL viral load was more predictive of CMV lymphocytes test kits are now commercially available

which allow intracellular cytokine staining. Using such References and recommended reading kits, stem cell transplant (SCT) recipients can be Papers of particular interest, published within the annual period of review, have been highlighted as: followed, despite lymphopenia [13]. Using an assay of special interest combining T cell stimulation by HCMV infected auto- of outstanding interest logous dendritic cells with an interferon assay, Lilleri Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 443–444). et al. [14] found that stem cell recipients with delayed specific immune reconstitution experienced recurrent 1 Piiparinen H, Helantera I, Lappalainen M, et al. Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treat- episodes of HCMV infection. From this paper, it ment in renal transplant patients. J Med Virol 2005; 76:367–372. would appear that these immunologic assays do not 2 Lengerke C, Ljubicic T, Meisner C, et al. Evaluation of the COBAS Amplicor assist detection of CMV infection/disease but instead, HCMV Monitor for early detection and monitoring of human cytomegalovirus infection after allogenic stem cell transplantation. Bone Marrow Transplant may define a population at continuing risk of CMV 2006; 38:53–60. infection/disease. The Cobas Amplicor HCMV assay compares favorably in sensitivity and specificity to an ’in house’ PCR. This is useful since the former is commercially available and can be used by many laboratories that have not developed an in house CMV The same group addressed the kinetics of cellular assay. immune responses in a cohort of solid organ transplant 3 Allice T, Enrietto M, Pittaluga F, et al. Quantitation of cytomegalovirus DNA by real-time polymerase chain reaction in peripheral blood specimens of patients recipients. Using their novel assay described above at with solid organ transplants: comparison with end-point PCR and pp65 antigen test. J Med Virol 2006; 78:915–922. 30 days posttransplant, they found that 85% of early This provides evidence that a real time PCR assay for CMV DNA quantitation has responders, that is, with CMV specific CD4þ and better performance characteristics than the Cobas amplicor assay when com- CD8þ cell counts of over 0.4/ml blood at 30 days post- pared to a quantitative antigenemia assay. 4 Pumannova M, Roubalova K, Vitek A, Sajdova J. Comparison of quantitative transplant had self-resolving CMV infection while 76% competitive polymerase chain reaction–enzyme-linked immunosorbent assay of late responders required antiviral therapy [15]. Again, with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation. Diagn these data would seem to identify those at high versus Microbiol Infect Dis 2006; 54:115–120. low risk of CMV disease. The authors suggest that ‘early’ The authors describe an in-house real time PCR assay for CMV DNA. responders might not require subsequent virologic 5 Yaghobi R, Behzad-Behbahani A, Sabahi F, et al. Comparative analysis of a double primer PCR assay with plasma leukocytes and antigenemia for monitoring while the late responders might require diagnosis of active human cytomegalovirus infection in bone marrow trans- CMV prophylaxis or preemptive monitoring [15]. This plant recipients. Bone Marrow Transplant 2005; 35:595–599. is a very provocative suggestion in an era where there is 6 Martin-Davila P, Fortun J, Gutierrez C, et al. Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the considerable ongoing debate over whether solid organ optimal cut-off value. J Clin Virol 2005; 33:138–144. transplant recipients should receive prophylaxis or 7 Hohenthal U, Itala M, Salonen J, et al. Bronchoalveolar lavage in immuno- preemptive management. compromised patients with haematological malignancy: value of new microbiological methods. Eur J Haematol 2005; 74:203–211. 8 Chemaly RF, Yen-Lieberman B, Chapman J, et al. Clinical utility of cyto- Conclusion megalovirus viral load in bronchoalveolar lavage in lung transplant recipients. Real time PCR to detect CMV DNA in blood compares Am J Transplant 2005; 5:544–548. favorably to conventional PCR assays, especially the 9 Marfori JE, Exner MM, Marousek GI, et al. Development of new cytomegalovirus UL 97 and DNA polymerase mutations conferring drug resis- most standardized assay, the Cobas Amplicor quantita- tance after valganciclovir therapy in allogenic stem cell recipients. J Clin tive assay. In comparison to antigenemia assays, PCR Virol 2007; 38:120–125. assays can be performed in profoundly neutropenic 10 Chou S, Waldemer RH, Senters AE, et al. Cytomegalvirus UL 97 phospho- transferase mutations that affect susceptibility to ganciclovir. J Infect Dis patients, using plasma and specimen handling for 2002; 185:162–169. PCR, especially plasma, is more convenient and not 11 Chou S, Lurain NS, Thompson KD, et al. Viral DNA polymerase mutations time constrained. The clinical utility to define infection associated with drug resistance in human cytomegalovirus. J Infect Dis 2003; 188:32–39. or the need for therapy, however, is equivalent for PCR 12 Jabs DA, Martin BK, Ricks MO, Forman MS. Cytomegalovirus retinitis and viral and antigenemia. resistance study group. Detection of ganciclovir resistance in patients with AIDS and cytomegalovirus retinitis: correlation of genotypic methods with viral phenotype and clinical outcome. J Infect Dis 2006; 193:1728–1737. Quantitative PCR of BAL, especially epithelial lining This documents a similarity of sequence results for detecting CMV resistance to fluid may offer advantages over other, qualitative assays ganciclovir when using clinical samples directly versus cultured isolates. but the data are scanty and need further confirmation. 13 Buxbaum S, Kraus FB, Hahn A, et al. Flow cytometric analysis of virus-specific T lymphocytes: practicability of detection of HCMV-specific T lymphocytes in whole blood in patients after stem cell transplantation. J Immunol Methods Direct sequencing of blood and other body fluids for 2006; 311:164–173. resistance mutations in both UL 97 and CMV DNA 14 Lilleri D, Gerna G, Fomara C, et al. Prospective simultaneous quantification of human cytomegalovirus-specific CD4þ and CD8þ T-cell reconstitution in polymerase is a practical and preferred approach. young recipients of allogenic hematopoietic stem cell transplants. Blood 2006; 108:1406–1412. This describes the kinetics of a CMV specific T cell reconstitution following stem Assays of CMV specific lymphocytes after trans- cell transplantation and its role in monitoring CMV infection as well as the utilization plant can identify patients at higher risk for CMV of antiviral therapy. disease who require more intensive monitoring or pro- 15 Gerna G, Lilleri D, Fornara C, et al. Monitoring of human cytomegalovirus- specific CD4þ and CD8þ T-cell immunity in patients receiving solid organ phylaxis. transplantation. Am J Transplant 2006; 6:2356–2364.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Late-onset cytomegalovirus disease in patients with solid organ transplant Pascal R. Meylana and Oriol Manuelb

Purpose of review Introduction: a historical perspective of To review existing data regarding late cytomegalovirus cytomegalovirus management disease occurring after antiviral prophylaxis. In the early days of organ transplantation, it was soon Recent findings realized that cytomegalovirus (CMV) was the number There is a continued debate as to the respective merits of one cause for morbidity and mortality, leading H.H. the preemptive and the prophylactic approach to prevent Balfour to dub it the ‘transplant Troll’ [1]. The spectrum cytomegalovirus disease after transplantation. Arguably, by of clinical syndrome was characterized, as was the timing of allowing some infection, the preemptive approach helps infection and disease, with close to 90% occurring within build immunity in contrast to prophylaxis, explaining the the first 3 months after transplantation [2]. In addition, the occurrence of late cytomegalovirus disease in the latter major risk factors for CMV disease were soon identified, approach. No study comparing directly both approaches is including, in addition to primary infection, the type of large enough to definitely determine whether the organ transplant and intensity of immunosuppressive preemptive approach leads to a faster development of treatment, and the appearance of CMV viremia [3,4]. immune response protective from late disease nor whether late disease is clinically different after prophylaxis compared In 1986, ganciclovir became available as the first antiviral to early cytomegalovirus diseases. While risk factors for late efficacious against CMV. Its efficacy in the treatment of cytomegalovirus disease all point to a delay in mounting CMV disease was established in several small-scale open immune responses, there are no identified markers that studies [5]. Simultaneously, it was soon realized that a would help predict the risk for late disease at the time of fraction of patients with CMV disease fared poorly prophylaxis discontinuation. Various approaches to prevent despite ganciclovir treatment [5]. In addition, suspicion late cytomegalovirus disease have been developed: that CMV infection/disease was a risk factor for fungal or prolonged prophylaxis, microbiological surveillance and other opportunistic infection and acute rejection [6] preemptive treatment after prophylaxis discontinuation. fueled a drive to develop more aggressive, preventive Considering the identifying risk factors for late disease, it approaches to limit the morbidity associated with CMV. would also make sense to envision vaccinating cytomegalovirus-seronegative recipients. On one hand, the preemptive approach was designed as Summary an early therapeutic intervention aimed at curtailing The best approach to prevent or manage late CMV replication before the appearance of symptoms cytomegalovirus disease associated with cytomegalovirus in patients identified as at high risk for disease by the prophylaxis remains to be defined. detection of (rising) viremia [7] or due to the administration of immunosuppressive agents such as OKT3 [8,9]. Keywords This approach therefore mandates microbiological sur- acute rejection, chronic allograft injury, cytomegalovirus, veillance for viremia. late disease, preemptive, prophylaxis, transplantation On the other hand, the prophylactic approach consists of Curr Opin Infect Dis 20:412–418. ß 2007 Lippincott Williams & Wilkins. prescribing antiviral agents for the period at highest risk for CMV disease, i.e. the first months after transplan- aMicrobiology Institute and Infectious Diseases Service, University Hospital, Lausanne, Switzerland and bTransplant Infectious Diseases, University of Toronto, tation. Early on, suboptimal oral drugs such as acyclovir Toronto, Ontario, Canada [10] or suboptimal duration (1 month) of prophylaxis Correspondence to Dr Pascal R. Meylan, Institut de Microbiologie, Centre using intravenous ganciclovir [11] were used with some Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland success. Later on, the use of drugs with improved anti- Tel: +41 21 314 4098; fax: +41 21 314 4095; e-mail: [email protected] viral efficacy, such as oral ganciclovir in liver transplant Current Opinion in Infectious Diseases 2007, 20:412–418 patients [12], bioavailability, such as valaciclovir in Abbreviation kidney transplant recipients [13], or both, such as CMV cytomegalovirus valganciclovir [14,15,16 ], have established a 3-month prescription of this latter drug as the state-of-the-art

ß 2007 Lippincott Williams & Wilkins prophylactic regimen, with the capacity to completely 0951-7375 suppress breakthrough viremia and disease while on

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therapy, and overall to reduce CMV disease incidence Burg et al. (Transforum, 27–29 September 2006, by about two-thirds over the first 6 months to 1 year Valencia, Spain) also compared prophylactic oral posttransplantation compared to placebo [17,18,19]. ganciclovir and preemptive intravenous ganciclovir in 148 kidney recipients. Results are not available at the Incidence of late cytomegalovirus disease time of writing. Simultaneously, these studies clearly demonstrated that compared to placebo, prophylaxis postponed CMV repli- While there is no doubt that prophylaxis is fraught with cation. Therefore, while placebo-treated patients experi- late disease compared with placebo, there is as yet enced the majority of CMV viremia episodes during the only scarce clinical data regarding the incidence of first 3 months after replication, prophylaxed patients (in late CMV disease after preemptive therapy. On one particular seronegative recipients of organs from seropo- hand, the study by Khoury et al. [27] suggests that it sitive donors) experienced CMV viremia episodes after might be a rare occurrence. On the other hand, the same prophylaxis discontinuation, i.e. past 3 months after study demonstrated that the time to CMV DNAemia transplantation. In fact, late disease occurred in up to clearance was similarly prolonged among high-risk CMV 26% in high-risk patients [20]. Inspection of figures Dþ/R– patients whether attended to prophylactically demonstrating the proportion of patients developing or preemptively despite continued valganciclovir treat- CMV disease over time shows that the vast majority of ment. This is reminiscent of our observation of pro- late disease occurs within 2 months after prophylaxis tracted CMV infection in high-risk Dþ/R– patients discontinuation, [13,14,16,20,21] with only a few treated preemptively [29] – perhaps another expression scattered cases later on [22–24]. This observation has of the fact that CMV clearance is delayed anyway in fueled the debate about which of the preemptive and this high-risk group of patients. prophylactic approaches best serves the transplant patient [25,26]. Few randomized studies have directly Clinical presentation of late cytomegalovirus compared the two approaches (see, e.g. [27]). In disease addition, these studies were underpowered to detect Some authors have suggested that late-onset CMV dis- minor differences in efficacy between both approaches, ease may differ from early CMV disease in the absence of particularly when stratifying patients into different prophylaxis [20,30]. Reportedly, it was associated with risk groups. To fulfill the gap of evidence, several a higher incidence of tissue-invasive disease, i.e. CMV meta-analyses comparing the results of the prophylactic colitis, CMV hepatitis [14,31] and, especially in lung and preemptive approaches have been published transplant recipients [32], CMV pneumonitis. Although it [17,18,19,28]; however, the limited number and size is evident that the onset of CMV is delayed with the of available studies, and the wide variety of methodo- introduction of antiviral prophylaxis, it is still debated logical approaches for virological surveillance and whether late-onset CMV disease is really a more severe antiviral intervention, especially for the preemptive disease than once seen before. Comparison between approaches, limit the strength of their conclusions. studies is challenging due to methodological disparities, including definition of CMV disease, approach to diag- More importantly for the purpose of the present review, nose tissue-invasive disease and difference in immuno- CMV disease incidence could not be analyzed separately suppressive regimens. As noted before, in one of the few in early (less than 3 months) and late onset (more than studies directly comparing a prophylactic vs. preemptive 3 months after transplantation) in these meta-analyses approach with valganciclovir in 98 kidney recipients quoted above. There is therefore very little data regard- [27], five patients had CMV disease, four in the pro- ing the differential incidence of late CMV disease in a phylactic group and one in the preemptive group. In preemptive vs. prophylactic approach, with the exception placebo-controlled studies of CMV prophylaxis, even of the paper by Khoury et al. [27] directly comparing a though they were not designed to assess comparatively prophylactic vs. a preemptive approach with valganciclo- severity of early CMV disease in placebo recipients with vir in 98 renal transplant recipients and appropriately late CMV disease in prophylaxed patients, there is no reporting the time-dependent occurrence of CMV hint that the latter may be more severe [13,14,17,18,19]. DNAemia. While this study demonstrates clearly that late DNAemia occurs much more frequently in the Our experience with late-onset CMV infection has been prophylaxis than in the preemptive setting, indeed in published in part [33,34]. We recorded 19 consecutive all seroconstellation risk groups, only five patients had patients (10 kidney, three lung, five heart and one CMV disease, four presenting as late disease in the liver recipients) with CMV disease after the discontinu- prophylactic group and one as early disease 62 days after ation of a 3-month prophylaxis with valganciclovir. transplantation in the preemptive group, a nonsignificant Twelve patients presented with viral syndrome, four difference. Of note, only one patient had tissue-invasive patients with gastrointestinal disease, one patient with disease (nephritis). In a yet to be published study, pneumonitis and one patient with retinitis. Mean CMV

viralloadatpresentationwas4.67logcopies/106 cells. CMV disease was associated with a higher mortality Fifteenoutof19occurredinpatientswithaDþ/R– (hazard ratio 5.1, P ¼ 0.002), even if CMV disease was serostatus pattern. All patients were treated with oral not directly responsible for any patient death. It is not valganciclovir on an outpatient basis, underlining the known, however, if the association of late-onset CMV fact that late-onset CMV disease, in our experience, is disease with mortality can be generalized to other organ not associated with more severe disease. Indeed this transplants. proportion is grossly similar to the proportion of CMV syndrome and end-organ disease observed previously at Risk factors for late cytomegalovirus disease this center during an era when patients were managed By far the most obvious and consistent risk marker for late preemptively or treated for established disease [35]. CMV disease is the Dþ/R– CMV serostatus pattern, which exposes the donor recipient to a primary infection. Probably the most informative recent study to evaluate In fact, late disease occurs almost exclusively [13,36,37] the clinical characteristics of late-onset CMV disease is or at least with a much higher incidence compared to the PV16000 study [14]. In this trial, 364 high-risk organ CMV seropositive recipients. Limaye et al. [31] thus transplant recipients (Dþ/R–) were randomized to indeed reported a hazard ratio of 6.4 (95% confidence receive valganciclovir or oral ganciclovir for 100 days as interval 3.3–13) after oral ganciclovir prophylaxis. In fact, CMV prophylaxis. After a 12-month follow-up, CMV these observations point to the role of primary immune disease was diagnosed in 17.2% in the valganciclovir responses in the control of CMV infection as a major group compared to 18.4% in the ganciclovir group. Some determinant of late CMV disease. interesting particularities of the results in this study should be underlined. First, there were major differences Other predictors of late disease have been studied. Thus, in the incidence of CMV disease between groups Freeman et al. [38] reanalyzed 20 demographic and depending of the organ type (by 6 months of follow- clinical variables data from the PV16000 study (which up, 19% in the valganciclovir group and 12% in the included only high-risk Dþ/R– patients) [14], and ident- ganciclovir group had CMV disease in liver recipients, ified low creatinine clearance, blood group A recipient compared to 6 and 23% in kidney recipients, 6 and 10% in and female sex as significant risk factors. In this study, heart recipients, and 0% and 17% in kidney/pancreas neither the prophylactic drug, oral ganciclovir vs. valgan- recipients, respectively). The high incidence of late- ciclovir, nor the type of organ transplant predicted late onset CMV disease in liver recipients after valganciclovir CMV disease, although there was a (nonsignificant) trend prophylaxis observed in this randomized trial is indeed for a higher incidence among liver transplant recipients. consistent with the incidence of 26% reported in Dþ/R– Of note, no lung transplants were included in this study. patients reported by Limaye et al. [20], with six of 10 Akalin et al. [21] also retrospectively analyzed predictors patients having tissue-invasive disease. Second, the val- of CMV disease in kidney and/or pancreas recipients, ganciclovir group had a higher incidence (9.2 vs. 6.4%) of and apart from the Dþ/R– serostatus pattern, found a tissue-invasive disease, most of the cases due to gastro- higher incidence of late disease among recipients intestinal tract disease. It has been hypothesized that the with thymoglobulin induction, cadaveric donor, Afri- most potent viral inhibition reached with valganciclovir can-American or over 50 years old. In this study, neither compared with oral ganciclovir may hamper a proper sex nor oral ganciclovir vs. valganciclovir as a prophylaxis CMV-specific immune reconstitution [25]. Third, there had an influence on the risk for late CMV disease. was a trend towards a lower peak of CMV viral load in Lately, no effect of serostatus was reported on the patients with suspected CMV disease in the valganciclo- occurrence of late CMV disease after oral ganciclovir vir group compared to the ganciclovir group. Although or reduced dose valganciclovir prophylaxis among rarely reported in the literature, a profile of late-onset kidney and/or pancreas recipients [16], in contrast to CMV disease presenting with gastrointestinal symptoms all other reports. and low viral loads may be more frequent according to our experience [34]. Finally, we recently reported that mannose-binding lectin deficiency was associated with CMV late disease after Finally, in a cohort of liver transplant recipients receiving valganciclovir prophylaxis [39], raising interesting hy- CMV prophylaxis (oral ganciclovir or valganciclovir) potheses as to the role of this humoral innate immunity for 3 months after transplantation [31], incidence of component in the control of CMV infection, either as an late-onset CMV disease was 8.5% (28.6% in Dþ/R–, effector or as a signaling transducer necessary for the 4.8% in Rþ). Eighteen patients out of 37 had tissue- build-up of effective cellular immune responses. invasive disease (13 in Dþ/R–, five in Rþ), i.e. six hepatitis, five pneumonia and seven enteritis. No While the exact mechanisms by which these risk factors CMV disease occurred while antiviral prophylaxis. The might influence CMV infection and favor late disease is most interesting finding of this study is that late-onset unknown, they all point to a delay in mounting anti-CMV

immunity not only at the time of transplantation, but also Even more importantly, observational studies have at the end of prophylaxis as a determinant for late disease. shown that CMV serostatus pattern predicts transplant survival, with high-risk CMV Dþ/R– serostatus pattern In 1999, Reusser et al. [40] demonstrated an inverse predicting the worst outcome and prophylaxis improving correlation between CMV-specific cytotoxic T lympho- the graft survival in high-risk patients approximately to cyte responses and antigenemia in 20 kidney recipients the level of intermediate risk (Dþ/Rþ and D–/Rþ) using in-vitro expansion of peripheral blood mononuclear among kidney, heart, lung or heart/lung, but not liver cells on infected fibroblasts and chromium-release assay. recipients. This raises the issue of the relationship This study did not, however, analyze separately seropo- between early and late CMV disease, and chronic allo- sitive and seronegative recipients. graft injury. While there are no data in the literature to our knowledge analyzing such a relationship specifically More recently, investigators have used newer, with late disease, Helantera et al. [46] have recently more informative tests to assess CMV-specific T cell reported that the persistence of CMV DNA in kidney responses after transplantation. Bunde et al. [41], using allografts is associated with a reduced graft survival, a fact intracellular cytokine-staining assays after peptide pool reminiscent of the association between CMV DNA per- stimulation, showed in seropositive heart and lung reci- sistence and chronic rejection in liver transplants [47,48], pients that IE-1-, but not pp65-specific T cell responses raising the issue of the relationship between persistent correlated with protection from CMV disease. Gerna et al. CMV DNA in the allograft, late disease and chronic [42] used a similar assay after CMV-infected autologous allograft injury. Evidence for a similar relationship dendritic cell stimulation in seropositive heart, lung between CMV infection and chronic allograft vasculo- and kidney recipients, classified them as early and late pathy has been reported in heart transplant recipients responders according the time required to recover pre- [49,50]. transplant levels of CMV-specific T cell responses, and showed that late responses correlated with symptomatic Management of late cytomegalovirus disease CMV disease. In both these latter studies, patients were and research agenda attended to preemptively for CMV infection. Shlobin Several options exist regarding the management of late et al. [43] studied seronegative recipients of seropositive CMV disease. First, it has been proposed that managing lung donors at various times after transplantation, and high-risk CMV patients with a preemptive approach found that acquisition of T cell responses against CMV would eliminate late CMV disease [25,51]. This remains correlated with freedom from CMV disease and preser- to be demonstrated in a study randomizing high-risk Dþ/ vation of allograft function. Only very recently were R– patients to preemptive vs. prophylaxis approaches. similar data generated in high-risk Dþ/R– patients after Given the rate of late disease after prophylaxis, this antiviral prophylaxis [44]. These authors could not, would require a quite large study. Major endpoints however, identify any pattern of T cell response that in such a study would be incidence of early and late may correlate with late CMV disease. infection and disease, acute rejection as well as long-term allograft outcome (function and survival). Additionally, Thus, as underlined in an accompanying editorial [45], an immunological substudy should aim at comparing we still lack an understanding of the generation of immune responses in patients experiencing early CMV protective immune responses in patients at risk for late infection in the preemptive arm vs. late infection after CMV disease and consequently cannot design rational prophylaxis discontinuation in the prophylactic arm. It is approaches for its prevention. our prejudice that in the end the rate of late disease in the prophylactic arm may be close to be being balanced by the rate of initial failure in the preemptive arm [52] Late cytomegalovirus disease and indirect (i.e. rate of patients presenting with CMV disease con- effects currently to the first positive viremia test). One of the While early CMV disease has been shown to promote expected answers of such a study would be whether acute rejection, and secondary fungal and protozoal infec- preemptive management is as capable as prophylaxis tions, no study has addressed these issues regarding late in preventing acute rejection in Dþ/R– high-risk CMV disease. In fact, the only available data in this patients. respect can be found in the study by Lowance et al. [13]. While prophylaxis during the first 3 months about Second, CMV infection might be managed in a preemp- halved the incidence of acute rejection in high-risk tive manner in the months after prophylaxis discontinu- seronegative recipients, the recrudescence of CMV dis- ation. Thus, Kuypers et al. [53] proposed a simplified ease in this population did not translate into a concurrent strategy for the surveillance and preemptive manage- increased rate of acute rejection. This suggests that late ment of late CMV disease after oral ganciclovir prophy- disease may differ in this respect from early disease. laxis in kidney recipients. This study included only

13 high-risk patients, however, of whom three developed study, demonstrating the need for the organization of late CMV disease. Thus, the optimal strategy to pre- large multicenter studies to address the questions ident- emptivelymanagelatediseaseremainstobedefined ified above. In the current debate between the promoters with respect to schedule of surveillance tests and of the prophylactic and preemptive management of organ preemptive treatment. recipients, there are arguably more reviews and editorials (including the present one!) than studies. Let us go back Third, investigators have tested whether prolonged pro- to work, and organize, perform, analyze and publish phylaxis might be more efficacious at preventing late clinical studies! CMV disease. Thus, Doyle et al. [54] compared a longer duration of antiviral prophylaxis (24 weeks) to a standard References and recommended reading 12-week prophylaxis with oral ganciclovir in a cohort of Papers of particular interest, published within the annual period of review, have 70 high-risk (Dþ/R– CMV serostatus receiving cytolytic been highlighted as: of special interest induction therapy) kidney transplant recipients. Only one of outstanding interest case of tissue-invasive CMV disease was observed (in the Additional references related to this topic can also be found in the Current 12-week prophylaxis group). The 24-week prophylaxis World Literature section in this issue (p. 444). group was associated with a reduction in the incidence of 1 Balfour HH. Cytomegalovirus: the ‘transplant Troll’ takes its toll. Clin Microbiol symptomatic CMV infection (odds ratio 0.15, P ¼ 0.039). Newslett 1980; 2:1–3. In lung recipients receiving sequential intravenous gan- 2 Peterson PK, Balfour HH, Marker SC, et al. Cytomegalovirus disease in renal allograft recipients: a prospective study of the clinical features, risk ciclovir and valganciclovir, Zamora et al. [55] observed a factors and impact on renal transplantation. Medicine 1980; 59:283– striking reduction in CMV infection and disease in those 300. receiving prophylaxis for at least 180 days. These data 3 Paya CV, Razonable RR. Cytomegalovirus infection after solid organ transplantation. In: Bowden RA, Ljungman P, Paya CV, editors. Trans- suggest that prolonged prophylaxis may in the end defeat plant infections, 2nd ed. Philadelphia: Lippincott Williams & Wilkins; late CMV disease. At that juncture, one cannot resist 2003. pp. 298–325. mentioning the remarkable observation of a late disease 4 Falagas ME, Snydman DR, George MJ, et al. Incidence and predictors of cytomegalovirus pneumonia in orthotopic liver transplant recipients. Boston occurring in a lung recipient after more than 5 years of Center for Liver Transplantation CMVIG Study Group. Transplantation 1996; continued oral ganciclovir prophylaxis [56]! Finally, an 61:1716–1720. 5 Paya CV, Hermans PE, Smith TF, et al. Efficacy of ganciclovir in liver ongoing study is currently randomizing high-risk Dþ/R– and kidney transplant recipients with severe cytomegalovirus infection. kidney recipients (http://www.roche-trials.com/patient/ Transplantation 1988; 46:229–234. trials/trial110566.html) to 100 vs. 200 days of valganci- 6 Rubin RH. The indirect effects of cytomegalovirus infection on the outcome of clovir prophylaxis with the proportion of patients who organ transplantation. JAMA 1989; 261:3607–3609. 7 Emery VC, Sabin CA, Cope AV, et al. Application of viral-load kinetics to develop CMV disease within the first 52 weeks post- identify patients who develop cytomegalovirus disease after transplantation. transplant as a primary outcome. In addition, it would be Lancet 2000; 355:2032–2036. desirable to complement this study with a long-term 8 Singh N, Yu VL, Mieles L, et al. High-dose acyclovir compared with short- course preemptive ganciclovir therapy to prevent cytomegalovirus disease in (several years) assessment of graft function and survival. liver transplant recipients. Ann Intern Med 1994; 120:375–381. In fact, the exact optimal duration and dosage of valgan- 9 Hibberd PL, Tolkoff-Rubin NE, Conti D, et al. Preemptive ganciclovir therapy ciclovir prophylaxis remain to be determined for the to prevent cytomegalovirus disease in cytomegalovirus antibody-positive renal different organ transplant populations. transplant recipients. Ann Intern Med 1995; 123:18–26. 10 Balfour HH, Chace BA, Stapleton JT, et al. A randomized, placebo-controlled trial of oral acyclovir for the prevention of cytomegalovirus disease in reci- Finally, as late disease most probably reflects the delay in pients of renal allografts. N Engl J Med 1989; 320:1381–1387. mounting anti-CMV immune responses in seronegative 11 Merigan TC, Renlund DG, Keay S, et al. A controlled trial of ganciclovir to prevent cytomegalovirus disease after heart transplantation. N Engl J Med recipients secondary to antiviral prophylaxis, a logical 1992; 326:1182–1186. approach would be to vaccinate CMV seronegative 12 Gane E, Saliba F, Valdecasas GJC, et al. Randomised trial of efficacy and transplant candidates. A CMV vaccine was assigned safety of oral ganciclovir in the prevention of cytomegalovirus disease in liver- transplant recipients. Lancet 1997; 350:1729–1733. the highest development priority in 1999 by the Institute 13 Lowance D, Neumayer HH, Legendre CM, et al. Valacyclovir for the preven- of Medicine, geared toward alleviating the burden of tion of cytomegalovirus disease after renal transplantation. International congenital disease by targeting preconceptual child-bear- Valacyclovir Cytomegalovirus Prophylaxis Transplantation Study Group. N Engl J Med 1999; 340:1462–1470. ing age women [57,58]. While it may be hard to justify 14 Paya C, Humar A, Dominguez E, et al. Efficacy and safety of valganciclovir vs. testing a vaccine in seronegative organ recipients con- oral ganciclovir for prevention of cytomegalovirus disease in solid organ sidering the risk of early CMV disease, late CMV disease transplant recipients. Am J Transplant 2004; 4:611–620. may serve as an ethically acceptable outcome for demon- 15 Humar A, Kumar D, Preiksaitis J, et al. A trial of valganciclovir prophylaxis for cytomegalovirus prevention in lung transplant recipients. Am J Transplant strating the induction of protective immunity in this 2005; 5:1462–1468. setting, at least as an intermediate step. 16 Weng FL, Patel AM, Wanchoo R, et al. Oral ganciclovir versus low-dose valganciclovir for prevention of cytomegalovirus disease in recipients of kidney and pancreas transplants. Transplantation 2007; 83:290–296. Conclusion This historically controlled study provides data similar to PV16000 [14] in kidney As a relatively rare event occurring in the high-risk subset and/or pancreas transplant recipients regarding valganciclovir prophylaxis. Curiously, it is the only study to not identify Dþ/R– serostatus pattern as a risk of organ recipients, late CMV disease is quite difficult to marker for late disease.

17 Small LN, Lau J, Snydman DR. Preventing postorgan transplantation cyto- 37 Murray BM, Subramaniam S. Late cytomegalovirus infection after oral ganci- megalovirus disease with ganciclovir: a meta-analysis comparing prophylactic clovir prophylaxis in renal transplant recipients. Transpl Infect Dis 2004; 6: and preemptive therapies. Clin Infect Dis 2006; 43:869–880. 3–9. This meta-analysis compared ‘active prophylaxis’ (i.e. ganciclovir or valganciclovir) 38 Freeman RB, Paya C, Pescovitz MD, et al. Risk factors for cytomegalovirus with placebo or suboptimal prophylaxis (including acyclovir) and the preemptive viremia and disease developing after prophylaxis in high-risk solid-organ approach with deferred therapy, and then compared the risk reduction conferred transplant recipients. Transplantation 2004; 78:1765–1773. by each approach. 39 Manuel O, Pascual M, Trendelenburg M, Meylan PR. Association between 18 Kalil AC, Levitsky J, Lyden E, et al. Meta-analysis: the efficacy of strategies to mannose-binding lectin deficiency and cytomegalovirus infection after kidney prevent organ disease by cytomegalovirus in solid organ transplant recipients. transplantation. Transplantation 2007; 83:359–362. Ann Intern Med 2005; 143:870–880. A preliminary evidence pointing to a humoral innate immunity effector deficiency as 19 Hodson EM, Jones CA, Webster AC, et al. Antiviral medications to prevent a risk factor for late CMV disease. cytomegalovirus disease and early death in recipients of solid-organ trans- 40 Reusser P, Cathomas G, Attenhofer R, et al. Cytomegalovirus (CMV)-specific plants: a systematic review of randomised controlled trials. Lancet 2005; T cell immunity after renal transplantation mediates protection from CMV 365:2105–2115. disease by limiting the systemic virus load. J Infect Dis 1999; 180:247– 20 Limaye AP, Bakthavatsalam R, Kim HW, et al. Late-onset cytomegalovirus 253. disease in liver transplant recipients despite antiviral prophylaxis. Transplan- 41 Bunde T, Kirchner A, Hoffmeister B, et al. Protection from cytomegalovirus tation 2004; 78:1390–1396. after transplantation is correlated with immediate early 1-specific CD8 T cells. 21 Akalin E, Sehgal V, Ames S, et al. Cytomegalovirus disease in high-risk J Exp Med 2005; 201:1031–1036. transplant recipients despite ganciclovir or valganciclovir prophylaxis. Am J 42 Gerna G, Lilleri D, Fornara C, et al. Monitoring of human cytomegalovirus- Transplant 2003; 3:731–735. specific CD4 and CD8 T-cell immunity in patients receiving solid organ 22 Shibolet O, Ilan Y, Kalish Y, et al. Late cytomegalovirus disease following liver transplantation. Am J Transplant 2006; 6:2356–2364. transplantation. Transpl Int 2003; 16:861–865. A description of CMV-specific T cell responses after organ transplantation in seropositive recipients, showing a correlation between the timing of T cell 23 Jochimsen F, Westhoff T, Engelmann E, et al. Late-onset cytomegalovirus response restoration and CMV reactivation. reactivation in critically ill renal transplant patients. Transplantation 2003; 76:430–432. 43 Shlobin OA, West EE, Lechtzin N, et al. Persistent cytomegalovirus-specific memory responses in the lung allograft and blood following primary infection 24 Slifkin M, Tempesti P, Poutsiaka DD, Snydman DR. Late and atypical in lung transplant recipients. J Immunol 2006; 176:2625–2634. cytomegalovirus disease in solid-organ transplant recipients. Clin Infect A description of CMV-specific T cell responses transversally after lung transplanta- Dis 2001; 33:E62–E68. tion in Dþ/R– recipients, characterizing CMV-specific cell responses in blood and 25 Singh N. Antiviral drugs for cytomegalovirus in transplant recipients: advan- bronchoalveolar lavage samples, and correlating these responses with freedom of tages of preemptive therapy. Rev Med Virol 2006; 16:281–287. CMV infection and preservation of lung function. This and [26] are the most recent duet of pros and cons of the prophylactic and 44 La Rosa C, Limaye AP, Krishnan A, et al. Longitudinal assessment of preemptive approaches. cytomegalovirus (CMV)-specific immune responses in liver transplant re- 26 Snydman DR. The case for cytomegalovirus prophylaxis in solid organ cipients at high risk for late CMV disease. J Infect Dis 2007; 195:633– transplantation. Rev Med Virol 2006; 16:289–295. 644. This and [25] are the most recent duet of pros and cons of the prophylactic and The first paper analyzing T cell responses longitudinally in high-risk CMV Dþ/R– preemptive approaches. recipients: fails to identify a response or lack thereof predictive of CMV disease or 27 Khoury JA, Storch GA, Bohl DL, et al. Prophylactic versus preemptive oral viremia. valganciclovir for the management of cytomegalovirus infection in adult renal 45 Boeckh M, Riddell SR. Immunologic predictors of late cytomegalovirus transplant recipients. Am J Transplant 2006; 6:2134–2143. disease after solid organ transplantation – an elusive goal? J Infect Dis The largest and only recent study randomizing patients in a prophylactic vs. a 2007; 195:615–617. preemptive valganciclovir arm. It does not demonstrate a significant difference in 46 Helantera I, Koskinen P, Finne P, et al. Persistent cytomegalovirus infection in the rate of symptomatic CMV disease, perhaps for lack of power. kidney allografts is associated with inferior graft function and survival. Transpl 28 Strippoli GF, Hodson EM, Jones C, Craig JC. Preemptive treatment for Int 2006; 19:893–900. cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ An observation linking persistence of detectable CMV DNA in kidney allograft with transplant recipients. Transplantation 2006; 81:139–145. function loss: raises the issue of the relationship between such persistence and 29 Muheim C, Vogel G, Seydoux C, et al. Determinants of protracted cytome- late CMV disease. galovirus infection in solid-organ transplant patients. Transplantation 2002; 47 Arnold JC, Portmann BC, O’Grady JG, et al. Cytomegalovirus infection 74:226–236. persists in the liver graft in the vanishing bile duct syndrome. Hepatology 30 Singh N. Late-onset cytomegalovirus disease as a significant complication in 1992; 16:285–292. solid organ transplant recipients receiving antiviral prophylaxis: a call to heed 48 Lautenschlager I, Hockerstedt K, Jalanko H, et al. Persistent cytomegalovirus the mounting evidence. Clin Infect Dis 2005; 40:704–708. in liver allografts with chronic rejection. Hepatology 1997; 25:190–194. 31 Limaye AP, Bakthavatsalam R, Kim HW, et al. Impact of cytomegalovirus in 49 Potena L, Grigioni F, Ortolani P, et al. Relevance of cytomegalovirus infection organ transplant recipients in the era of antiviral prophylaxis. Transplantation and coronary-artery remodeling in the first year after heart transplantation: a 2006; 81:1645–1652. prospective three-dimensional intravascular ultrasound study. Transplantation Careful description of the impact of CMV in liver recipients receiving antiviral 2003; 75:839–843. prophylaxis, evidencing the timing of and the role of CMV recipient seronegativity as a risk factor for late disease, as well as the significance of CMV disease as a 50 Potena L, Holweg CT, Chin C, et al. Acute rejection and cardiac allograft predictor for mortality. vascular disease is reduced by suppression of subclinical cytomegalovirus infection. Transplantation 2006; 82:398–405. 32 Chemaly RF, Yen-Lieberman B, Chapman J, et al. Clinical utility of cytome- An observation linking persistence of detectable CMV viremia with cardiac galovirus viral load in bronchoalveolar lavage in lung transplant recipients. Am allograft vascular disease: raises also the issue of the relationship between such J Transplant 2005; 5:544–548. persistence and late CMV disease. 33 Fellay J, Venetz JP, Aubert JD, et al. Treatment of cytomegalovirus infection or 51 Singh N, Wannstedt C, Keyes L, et al. Efficacy of valganciclovir administered disease in solid organ transplant recipients with valganciclovir. Transplant as preemptive therapy for cytomegalovirus disease in liver transplant Proc 2005; 37:949–951. recipients: impact on viral load and late-onset cytomegalovirus disease. 34 Manuel O, Meylan PR, Rotman S, et al. Oral valganciclovir for cytomegalovirus Transplantation 2005; 79:85–90. colitis after liver transplantation. Transplantation 2007; 83:239–240. 52 Meylan PR, Pascual M. Preemptive versus prophylactic approaches in the 35 Kunzle N, Petignat C, Francioli P, et al. Preemptive treatment approach to management of cytomegalovirus disease in solid organ transplant recipients: cytomegalovirus (CMV) infection in solid organ transplant patients: relation- what we know and what we do not know. Clin Infect Dis 2006; 43:881– ship between compliance with the guidelines and prevention of CMV 883. morbidity. Transpl Infect Dis 2000; 2:118–126. 53 Kuypers DR, Claes K, Evenepoel P, et al. Asimplified strategy for 36 Taber DJ, Ashcraft E, Baillie GM, et al. Valganciclovir prophylaxis in patients at clinical management of late cytomegalovirus infection after oral ganciclovir high risk for the development of cytomegalovirus disease. Transpl Infect Dis prophylaxis in renal recipients. J Antimicrob Chemother 2005; 55:391– 2004; 6:101–109. 394.

54 Doyle AM, Warburton KM, Goral S, et al. 24-week oral ganciclovir prophylaxis 56 Kijpittayarit S, Deziel P, Eid AJ, Razonable RR. Primary cytomegalovirus in kidney recipients is associated with reduced symptomatic cytomegalovirus disease after ﬁve years of antiviral prophylaxis. Transplantation 2006; 81: disease compared to a 12-week course. Transplantation 2006; 81:1106– 137–138. 1111. A prospective study of prolonged oral ganciclovir prophylaxis compared to 57 Arvin AM, Fast P, Myers M, et al. Vaccine development to prevent cytome- historical controls: diminishes the incidence of symptomatic CMV infection. galovirus disease: report from the National Vaccine Advisory Committee. 55 Zamora MR, Nicolls MR, Hodges TN, et al. Following universal prophylaxis Clin Infect Dis 2004; 39:233–239. with intravenous ganciclovir and cytomegalovirus immune globulin, valganciclovir is safe and effective for prevention of CMV infection following lung 58 Schleiss M. Progress in cytomegalovirus vaccine development. Herpes 2005; transplantation. Am J Transplant 2004; 4:1635–1642. 12:66–75.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Prophylaxis and treatment of cytomegalovirus disease in recipients of solid organ transplants: current approach and future challenges Yoram A. Puius and David R. Snydman

Purpose of review Abbreviations Cytomegalovirus infection is a major cause of morbidity and ACV acyclovir mortality in solid-organ transplant recipients, in terms of CMV cytomegalovirus CMV-IG cytomegalovirus immune globulin cytomegalovirus disease itself and the associated GCV ganciclovir outcomes of organ rejection and death. This review focuses SOT solid organ transplant VACV valacyclovir on recent literature concerning prevention and treatment of VGCV valganciclovir cytomegalovirus disease in this population. Recent findings ß 2007 Lippincott Williams & Wilkins Two major strategies for the prevention of cytomegalovirus 0951-7375 infection in solid-organ transplant recipients – preemptive and prophylactic treatment – are reviewed. Both strategies result in a lower incidence of cytomegalovirus Introduction disease when compared to a ‘wait and treat’ approach, Cytomegalovirus (CMV) remains one of the most common and are generally considered cost-effective. Neither pathogens to affect the solid-organ transplant (SOT) prophylaxis nor preemption has yet been shown to recipient. Since CMV infection has been associated with be superior. Newer trials are also reviewed, which other infectious complications, graft loss, and overall are beginning to evaluate protocols of preemption or mortality, control and prevention of CMV disease remains prophylaxis representative of current practice, as well a high priority. Here, we address recent data pertaining as to explore alternative dosing strategies, the benefits of to prevention of CMV disease, most notably prophylactic cytomegalovirus immune globulin, and the potential benefit and preemptive strategies, both meta-analyses and new of a longer course of prophylaxis. Concerns for the selection primary data. We will also touch upon length of prophy- of ganciclovir-resistant strains of cytomegalovirus are also laxis, use of CMV immune globulin (CMV-IG), and issues addressed. of cost-effectiveness and resistance. Summary The consensus is that there is benefit for the treatment Definitions and overview: preemptive and of solid-organ transplant patients with an antiviral agent prophylactic strategies before clinical evidence of cytomegalovirus disease. Regarding the differing approaches to CMV prevention, So far, there has been no demonstration of the superiority there has been some evolution in terminology [1]. Stan- of prophylactic or preemptive regimens, nor has the exact dard therapy involves deferring therapy until clinical signs nature and dosing of the oral antiviral agent of choice been and symptoms are evident. Prophylaxis refers to treatment established. of all patients prior to detectable CMV viremia during the highest-risk period, usually for a predetermined time after Keywords transplantation [2]. Preemptive therapy is the detection antiviral therapy, cytomegalovirus, ganciclovir, transplant, and treatment of CMV viremia before the patient is valganciclovir symptomatic [3,4]. We define targeted prophylaxis as selective prophylaxis to those entering a high-risk state Curr Opin Infect Dis 20:419–424. ß 2007 Lippincott Williams & Wilkins. [5], formerly called preemptive therapy.

Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Boston, Massachusetts, USA The debate over the advantages of prophylactic versus Correspondence to David R. Snydman, MD, Division of Geographic Medicine and preemptive therapy is encapsulated in two pairs of point– Infectious Diseases, Tufts-New England Medical Center, 750 Washington Street, counterpoint editorials published in 2005 [6,7] and 2006 Box 41, Boston, MA 02111, USA Tel: +1 617 636 7001; fax: +1 617 636 8525; [1,8]. The risks of prophylaxis cited are drug toxicity e-mail: [email protected] and cost, antiviral resistance, delayed recovery of CMV-

Current Opinion in Infectious Diseases 2007, 20:419–424 speciﬁc T-cell responses, and late-onset CMV disease [6]. The potential beneﬁts of prophylaxis are prevention of end-organ disease, the improvement of survival, reduction in organ rejection, and reduction in the incidence of

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other opportunistic infections [7]. Much of this review The data evaluating preemptive therapy are sparse, and will focus on the risks and benefits of one preventive also depend on variable criteria for CMV viremia. It strategy versus the other. should also be noted that 17.8% (64 of 358) patients in the meta-analysis were excluded because of early CMV Benefits of therapy prior to symptomatic disease occurring in days 0–10. It is tempting to speculate cytomegalovirus disease that inclusion of these patients in an intent-to-treat This section will address evidence suggesting why stan- analysis may have resulted in a more definitive advantage dard therapy is less likely to be considered the standard of of preemptive therapy. care when compared to preemption or prophylaxis. The main endpoints addressed will be symptomatic CMV Comparisons of preemptive and prophylactic disease, early rejection, and overall survival. strategies To address the merits of one approach versus the other, we Prophylaxis summarize here the most recent meta-analyses, the rela- Hodson et al. [9] performed a meta-analysis of randomized tively new head-to-head trials, and the pharmacoeconomic controlled trials (RCTs) comparing prophylaxis to stan- comparisons between preemption and prophylaxis. dard care among liver, kidney, and heart SOT recipients, who were treated with either oral acyclovir (ACV), oral Meta-analyses valacyclovir (VACV), or oral ganciclovir (GCV). Table 1 A small sub-analysis within the preemptive meta-analysis summarizes the aggregate outcomes. Overall, the risk of discussed above [10] compared the two approaches. CMV disease was reduced by 60%. Mortality was Symptomatic and acute rejection were decreased more decreased by 40% compared to standard therapy, although in the preemptive group, whereas an overall mortality the magnitude of this benefit was not evident from any benefit favored prophylaxis, but none of these results individual trial. No clear effect was seen on acute rejection, approached statistical significance. although there was a trend favoring prophylaxis. A larger meta-analysis by Small et al. [11] com- This analysis combined patients who were heterogeneous pared outcomes from prophylaxis and preemption trials, in donor and recipient serostatus, immunosuppressive breaking down the results by organ, serostatus, trial regimens, antiviral drug employed, and length of therapy. methodology, and antiviral drug. Indirect comparisons Given the consistent trends favoring prophylaxis, it is of outcomes were used: prophylaxis versus standard unlikely that controlling for these variables would yield therapy as compared to preemption versus standard qualitatively different results, but larger effects might have therapy. Among interventions based solely on GCV been hidden by this variation. (Table 2), no significant difference was seen in CMV disease, graft loss, or mortality. Preemption In a complementary meta-analysis, Strippoli et al. [10] Kalil et al. [12] performed a meta-analysis using many of pooled trials examining preemptive therapy, including the same trials, but pooling all antiviral agents [ACV, GCV, RCTs comparing preemption to standard care; overall and valganciclovir (VGCV)]. Both strategies were signifi- outcomes are summarized in Table 1. Preemptive cantly more effective than standard therapy or placebo at therapy was better than standard care for the prevention reducing CMV disease and rejection, although neither of CMV disease, but did not clearly improve rejection approach demonstrated superiority. In a sub-group or mortality. analysis of the highest risk group, donor seropositive/

Table 1 Selected meta-analysis data for outcomes of prophy- Table 2 Meta-analysis by Small et al. [11] comparing out- lactic and preemptive therapy comes of prophylactic and preemptive therapy with ganciclovir Prophylaxis [9] Preemption [10] Prophylaxis Preemption P

CMV disease CMV disease 0.07 Trials 19 5 Trials 16 9 Patients (antiviral/control) 1006/975 142/146 Patients (GCV/control) 750/759 235/222 RR (95% CI) 0.42 (0.34–0.52) 0.29 (0.11–0.80) RR (95% CI) 0.49 (0.39–0.60) 0.30 (0.15–0.60) Rejection Rejection 0.17 Trials 13 3 Trials 13 4 Patients (antiviral/control) Not available 92/93 Patients (GCV/control) 632/627 108/108 RR (95% CI) 0.90 (0.78–1.05) 1.06 (0.64–1.76) RR (95% CI) 0.90 (0.79–1.01) 0.54 (0.29–1.01) Overall mortality Overall mortality 0.55 Trials 17 3 Trials 12 4 Patients (anitviral/control) 933/905 87/89 Patients (GCV/control) 671/651 108/100 RR (95% CI) 0.63 (0.63–0.92) 1.23 (0.35–4.30) RR (95% CI) 0.99 (0.68–1.43) 0.94 (0.43–2.07) CMV, cytomegalovirus. CMV, cytomegalovirus; GCV, ganciclovir.

recipient seronegative, prophylaxis reduced end-organ GCV, except in cases where both donor and recipient are disease signiﬁcantly, but the decrease in the preemptive seronegative. When prophylaxis is employed, it is usually group was not statistically signiﬁcant for a minimum of 90 days.

Head-to-head trials Prophylaxis A small, early head-to-head trial [13] compared 36 patients A retrospective analysis examined outcomes of prophy- receiving preemptive therapy with 34 patients receiving laxis in 437 liver transplant recipients [17]. In cases with oral GCV for 90 days beginning week 2 posttransplant. No seropositive recipients or donors, the recipients received difference was seen in symptomatic CMV disease, and the intravenous GCV for 7 days posttransplant, followed by endpoints of rejection and mortality were not addressed. oral GCV or VGCV for 3 months; in cases where both donor and recipient were seronegative, the recipients received A somewhat larger, more recent trial [14] was the first to prophylactic VACV instead. CMV disease developed in address the choice between prophylaxis and preemption 8.5% of the subjects within 1 year, with only one case in a employing oral VGCV, which is becoming the antiviral donor seronegative/recipient seronegative transplant; the agent of choice. Each arm contained 49 patients, with the median onset of disease was 4.5 months. The mortality prophylactic arm receiving VGCV 900 mg by mouth daily associated with infections was 49%. No difference in out- for 100 days, whereas the preemptive arms had viremia comes was seen between VGCV and oral GCV. measured at intervals for up to 12 months. Viremic patients were treated with VGCV 900 mg by mouth twice This protocol may be most representative of current a day for 21 days. Since the study was geared to be a practice. This study demonstrated a strong association pharmacoeconomic analysis, the numbers relevant to between mortality and CMV disease, which does not hard clinical endpoints were small: CMV infection was answer the question of causality. The lack of difference seen in 4 of 49 in the preemptive arm, 1 of 49 in the between GCV and VGCV is deserving of further study prophylactic arm; rejection was seen in 4 of 49 in the given the cost differential. preemptive arm, 1 of 49 in the prophylactic arm; and no deaths occurred during the 1 year follow-up. Preemption A trial focusing on VGCV alone [18] employed a com- Cost analyses bination of targeted prophylaxis and preemptive therapy. The determination of the cost-effectiveness of either The highest risk population of recipient seronegative approach is complex, since the prophylactic drugs (the patients received VGCV prophylaxis, and were then backbone of prophylaxis), the tests to monitor for CMV excluded from further study. The remaining 301 heart, viremia (the backbone of preemption), and treatments for kidney, kidney/pancreas, and liver recipients had anti- CMV disease, rejection, and other co-morbidities are genemia measured until 6 months posttransplant. all costly. Thus, all analyses will be subject to future variations in cost. Antigenemia was seen in 7.9% of patients, although none developed symptomatic disease. Another 4.6% received A French model [15] compared oral GCV prophylaxis, oral VGCV in conjunction with a high-dose immunosuppres- VACV prophylaxis, preemptive intravenous GCV, and sive regimen. VGCV was started at an average of 58 days standard therapy in renal transplants. Prophylaxis with oral after transplantation. VACV appeared to be the most cost-effective, suggesting that the greater cost of VACV was not outweighed by the This trial further demonstrated the viability of preemptive modest advantage in clinical efficacy of GCV. A similar treatment, but the targeted prophylaxis of the recipient conclusion was reached in a Czech study [16]. seronegative group did not allow for the study of preemption in this high-risk subgroup. It is interesting to note In the study by Khoury et al. [14], a side-by-side cost that the average time of starting VGCV is well within the analysis showed no significant difference in the cost of usual 90 days of treatment employed in most prophylactic preemptive or prophylactic therapy. Surprisingly, the cost regimens. of either regimen was relatively insensitive to variations in the cost of the CMV PCR test or the VGCV pills. Low-dose valganciclovir prophylaxis Given the expense of VGCV, and its excellent bioavail- Newer trials ability, the question of whether a lower dose could be The most recent trials published are beginning to reflect employed has been posed in two recent studies. Park and the most common protocols in current practice. The de- co-workers [19] compared 90 days of prophylaxis with facto standard of care is evolving into a choice of either either GCV 1 g three times daily or VGCV 450 mg once prophylactic or preemptive therapy, where the primary daily in 109 liver transplant patients. Results were similar antiviral agents employed now are usually VGCV or oral for rates of disease (4% for GCV versus 3% for VGCV)

and toxicity profiles in a 1-year follow-up. A similar at fixed intervals. By week 14 (i.e. within 2 weeks of retrospective study of 500 kidney and pancreas recipients stopping prophylaxis) eight of 54 (14.8%) were viremic, [20] yielded almost identical rates of disease (5.4% for and three of these eight developed symptomatic, biopsy- GCV versus 3.0%, for VGCV). The theoretical risk of a confirmed gastrointestinal disease. One of the 45 PCR- higher rate of resistant virus or worse clinical outcomes negative patients developed CMV disease at week 25. with the lower dose of VGCV has yet to be addressed. For this approach, the specificity was calculated to be 88%, negative predictive value of 98%, sensitivity 75%, Use of cytomegalovirus immune globulin in prophylaxis and positive predictive value 25%. Since CMV-IG is expensive, in short supply, and constitutes a large load of colloid, its use in prophylaxis has been This approach effectively functions as a combination of controversial. The mechanistic basis for its use may lie in preemptive and prophylactic methods, serving to identify the high incidence of hypogammaglobulinemia in SOT patients for whom closer monitoring for late disease may recipients, including liver [21] and lung [22] recipients. be indicated. The prompt occurrence of viremia after Two recent studies have further examined how allograft stopping prophylaxis does pose the question of whether outcome may be improved by CMV prophylaxis involving more aggressive prophylaxis is warranted. CMV-IG. The first trial to prospectively address the length of In a retrospective study of a series of donor seropositive prophylaxis was reported by Doyle et al. [28]. A cohort lung transplant recipients [23], a control group of 30 pa- of 31 donor seropositive/recipient seronegative kidney tients receiving 100 days of GCV prophylaxis was com- recipients receiving 24 weeks of oral ganciclovir was pared with a study group of 38 patients additionally compared to 39 historical controls receiving 12 weeks receiving seven doses of CMV-IG between posttransplant over a 1-year follow-up period. Significantly better out- days 1 and 28. Patients receiving augmented CMV comes were seen in the 24-week group with respect to prophylaxis had significant benefits in terms of survival, CMV infection and delayed graft function, but not for CMV disease, and the incidence of bronchiolitis acute or delayed rejection, without any additional tox- obliterans. icities. Contrary to the conventional wisdom that a longer course of prophylaxis merely delays the first occurrence of A prospective cohort study [24] of heart transplant patients CMV, the Kaplan–Meier curves show a longer-term employed a regimen in which 21 high-risk donor seropo- benefit for the 24-week course, extending well past the sitive/recipient seronegative patients received an ‘aggres- end of prophylaxis. sive’ regimen of 3 months of GCV/VGCV prophylaxis and CMV-IG at intervals up until posttransplant week 16; the Ganciclovir resistance as a consequence of control arm consisted of 45 recipient seropositive patients prophylaxis receiving only 4 weeks of prophylaxis. Patients receiving The concern for the emergence of antiviral resistance due the aggressive regimen had a significant reduction in to exposure to antiviral drugs is often cited as an argu- CMV viremia, rejection, and allograft vascular disease ment against prophylaxis [6]. Much of the early data came over the 1-year follow-up. Since the control regimen out of studies of HIV patients, in whom CMV viral involved a short course of antiviral prophylaxis, the dynamics and the correlation between viral load and contribution of CMV-IG to the improved outcomes clinical disease are different than in SOT recipients. remains uncertain. A trial involving preemptive VGCV [29] did not demon- Duration of prophylaxis strate the emergence of resistance, although it was not The current practice of 90–100 days of prophylaxis is explicitly designed to measure this endpoint. A multi- thought to have shifted the peak incidence of CMV disease center trial comparing prophylactic oral GCV with VGCV to 4–6 months posttransplant [25]. A longer length of [30] showed a low incidence of resistance, 0% in the VGCV therapy, however, is no guarantee against late CMV dis- arm, 1.9% in the GCV arm, with an incidence of 6.1% in the ease, as shown in the example of a lung transplant patient subgroup of GCV patients with suspected CMV disease (as on oral GCV prophylaxis for 5 years, who developed opposed to viremia). symptomatic CMV disease within 6 weeks of stopping therapy [26]. Genotypic evidence of resistance was found in a series of kidney and pancreas–kidney transplants who presented A prophylactic approach with surveillance was employed with persistent viremia during GCV therapy for CMV in a prospective series of 54 renal allograft recipients who disease [31], none of whom had received prophylaxis. were donor seropositive and/or recipient seronegative These patients had a longer persistence of viremia com- [27]. The subjects were given oral GCV prophylaxis from pared to those without genotypic resistance, although no posttransplant weeks 2–12, with CMV PCR performed comment was made on differences in clinical outcomes.

The study could not determine whether any of the 5 Hibberd PL, Tolkoff-Rubin NE, Conti D, et al. Preemptive ganciclovir therapy to prevent cytomegalovirus disease in cytomegalovirus antibody-positive renal mutations were preexistent in the CMV strains. The transplant recipients. A randomized controlled trial. Ann Intern Med 1995; case series does show, however, that resistance can occur 123:18–26. in the absence of prophylaxis. 6 Singh N. Late-onset cytomegalovirus disease as a significant complication in solid organ transplant recipients receiving antiviral prophylaxis: a call to heed the mounting evidence. Clin Infect Dis 2005; 40:704–708. Evidence for the evolution of resistance during prophy- 7 Snydman DR. Counterpoint: prevention of cytomegalovirus (CMV) infection and CMV disease in recipients of solid organ transplants: the case for laxis was seen in a series of SOT recipients [32], taken prophylaxis. Clin Infect Dis 2005; 40:709–712. from an original pool of 301 nonlung [30] and 80 lung 8 Singh N. Antiviral drugs for cytomegalovirus in transplant recipients: advantages donor seropositive/recipient seronegative transplants of preemptive therapy. Rev Med Virol 2006; 16:281–287. receiving GCV/VGCV prophylaxis. Resistance mutations 9 Hodson EM, Jones CA, Webster AC, et al. Antiviral medications to prevent cytomegalovirus disease and early death in recipients of solid-organ transplants: were seen in three liver, one kidney, and three lung a systematic review of randomised controlled trials. Lancet 2005; 365:2105– transplants, with rejection in one kidney and one lung 2115. patient, relapse in two lung patients, and asymptomatic 10 Strippoli GF, Hodson EM, Jones C, Craig JC. Preemptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ viremia in three liver patients. The correlation between transplant recipients. Transplantation 2006; 81:139–145. genotypic resistance and clinical outcome was not The investigators pooled a number of trials involving preemptive therapy, including six RCTs including 288 patients, comparing preemption to standard care, where addressed. the benefit was clearest in prevention of CMV disease. A sub-analysis also compared preemption to prophylaxis. The question of a clinically significant pool of CMV 11 Small LN, Lau J, Snydman DR. Preventing postorgan transplantation cytomegalovirus disease with ganciclovir: a meta-analysis comparing prophylactic isolates with preexisting resistance mutations is raised and preemptive therapies. Clin Infect Dis 2006; 43:869–880. by the case of a 24-year-old seronegative woman who This large meta-analysis compared outcomes from 17 prophylaxis trials containing 1560 subjects against nine preemption trials containing 457 subjects, showing no underwent lung transplantation from a seropositive clear difference in outcomes. donor, experienced GCV-resistant CMV during post- 12 Kalil AC, Levitsky J, Lyden E, et al. Meta-analysis: the efficacy of strategies to operative prophylaxis, and ultimately required treatment prevent organ disease by cytomegalovirus in solid organ transplant recipients. Ann Intern Med 2005; 143:870–880. with foscarnet [33]. The existence of other early-onset 13 Jung C, Engelmann E, Borner K, Offermann G. Preemptive oral ganciclovir cases of CMV resistance also raises the question as to therapy versus prophylaxis to prevent symptomatic cytomegalovirus infection whether cumulative GCV exposure is the main reason for after kidney transplantation. Transplant Proc 2001; 33:3621–3623. antiviral resistance. 14 Khoury JA, Storch GA, Bohl DL, et al. Prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients. Am J Transplant 2006; 6:2134–2143. Conclusion This study demonstrated similar outcomes and cost-effectiveness in renal transplant patients receiving VGCV-based preemptive or prophylactic regimens. The morbidity and mortality of CMV-associated disease 15 Legendre C, Beard SM, Crochard A, et al. The cost-effectiveness of prophylaxis in SOT recipients has greatly been improved by the use with valaciclovir in the management of cytomegalovirus after renal transplanta- of antiviral agents before clinical disease is apparent. tion. Eur J Health Econ 2005; Mar 12 [Epub ahead of print]. 16 Reischig T, Opatrny K Jr, Treska V, et al. Prospective comparison of valacyclovir The determination of the optimal regimen remains to be and oral ganciclovir for prevention of cytomegalovirus disease in high-risk renal seen, since issues pertaining to the antiviral agent transplant recipients. Kidney Blood Press Res 2005; 28:218–225. employed, preemption versus prophylaxis, length of 17 Limaye AP, Bakthavatsalam R, Kim HW, et al. Impact of cytomegalovirus in organ transplant recipients in the era of antiviral prophylaxis. Transplantation treatment, dosage, and cost-effectiveness are all in flux. 2006; 81:1645–1652. It will be interesting to see if one strategy emerges as This retrospective analysis of outcomes in 437 liver transplant recipients used targeted GCV or VGCV prophylaxis in high-risk patients, VACV for others, with a clear front-runner in terms of clinical outcome or cost- favorable results. effectiveness. 18 Diaz-Pedroche C, Lumbreras C, San Juan R, et al. Valganciclovir preemptive therapy for the prevention of cytomegalovirus disease in high-risk seropositive solid-organ transplant recipients. Transplantation 2006; 82:30–35. References and recommended reading This combination of targeted prophylaxis with VGCV in high-risk patients, with VACV Papers of particular interest, published within the annual period of review, have for donor seronegative/recipient seronegative cases, showed a low incidence of been highlighted as: CMV disease in a commonly employed regimen. of special interest of outstanding interest 19 Park JM, Lake KD, Arenas JD, Fontana RJ. Efficacy and safety of low-dose Additional references related to this topic can also be found in the Current valganciclovir in the prevention of cytomegalovirus disease in adult liver World Literature section in this issue (p. 444). transplant recipients. Liver Transpl 2006; 12:112–116. In liver SOT recipients, prophylaxis with half-dose VGCV has similar outcomes to 1 Snydman DR. The case for cytomegalovirus prophylaxis in solid organ standard-dose oral GCV, suggesting the possibility of using lower doses of VGCV. transplantation. Rev Med Virol 2006; 16:289–295. 20 Weng FL, Patel AM, Wanchoo R, et al. Oral ganciclovir versus low-dose 2 Gane E, Saliba F, Valdecasas GJ, et al. Randomised trial of efficacy and safety of valganciclovir for prevention of cytomegalovirus disease in recipients of kidney and pancreas transplants. Transplantation 2007; 83:290–296. oral ganciclovir in the prevention of cytomegalovirus disease in liver-transplant recipients. The Oral Ganciclovir International Transplantation Study Group Similarly to [19 ], prophylaxis with half-dose VGCV has similar outcomes to [corrected]. Lancet 1997; 350:1729–1733. standard-dose oral GCV in renal and pancreas SOT recipients as well. 3 Koetz AC, Delbruck R, Furtwangler A, et al. Cytomegalovirus pp65 antigen- 21 Doron S, Ruthazer R, Werner BG, et al. Hypogammaglobulinemia in liver guided preemptive therapy with ganciclovir in solid organ transplant recipients: transplant recipients: incidence, timing, risk factors, and outcomes. Transplan- a prospective, double-blind, placebo-controlled study. Transplantation 2001; tation 2006; 81:697–703. 72:1325–1327. 22 Yip NH, Lederer DJ, Kawut SM, et al. Immunoglobulin G levels before and 4 Singh N, Paterson DL, Gayowski T, et al. Cytomegalovirus antigenemia directed after lung transplantation. Am J Respir Crit Care Med 2006; 173:917–921. preemptive prophylaxis with oral versus I.V. ganciclovir for the prevention of 23 Ruttmann E, Geltner C, Bucher B, et al. Combined CMV prophylaxis improves cytomegalovirus disease in liver transplant recipients: a randomized, controlled outcome and reduces the risk for bronchiolitis obliterans syndrome (BOS) trial. Transplantation 2000; 70:717–722. after lung transplantation. Transplantation 2006; 81:1415–1420.

24 Potena L, Holweg CT, Chin C, et al. Acute rejection and cardiac allograft 29 Singh N, Wannstedt C, Keyes L, et al. Efficacy of valganciclovir administered as vascular disease is reduced by suppression of subclinical cytomegalovirus preemptive therapy for cytomegalovirus disease in liver transplant recipients: infection. Transplantation 2006; 82:398–405. impact on viral load and late-onset cytomegalovirus disease. Transplantation 2005; 79:85–90. 25 Razonable RR. Epidemiology of cytomegalovirus disease in solid organ and hematopoietic stem cell transplant recipients. Am J Health Syst Pharm 2005; 30 Boivin G, Goyette N, Gilbert C, et al. Absence of cytomegalovirus-resis- 62:S7–S13. tance mutations after valganciclovir prophylaxis, in a prospective multicenter study of solid-organ transplant recipients. J Infect Dis 2004; 189:1615– 26 Kijpittayarit S, Deziel P, Eid AJ, Razonable RR. Primary cytomegalovirus disease 1618. after five years of antiviral prophylaxis. Transplantation 2006; 81:137–138. 31 Nogueira E, Ozaki KS, Tomiyama H, et al. The emergence of cytomegalovirus 27 Kuypers DR, Claes K, Evenepoel P, et al. A simplified strategy for clinical resistance to ganciclovir therapy in kidney transplant recipients. Int Immuno- management of late cytomegalovirus infection after oral ganciclovir prophylaxis pharmacol 2006; 6:2031–2037. in renal recipients. J Antimicrob Chemother 2005; 55:391–394. 32 Boivin G, Goyette N, Gilbert C, et al. Clinical impact of ganciclovir-resistant 28 Doyle AM, Warburton KM, Goral S, et al. 24-week oral ganciclovir prophylaxis in cytomegalovirus infections in solid organ transplant patients. Transpl Infect kidney recipients is associated with reduced symptomatic cytomegalovirus Dis 2005; 7:166–170. disease compared to a 12-week course. Transplantation 2006; 81:1106– 1111. 33 Chen F, Bando T, Hanaoka N, et al. Early onset of ganciclovir-resistant This is the first head-to-head comparison of 24 versus 12 weeks of prophylaxis, cytomegalovirus infection in a lung-transplant recipient. Jpn J Thorac Cardiovasc demonstrating long-term benefit for the longer course. Surg 2005; 53:562–564.

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. Cytomegalovirus-associated allograft rejection in heart transplant patients Luciano Potenaa and Hannah A. Valantineb

Purpose of review Abbreviations Modern antiviral strategies are effective in controlling the ADMA asymmetric dimethylarginine clinical syndromes associated with acute cytomegalovirus CAV cardiac allograft vasculopathy CMV cytomegalovirus infection in heart transplant recipients. Despite this CMVIG cytomegalovirus hyperimmune globulin effectiveness, subclinical cytomegalovirus infection is a NF-kB nuclear factor kB TLR toll-like receptor common finding in these patients and its impact on long- term graft outcome is currently underestimated. Recent findings ß 2007 Lippincott Williams & Wilkins Recent studies provide evidence implicating subclinical 0951-7375 cytomegalovirus infection in the pathogenesis of allograft rejection and cardiac allograft vasculopathy. In this process, cytomegalovirus interacts with local inflammatory pathways, Introduction and systemic immune-regulation mechanisms, which may Despite major advancements in immunosuppression lead to graft damage, even in the absence of therapy, acute rejection and cardiac allograft vasculopa- cytomegalovirus replication within the graft. Consequently, thy (CAV) remain important causes of early and late in addition to pharmacologic strategies that inhibit viral failure of the transplanted heart [1]. Although numerous replication, immune-based therapies that abrogate host immune-mediated and metabolic risk factors have been immune response may provide an effective tool to prevent implicated in the pathogenesis of CAV [2], to date no the indirect impact of cytomegalovirus on graft function. effective treatment is available to fully eliminate CAV Summary and its related adverse outcomes. Therefore, the main Current evidence suggests that subclinical cytomegalovirus therapeutic strategy against CAV is the prevention and infection plays an important role in the pathogenesis of long- treatment of the factors known to trigger or accelerate the term graft dysfunction in heart transplant recipients and in disease [3 ]. Among these known risk factors is cytome- other solid organ transplant recipients. Pending the galovirus (CMV) infection, which plays a key role in CAV availability of definitive data from randomized trials, we progression, possibly through its complex interaction propose that the use of pharmacologic and immune-based with the host immune system [4,5]. Importantly, strat- approaches, directed at complete suppression of egies that target CMV offer the possibility of effective cytomegalovirus infection, represents the best strategy for prevention of CAV while also advancing our understand- prevention of cytomegalovirus-induced rejection, cardiac ing of its pathogenesis. The efficacy of distinct anti-CMV allograft vasculopathy and chronic allograft damage. strategies in limiting CAV, however, requires further evaluation, in concert with studies that identify the Keywords specific mechanisms by which CMV mediates graft coronary allograft vasculopathy, cytomegalovirus, heart injury. This knowledge is necessary to advance the field transplantation and settle the much-debated controversy of whether CMV is an innocent bystander or directly involved in

Curr Opin Infect Dis 20:425–431. ß 2007 Lippincott Williams & Wilkins. CAV pathogenesis [6].

aInstitute of Cardiology, University of Bologna, Bologna, Italy and bDivision of This article reviews recent studies that provide clear Cardiovascular Medicine, Stanford University, Stanford, California, USA evidence implicating CMV in the pathogenesis of Correspondence to Hannah A. Valantine, MD, MRCP, FACC, Senior Associate allograft rejection and CAV. Speciﬁcally discussed are Dean Professor of Medicine, Cardiovascular Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Falk CVRC, Stanford, CA 94305-5406, USA the results of new clinical data and mechanistic path- Tel: +1 650 723 2665; fax: +1 650 725 1599; e-mail: [email protected] ways potentially implicated in CMV-induced allograft Current Opinion in Infectious Diseases 2007, 20:425–431 damage. Also discussed are the implications of these data that point to the need for chronic suppression of subclinical viral activation as a long-term strategy to prevent acute rejection, CAV and chronic allograft damage.

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Clinical evidence linking cytomegalovirus to antibody positive, receives the antiviral drug prophyl- acute rejection and cardiac allograft actically. These two distinct approaches have allowed vasculopathy: from cytomegalovirus disease for the comparison of outcomes with respect to asympto- to subclinical infection matic infection. The association of CMV infection with cardiac allograft rejection was first reported over two decades ago in We have shown that in heart transplant recipients man- patients not receiving antiviral prophylaxis [7–9]. These aged by a preemptive strategy, asymptomatic CMV early observational studies were conducted at a time infection was associated with increased risk of developing when the methods for detection of CMV infection were CAV, defined as abnormal coronary remodeling 1 year relatively insensitive, relying predominantly on clinical after transplant [20]. In this study antiviral treatment manifestations of viral disease and confirmation by was administered only to patients who developed over histology or viral culture. From these observations devel- 30 pp65 positive cells per 105 polymorphonuclear cells, oped the concept that CMV may not only cause an organ- consistent with the preemptive approach. The clinical specific or systemic disease due to direct viral damage, relevance of subclinical infection as shown by these but is also capable of inducing immune activation that studies suggests that prevention of CMV activation targets the allograft, and thus indirectly results in acute and minimization of viral load could lead to a greater rejection and CAV [10]. protection from acute and chronic rejection. This concept has been addressed by a prospective study undertaken at The advent of antiviral drugs, particularly ganciclovir, led Stanford [21] where patients managed by a universal to effective therapeutic strategies for preventing CMV prophylaxis approach were monitored for CMV infection disease and to a reduction in virus-related graft failure, using serial measurements of CMV DNA in peripheral both in experimental models, and in heart transplant blood leukocytes, and for CAV development by serial recipients [11–13]. These advances in antiviral therapy coronary artery intravascular ultrasound examinations. In were paralleled by the development of highly sensitive this cohort, despite antiviral prophylaxis with ganciclovir, diagnostic tools to detect CMV infection, enabling the CMV DNA indicating active infection was detected in identification of a large number of patients who devel- over 90% of the patients, who however remained asymp- oped subclinical viral infection, hitherto unrecognized by tomatic. In the majority of patients developing infection prior methods [14,15]. By monitoring asymptomatic (80%), CMV was detected only after discontinuing CMV activation in peripheral blood, Emery and col- prophylaxis, raising the question of the importance of leagues [16] reported that risk of overt CMV disease is duration and type of CMV prophylaxis. To address this proportional to the level of viral DNA detected in question in the same cohort of patients [22], we peripheral blood. These observations linking viral load examined the outcomes in patients receiving a ‘standard with acute disease raised the question of whether asymp- regimen’ of intravenous ganciclovir for 28 days, compared tomatic viral replication may also predict the long-term with Dþ/R patients who received a more aggressive consequences of CMV infection. Several studies, regimen consisting of 3 months of (val-)ganciclovir and summarized below, provide confirmatory evidence for CMV hyperimmune globulin (CMVIG). Despite being at this link. higher risk for CMV activation because of the serological mismatch, recipients treated with the aggressive regimen In a large retrospective analysis including more than 400 showed delayed and reduced CMV infection rate, kidney recipients, Sagedal et al. [17] showed that in the compared with the patients treated with the standard absence of either prophylaxis or preemptive strategies, regimen. Most importantly, aggressive prophylaxis was CMV infection at a subclinical level as detected by pp65 associated with a reduced risk of acute rejection and antigenemia was associated with increased risk for overall CAV. Of note is that CMV-associated CAV was manifest and cardiovascular mortality. The authors additionally as a constrictive vascular remodeling, and not only intimal showed that subclinical CMV disease during the first thickening, as previously reported in patients managed 100 days after transplant increased the risk for sub- by the preemptive approach [20]. A key issue in this sequent rejection [18]. study was the highly sensitive [23] method used to detect viral loads as low as 5 DNA copies/105 cells. As opposed to a universal prophylaxis strategy, the pre- The association between low-grade infection and graft emptive strategy involves the administration of antiviral outcomes in the Stanford prospective study contrasts drugs only in those patients who reach a certain threshold with results from other investigators that have failed to level of viral activity [19]. Thus, patients managed by show any significant correlation. This discordance may a preemptive strategy develop significantly higher be explained, in part, by inadequate methods used to levels of subclinical CMV replication compared with detect low-grade CMV replication [24]. Similar results as those managed by a prophylaxis strategy, in which all those reported in the heart recipients were found by patients in whom either donor or recipient is CMV Ruttmann and colleagues in lung recipients [25]. Also

in this series of nonrandomized consecutive lung trans- has been ascribed to US28, a viral protein encoding for plant patients, aggressive prophylaxis with ganciclovir a G-protein-coupled chemokine receptor homologue and CMVIG was associated with suppression of CMV that is able to induce smooth muscle cell proliferation infection, and a reduced incidence of bronchiolitis [41]. In a rat heart transplant model, rat CMV lacking obliterans syndrome, the major cause of chronic lung R33, a functional homologue of US28 encoded by human allograft failure. CMV, has a significantly reduced ability to induce transplant vasculopathy as compared with wild type rat CMV Thus, the findings of these studies suggest a pathophy- encoding R33 [42]. siological role of CMV in chronic graft failure, limiting long-term outcome in heart transplant recipients. Most As a consequence of the activation of local inflammatory important, the recent data suggest that such chronic mediators in the graft vascular system, the endothelial damage to the graft may proceed unabated, even in layer rapidly become dysfunctional, reducing nitric oxide the absence of overt clinical CMV disease. Randomized synthesis and impairing nitric oxide-mediated vaso- clinical trials are required to confirm these observations. dilation [43]. This mechanism has been implicated in CAV development [44,45], further supported by recent ‘Direct’ biological mechanisms of injury studies in which asymmetric dimethylarginine (ADMA) leading to ‘indirect’ clinical effects: looking has been correlated with CAV [46]. ADMA is an for the smoking gun endogenous inhibitor of endothelial nitric oxide syn- The propensity for CMV to actively alter host cell func- thesis, increases conditions of intracellular oxidative tion by activating local inflammatory response and to stress and amplifies the disruption of endothelial homeo- modulate immune system responses to infection [4] stasis, and thus it may be regarded as a systemic marker of underscores the potential implications of CMV infection impaired endothelial function [43,47,48]. CMV infection in the pathophysiology of both acute rejection and is capable of increasing ADMA in cultured endothelial CAV. cells, and patients with CMV DNA detected in peripheral blood were shown to have higher ADMA plasma Histological signs of local inflammatory response were concentrations and were more likely to develop CAV than reported by Koskinen et al. [10,26] who first described recipients with no CMV detection [46]. In addition, signs of endothelialitis in endomyocardial biopsy speci- patients who experienced CMV infection were more mens from patients with CMV infection. The main likely to develop coronary endothelial dysfunction 1 year mechanism through which CMV is capable to activate after transplant [49]. It must be noted, however, that these inflammatory response is transactivation of nuclear factor CMV-dependent inflammatory mechanisms require allo- kB (NF-kB). This transcription factor is implicated in the geneic response to accelerate graft rejection, because regulation of many inflammatory cytokine genes and infected animals receiving syngeneic organs do not adhesion molecules and has recognition sites for CMV develop disease [50]. Therefore, an interplay between major immediate early promoter. Virus–cell interaction is CMV and host immune system appears to be a crucial sufficient for activation of NF-kB, which is necessary to factor in CMV-associated graft injury. initiate the CMV transcription machinery [27,28]. In addition, activation of NF-kB by immuno-inflammatory Despite these plausible local mechanisms for implicating stimuli (e.g. infections or allograft recognition) partici- CMV in the pathophysiology of CAV, the lack of con- pates in CMV activation in cells harboring latent infec- sistent data confirming the presence of the virus in the tion, such as monocytes differentiating into macrophages allograft challenges the hypothesis [51]. This apparent [29]. By contrast, pharmacological inhibition of NF-kB discrepancy may be addressed by hypothesizing that may reduce CMV replication in endothelial cells [20]. As allograft rejection is mediated not only by CMV’s ability a result of NF-kB activation [30], CMV infection induces to modulate local inflammatory pathways, but also by the expression of adhesion molecules on endothelial cells virus effects on adaptive and innate immune response, [31], major histocompatibility complex class I [32], and not requiring the active presence of the virus within the proinflammatory cytokines [33,34]. All these events have graft. In a recent review of this concept, Stassen et al. been implicated in CAV development [35–37]. Aside [52] point to the fact that an interaction between viral from these cellular inflammatory mediators induced by envelope glycoproteins with cell membrane receptors is the virus, CMV genome encodes for several chemokine able to induce NFkB activation, even in the absence of and chemokine receptor homologues [38,39], underscor- a complete viral particle. Among the possible cellular ing the importance of local inflammatory activation for receptors likely involved in this process, toll-like receptor CMV spreading. Chemokines promote cellular migration (TLR) 2 has been shown to interact with viral glyco- and immune system activation that may favor the protein B, a major surface antigen of viral envelope immune response against the allograft, thus predisposing [53,54,55]. TLRs are part of the innate immune system to acute rejection and CAV [40]. Particular importance and recognize pathogen-associated molecular patterns

present in microbes and products from cell injury. episodes compared with placebo [63]. This contrasted Their activation leads to the production of inflammatory with the observations from a metaanalysis of preemptive cytokines, costimulatory signals, and upregulation of strategy trials in which the preemptive approach was TLR gene expression, mainly (but not only) through shown to be effective in preventing CMV disease, but the NF-kB pathway. Even after its clearance, CMV failed to demonstrate any effect on survival or graft- infection may thus upregulate the expression of TLRs, related endpoints [64]. An important limitation of these thereby increasing cell responsiveness to other inflam- observations is that no preemptive strategy trials in heart matory stimuli potentially sustaining chronic allograft recipients were included in the analysis. Further studies rejection [52,56]. [65,66], however, have confirmed the superiority of universal prophylaxis over the preemptive strategy for In addition to mechanisms inducing inflammation, CMV prevention of allograft injury and ultimately graft loss, exerts several effects relevant to immune-system escape despite equivalent efficacy for prevention of acute CMV and suppression of cellular immunity [4], which, para- disease. This discordance between the two approaches is doxically, may be involved in acute rejection and CAV consistent with our observation linking subclinical infec- pathogenesis. Our group [57] recently analyzed the tion with acute rejection and CAV, the two determinants time course of CMV-specific cellular immunity in of chronic allograft loss in heart and other solid organ CMV seropositive heart recipients and its relationship transplant recipients [17,22]. This is further supported with viral and allograft outcomes. CMV infection by recent experimental studies indicating that very occurred earlier, at a higher level and in a greater pro- low viral burden, and even partial viral constituents, portion of recipients with a delayed CD4þ response are able to disrupt the delicate equilibrium between compared with those with an early CD4þ activation, graft, host and immune system [30,52,54,55,57]. consistent with similar findings by other investigators The observational and experimental data together under- [58,59]. Furthermore, recipients with a delayed score an important need for a randomized head-to-head CMV-specific immunity also had an increased incidence comparison of the two strategies, with the primary end- of acute rejection and a more accelerated progression of point being composite measures of graft injury such as CAV detected by intravascular ultrasound compared with acute rejection; CAV in heart recipients; chronic allograft those with an early CMV-specific immunity. Importantly, nephropathy in kidney transplants; bronchiolitis obliter- the magnitude of coronary lumen loss was proportional to ans in lung transplants; and allograft survival across all the delay of CMV-specific immunity. These findings transplant groups. Pending confirmation from such a trial, suggest that CMV-specific immunity is protective, we propose that the optimal prevention strategy should instead of harmful, for preserving graft function. More- be one that minimizes CMV replication right from the over, we may speculate that delayed CD4þ activation is outset after transplant, rather than one that allows for viral dependent on a successful CMV strategy of immune- replication to ‘preempt’ the initiation of therapy (Fig. 1). system escape that indirectly favors graft injury. Thus, Development of drug resistances and onset of delayed therapeutic strategies directed at increasing anti-CMV CMV disease are valid concerns of such an approach immunity and disrupting CMV’sinfluence on the [67,68]. The use of valganciclovir, however, a newly immune system may provide an additional benefitin developed derivative of ganciclovir with high oral bio- the long-term protection of graft function. availability, markedly reduces the likelihood of inducing CMV resistance [69,70]. Clinical implications for treatment: from the diatribe of prophylaxis versus preemptive to Immunomodulatory agents provide yet another strategy novel immune-related concepts for preventing acute CMV disease, and abrogating its A large body of literature demonstrates that both uni- effects on allograft injury. We and others [22,25,71] versal prophylaxis and preemptive strategies are effective have shown that the combination of ganciclovir with for prevention of acute CMV disease. The prevention of CMVIG appears superior to ganciclovir alone in prevent- CMV-associated graft dysfunction (acute rejection and ing acute rejection and CAV. Although we cannot CAV), however, has only been shown for universal pro- exclude that different durations of prophylaxis may be phylaxis and not for preemptive therapeutic strategy. even more important, there is evidence that hyperim- Considerable debate exists around the issue of the mune sera can provide an additional beneficial immune optimal strategy for prevention of acute CMV disease modulation of host responses, thereby reducing the risk versus CMV-associated graft injury [60,61,62]. For of both acute CMV disease and rejection [72,73]. In example, a recent metaanalysis of randomized trials addition to the effects of improved humoral immunomo- evaluating the effect of CMV prophylaxis in solid organ dulation associated with CMVIG therapy, modulation of recipients showed that in addition to a clear prevention of CMV-specific cellular immunity appears to play a role in CMV disease, the universal prophylaxis strategy was preventing CMV-mediated graft injury [57]. These associated with superior survival and lower rejection data suggest that increasing CMV-specific cellular immu-

Figure 1 Inﬂammation and immunosuppression favor cytome- viral ﬁngerprints have been detected on numerous ‘weap- galovirus (CMV) reactivation in the transplant recipient ons’ implicated in graft injury.

Inflammation Acknowledgements immunosuppression This work has partially been funded by a grant from the National Prophylaxis Institutes of Health (PO1 AI-50153), and by grants from the Italian Society of Cardiology and ‘Luisa Fanti Melloni’ foundation. CMV infection/reactivation 2 Preemptive strategy References and recommended reading Subclinical Papers of particular interest, published within the annual period of review, have been highlighted as: viremia 1 of special interest of outstanding interest Additional references related to this topic can also be found in the Current Acute CMV disease Allograft injury World Literature section in this issue (p. 446).

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61 Dummer S. Controlling the troll: management of cytomegalovirus infection 66 Kalil AC, Levitsky J, Lyden E, et al. Meta-analysis: the efficacy of strategies to after transplantation. Ann Intern Med 2005; 143:913–914. prevent organ disease by cytomegalovirus in solid organ transplant recipients. Ann Intern Med 2005; 143:870–880. 62 Meylan PR, Pascual M. Preemptive versus prophylactic approaches in the management of cytomegalovirus disease in solid organ transplant recipients: 67 Limaye AP. Ganciclovir-resistant cytomegalovirus in organ transplant recipi- what we know and what we do not know. Clin Infect Dis 2006; 43:881– ents. Clin Infect Dis 2002; 35:866–872. 883. 68 Limaye AP, Bakthavatsalam R, Kim HW, et al. Impact of cytomegalovirus in Focalized editorial underscoring the unmet questions of the studies investigating organ transplant recipients in the era of antiviral prophylaxis. Transplantation anti-CMV therapies in solid organ transplantation. 2006; 81:1645–1652. 63 Hodson EM, Jones CA, Webster AC, et al. Antiviral medications to prevent 69 Boivin G, Goyette N, Gilbert C, Covington E. Analysis of cytomegalovirus cytomegalovirus disease and early death in recipients of solid-organ trans- DNA polymerase (UL54) mutations in solid organ transplant patients receiving plants: a systematic review of randomised controlled trials. Lancet 2005; valganciclovir or ganciclovir prophylaxis. J Med Virol 2005; 77:425–429. 365:2105–2115. 70 Boivin G, Goyette N, Gilbert C, et al. Absence of cytomegalovirus-resistance 64 Strippoli GF, Hodson EM, Jones C, Craig JC. Preemptive treatment for mutations after valganciclovir prophylaxis, in a prospective multicenter study cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ of solid-organ transplant recipients. J Infect Dis 2004; 189:1615–1618. transplant recipients. Transplantation 2006; 81:139–145. This metaanalysis, which includes studies on the preemptive approach, confirms 71 Valantine H, Luikart H, Doyle R, et al. Impact of cytomegalovirus hyperimmune its efficacy in preventing CMV disease, but is unable to demonstrate any graft- globulin on outcome after cardiothoracic transplantation. Transplantation related benefit of this strategy as was demonstrated by Ref. [63]. 2001; 72:1647–1652. 72 Toyoda M, Petrosyan A, Pao A, Jordan SC. Immunomodulatory effects of 65 Small LN, Lau J, Snydman DR. Preventing postorgan transplantation cyto- combination of pooled human gammaglobulin and rapamycin on cell prolif- megalovirus disease with ganciclovir: a meta-analysis comparing prophylactic eration and apoptosis in the mixed lymphocyte reaction. Transplantation and preemptive therapies. Clin Infect Dis 2006; 43:869–880. 2004; 78:1134–1138. This important study partially makes up for the lack of direct comparisons between prophylaxis and the preemptive approach for prevention of CMV disease. While 73 Jordan SC, Vo A, Bunnapradist S, et al. Intravenous immune globulin treat- showing that the two strategies appear equally effective for prevention of CMV ment inhibits crossmatch positivity and allows for successful transplantation disease, prophylaxis appears to have an additional benefit related to patient of incompatible organs in living-donor and cadaver recipients. Transplantation survival. 2003; 76:631–636.

This bibliography is compiled by clinicians from Contents 443 Miscellaneous the journals listed at the end of this publication. It Infections of the immunocompromised is based on literature entered into our database Nosocomial and hospital related host between 1 April 2006 and 31 March 2007 infections (articles are generally added to the database 432 Advances in pathogenesis and 443 Laboratory diagnosis of cytomegalovirus about two and a half months after publication). In management of sepsis infection and disease in addition, the bibliography contains every paper immunocompromised patients annotated by reviewers; these references were 435 Clostridium difﬁcile: changing 444 Late-onset cytomegalovirus disease in obtained from a variety of bibliographic data- epidemiology and new treatment options bases and published between the beginning of patients with solid organ transplant the review period and the time of going to press. 436 Diagnosis and prevention of intravascular catheter infection 444 Prophylaxis and treatment of The bibliography has been grouped into topics cytomegalovirus disease in recipients of that relate to the reviews in this issue. 436 Measuring the impact of nosocomial solid organ transplants: current approach infection with multi-drug resistant and future challenges pathogens 444 Current approach to prophylaxis and Papers considered by the reviewers to be of 437 Biomarkers: useful tools for managing treatment of cytomegalovirus disease in special interest infection in critically-ill patients the setting of bone marrow Papers considered by the reviewers to be of transplantation outstanding interest 437 Glycopeptide-resistant enterococci: deciphering virulence, resistance 445 Indirect effects of cytomegalovirus The number in square brackets following a and epidemicity infection and disease in selected paper, e.g. [7], refers to its number in immunosuppressed patients: what have the annotated references of the corresponding 438 Extended-spectrum beta-lactamase we learned over the past thirty years? review. producing Gram-negative bacilli 446 Cytomegalovirus-associated allograft 439 New diagnostic and management rejection in heart transplant patients Current Opinion in Infectious Diseases approaches to nosocomial infection in 2007, 20:432–448 children 446 Fungal infection in immune conjuromised 439 Staphylococcal infectiosn including 447 Febrile neotrogenia # 2007 Lippincott Williams & Wilkins MRSA, prevention and treatment 447 Infetion secondary to malignant disease 0951-7375 strategies 447 Non CMV viral infection in immune 442 Nosocomial bacterial infections excluding conjuromised staphylococci 447 Parasule infection in immune 442 Health-care associated blood stream infections conjuromised 443 Nosocomial respiratory tract infections 448 Miscellaneous 443 Treatment of nosocomial infections 443 Neonatal immune responses to coagulase-negative staphylococci

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