Volume 123 , February 2020 ISSN 1386-6532

2017IMPACT FACTOR

3.020JOU

RNA

L CITATION REPORTS . 2019

CLARIVATE ANALYTICS©

Available online at www.sciencedirect.com

Reprinted from J Clin Virol. 2020;123:104211

CMV infection management in transplant patients in ItalyPaolo Antonio Grossi, Fausto Baldanti, Massimo Andreoni and Carlo Federico Perno

This material is provided by MSD as a professional service to the medical community. Information related to any product(s) may not be consistent with the prescribing information.Please consult the full prescribing information for approved information on any products discussed in this publication and before prescribing.

© 2019 Published by Elsevier B.V.

Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Reproduced by:Elsevier España, S.L.U.(A member of Elsevier)Av. Josep Tarradellas, 20-30 08029 Barcelona Tel.: 932 000 711Fax: 932 091 136

Contents lists available at ScienceDirect

Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Review

CMV infection management in transplant patients in ItalyPaolo Antonio Grossia,b, Fausto Baldantic, Massimo Andreonid,e,f, Carlo Federico Pernod,e,f,*a Infectious Diseases Section, Department of Medicine and Surgery, University of Insubria, VaresebNational Center for Transplantation, Rome, ItalycMolecular Virology Unit, Policlinic IRCCS Foundation San Matteo, Pavia, Italyd Infectious Disease Unit, Policlinic Foundation Tor Vergata, Rome, Italye Department of Oncology and Oncohematology, University of Milan, ItalyfDepartment of Clinical Virology, ASST Niguarda, Milan, Italy

A R T I C L E I N F O

Keywords:Pre-emptive therapyUniversal prophylaxisViral loadCMVReal time-PCR

A B S T R A C T

Transplant represents an effective strategy in the management of chronic organ dysfunction. Nonetheless, lifethreatening risks remain, especially in the post-transplant; among them, human cytomegalovirus (CMV) is amajor concern, currently causing active infections in at least one-third of transplant recipients. Microbiologistand transplant scientific societies redefined guidance on CMV disease prevention and the best use for universalprophylaxis and pre-emptive virological monitoring. Developments in molecular diagnostic supported the spreadof the pre-emptive strategy, and quantitative Real Time-PCR assays has unravelled the potential of viral loadmeasurement as a predictor of the infection development in CMV post-transplant management. However, despitethe WHO 1st CMV International Standard, the standardization of diagnostic and clinical practice has beenlimited by the absence of algorithms for calculating conversion factor to International Units and the lack ofshared monitoring procedure, both at national and international level. At a regional level, the Italian scientificsocieties, AMCLI (Italian Clinical Microbiologist Association), SITO (Organ Transplant Italian Society), GITMO(Italian Group for Bone Marrow Transplant), recently tried to define a consensus for post-transplant monitoring.The concerted practice encompasses molecular quantitative PCR assays technical aspects and endorses the re-levance of immunologic monitoring for improvement in patient risk stratification and prognosis. Here, weprovide an overview of the state of the art of CMV management strategies, with a specific focus on the clinicalpractices and on the scientific societies' initiatives that aim to implement international standardization guide-lines at a national level.

1. Introduction

CMV prevention represents a major concern across the transplantscenario, capable of strongly jeopardizing transplant outcomes. Despitethe advances in the clinical practice, immuno-suppression exposes pa-tients to the risk of developing CMV infection and severe CMV-relateddisease, at both organ and systemic level, and to graft rejection, withvariable risk rate of multiorgan damage and death. About 8–39% ofsolid organ transplant (SOT) patients [1] and 5–30% of haematopoieticstem cell transplant (HSCT) recipients (respectively for autologous andallogeneic transplant) face CMV infection [2] post-transplant. Mole-cular diagnostics currently constitutes a mainstay in the transplantmanagement strategy [3]. In European institutions, CMV infectionmonitoring in SOT patients is carried out by quantitative Real-TimePCR (qPCR) in about 99 % of centers [4]. Yet, broad inconsistencyamong procedures and relevant comparative elements are still present

and delay the agreement and sharing of best clinical practices [1].In 2010 the release of the first WHO International Standard for CMV

quantitation with nucleic acids amplification techniques (NAAT) en-dorsed DNAemia reporting in International Units (IU) rather than incopies [5,6]. Only two years later IU reporting was first implemented inthe SOT clinical practice [7] and then in HSCT [8,9].

In Italy, the first guideline on prophylaxis and pre-emptive therapyin renal transplant appeared in 2007 [10], but only recently the sci-entific community jointed efforts for establishing a consensus [11].Here, we provide an insight into the recent progress in the CMV man-agement strategies and the future perspective of therapies specificallyfocusing on the Italian approach.

1.1. Transplant: a successful solution to chronic dysfunction

Over the last decade organ transplantation has become extremely

https://doi.org/10.1016/j.jcv.2019.104211Received 19 January 2019; Received in revised form 1 October 2019; Accepted 4 November 2019

⁎ Corresponding author at: Department of Oncology and Hemato-Oncology, Università degli Studi di Milano, Milan, Italy.E-mail address: carlo.perno@unimi.it (C.F. Perno).

Journal of Clinical Virology 123 (2020) 104211

1386-6532/ © 2019 Published by Elsevier B.V.

T

cost-effective in the management of chronic organ dysfunction, max-imizing patient survival and expectation in life quality. Also in Italytransplants have seen a growing trend with 14 % more of HSCT [12,13]and 20 % more of SOT [14] (Fig. 1). Related national socioeconomicstudies demonstrated that transplant is also cost effective, largely out-weighs the costs for chronic treatment (Table 1). For instance, over afive-year period the kidney transplant costs are roughly one-third of thedialysis, [15] and the risk of death is 70 % less in transplant recipientsthan in coeval patients undergoing dialysis [16–18].

Certainly, the evolution of surgical techniques and main progressesin the management of the CMV post-transplant complications, havebeen decisive for such growth. In absence of a specific prophylaxis, theCMV infection occurs usually (with significative exceptions) within 2–3months after transplantation with variable rates depending on the typeof transplant, the transplanted organ, and serostatus [19–22] and age ofdonor and recipient [20–24] (Table 2). Thus, the timely identificationof CMV infection and the prevention of CMV disease development arepressing issues.

2. Current strategies of CMV management

Pre-emptive treatment and universal prophylaxis constitute thecurrent strategies of CMV disease management, with the first mostlyapplied in HSCT recipients [8,9] and low-risk SOT patients [1,7].

Both strategies relay on DNAemia monitoring and treatment, usingthe same antiviral drugs, under clinical procedures specifically con-solidated for each transplant type (Table 3) [8,9,25–27].

In the pre-emptive strategy, CMV DNAemia is measured accordingto a predetermined time schedule and the antiviral is administeredwhen the CMV DNA level reaches alert thresholds, but the infection isstill asymptomatic. Under such conditions, only a restricted cohort of

patients is treated for a reduced period. This limits the potential drug-toxicity effects (Table 3) [8,20,23] while exposing the patient’s immunesystem to low viral load levels that trigger the development of specificimmunity, which is crucial for the long-term control of CMV infection[25]. Hence, the viral load is pivotal to initiate the antiviral treatmentto prevent the disease development and therefore rapid detection andsensitive quantitation of the virus are key to the pre-emptive approachsuccess [8].

Differently, in the (universal) prophylaxis strategy the administra-tion of antiviral drugs, mostly Ganciclovir, or its derivativeValganciclovir, for 3 or more months, starts immediately after trans-plant [24,28] and the viral load is tested upon demand mainly fortherapy change or dismission. This approach drastically reduces theCMV infection rate in the early post-transplant phase, but may inducemyelotoxicity (Table 3) [27] and increase the risk of late-onset CMVdisease in the longer period, especially among those patients whocannot develop the CMV specific immunity because of severe andprolonged immunosuppression [7,29]. Moreover, the antiviral adverseeffects, such as fever and diarrhoea, may mask the symptoms of graftrejection in HSCT recipients, or the development of a CMV-resistancewhich may compromise the graft survival. Nevertheless, Letermovir, anew anti-CMV agent acting through a different mechanism and lackingside effects, has recently been approved for the prophylaxis of CMV (seebelow).

The decision on pursuing prophylaxis or pre-emptive therapy isbased on risk stratification upon analysis of donor and recipient ser-ostatus (Fig. 2) [22,30–36], transplant type, recipient's im-munosuppressive state, indirect risks triggered by CMV infection andpharmacological treatments toxicity (Table 4) [7–9,31]. Both

Fig. 1. Transplant rates in Italy in 2018. a) In 2018 in Italy were performed a total of 8.929 transplants, of which 58 % were stably represented by HSCT; b)increment of transplant activity in the last decade: compared to 2008, the increase has been 20 % in SOT, the majority of which are represented by kidney transplantand liver transplant (90 % of SOT performed in 2018) and 14 % in HSCT, where allotransplants are increasing more than autologous transplants. Abbreviations:HSCT: Hematopoietic stem cell transplantation; SOT: solid organ transplant. Source: Italian National Transplant Center (CNT): Annual activity report-2018 [14] andItalian Group of bone marrow transplantation (GITMO) 2018 activity [12].

Table 1SOT adult patient survival rate in Italy.

SOT Post-transplant survival rate

Graft 1 year 5 years

Patient survival% (range)

Organ survival% (range)

Patientsurvival %*

Organ survival%*

Kidney° 97.2 (89.7-97.4) 92.1 (89.0-92.1) 92.1 82.1Liver§ 86.6 (82.6-87.0) 82.2 (78.4-82.6) 75.1 70.5Heart 82.3 (80.3-82.4) 81.8 (79.7-81.9) 73.5 72.9

§ National Transplant Centre Data for 2000–2016 [16].° National Transplant Centre Data for 2000–2015 [17,18].* Range not available.

Table 2Rate of CMV infection and CMV disease by graft type. Rates for CMV infection(defined as evidence of CMV replication regardless of symptoms and differingfrom latent CMV) and CMV disease (defined as evidence of CMV infection withrelated symptoms [23]) vary mostly depending on transplanted organ and onserostatus and age of donor and recipient. The most updated data currentlyavailable in literature for SOT [24] and HSCT [21] are summarized herein.

Average Rate of CMV infection and CMV disease by graft type

Graft CMV infection rate CMV disease rate

Kidney 8-32% 8%Heart, heart/lung 9-35% 25%Liver 22-29% 29%Pancreas, pancreas/kidney 50% 50%HSCT transplant

(HSCT autologous transplant)7-37%(12%)

2-14%

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

2

Table3

Drugs

used

incurrentCM

Vdiseasetreatm

ent(adaptedfrom

McIntosh20

16[27]).

Drug

Adm

inistration

Indicatio

nSide

effects

Mecha

nism

ofAction

Ganciclovir

(firstlin

etreatm

ent)

Intravenou

sinfusion

Treatm

ent,prop

hylaxisa

ndpre-em

ptivetherapyof

CMV

diseasein

SOTandHSC

Thigh

-riskrecipients

Severe

leuk

openia,n

eutrop

enia,a

naem

ia,throm

bocytopenia,

pancytop

enia,bon

emarrowdepression

andrenalinsuffi

ciency

have

been

observed

inpatie

ntstreatedwith

ganciclovir.

Sensitive

human

virusesinclud

eCM

Vandallh

erpes

viruses.In

CMV-infected

cells,g

ancicloviris

phosph

orylated

toganciclovirtripho

spha

tewhich

isthen

metabolised

intracellularly.

Thevirustatic

activ

ityof

ganciclovirisaresultof

theinhibitio

nof

viralD

NA

synthesis

Valganciclovir

(firstlin

etreatm

ent)

Oral

Treatm

ent,prop

hylaxisa

ndpre-em

ptivetherapyof

CMV

diseasein

SOTandHSC

Thigh

-riskrecipients

Severe

leuk

openia,n

eutrop

enia,a

naem

ia,throm

bocytopenia,

pancytop

enia,b

onemarrowdepression

andrenalfailure

have

been

observed

inpatie

ntstreatedwith

valganciclovir

Valganciclovirisaprod

rugof

ganciclovir.In

CMV-infected

cells,g

anciclovirisph

osph

orylated

toganciclovir

tripho

spha

tewhich

isthen

metabolised

intracellularly.

The

virustatic

activ

ityof

ganciclovirisaresultof

theinhibitio

nof

viralD

NAsynthesis.

Foscarnet

(secon

dlin

etreatm

ent)

Intravenou

sinfusion

Treatm

ent,prop

hylaxisandpre-em

ptivetherapyof

gancicloviror

valganciclovir-resistant

CMVin

SOTand

HSC

Thigh

-riskrecipients

Themajor

toxicity

ofFo

scarnetisrenalim

pairment.Fo

scarnet

sodium

hasbeen

associated

with

changesin

serum

electrolytes.

Foscarnetexertsits

antiv

iral

activ

ityby

aselective

inhibitio

nat

thepy

roph

osph

atebind

ingsite

onvirus-

specificDNApo

lymerases

atconcentrations

that

dono

taff

ectcellu

larDNApo

lymerases.

Valaciclovir

(secon

dlin

etreatm

ent)

Oral

Treatm

ent,prop

hylaxisandpre-em

ptivetherapyof

gancicloviror

valganciclovir-resistant

CMVin

kidn

eytransplant

patie

nts

Leuk

openia,n

eutrop

enia,n

europsychiatriceff

ects.

Valaciclovirisaprod

rugof

acyclovir.Sensitive

human

virusesinclud

eCM

Vandallh

erpesviruses.Va

lacicloviris

rapidlyconv

ertedto

acyclovirandvalin

e;acycloviris

phosph

orylated

toacyclovirtripho

spha

te.T

hevirustatic

activ

ityof

acyclovirisa

resultof

theinhibitio

nof

viralD

NA

synthesis.

Cido

fovir

(secon

dlin

etreatm

ent)

Intravenou

sinfusion

Treatm

ent,prop

hylaxisin

HSC

Tor

SOTin

HIV

patie

nts

andsecond

arypre-em

ptivetreatm

entin

case

ofCM

Vganciclovirresistance.

Themostc

ommon

lyrepo

rted

adversereactio

nsareacute

renaltub

ular

necrosisthat

limits

itsuseespecially

inHSC

T.It

isassociated

with

crossresistance.

Cido

fovirisamon

opho

spha

tenu

cleotid

eanalogue

that

aftercellph

osph

orylationcompetitivelyinhibitsthe

incorporationof

deoxycytidinetripho

spha

teinto

viralD

NA

byviralD

NApo

lymerase.Incorporationof

thedrug

disrup

tsfurtherchainelon

gatio

n.Leterm

ovir

(new

treatm

entfor

HSC

T,un

dertrialfor

SOT)

Orala

ndintravenou

sinfusion

Prop

hylaxisof

cytomegalovirus

(CMV)

reactiv

ationand

diseasein

adultC

MV-seropo

sitiv

erecipients[R+]of

anallogeneic

haem

atop

oietic

stem

celltransplant

(HSC

T).

Diarrho

ea,n

ausea,

vomiting

Leterm

ovirinhibitstheCM

VDNAterm

inasecomplex

that

isrequ

ired

forcleavage

andpackagingof

viralp

rogeny

DNA.

Itaff

ectstheform

ationof

prop

erun

itleng

thgeno

mes

and

interferes

with

virion

maturation.

Maribavir

(new

treatm

ent,un

der

trial)

Oral

Phase3stud

iesareon

goingin

HSC

TandSO

Tpatie

nts

Nomyelotoxicity

effects.T

aste

disturbanceandpo

ssible

cross

resistance.Ineffe

ctiveon

UL9

7CM

Vmutants

MaribavirinhibitsUL9

7mediatedph

osph

orylationof

nuclearlam

inin

A/C

that

isrequ

ired

bynewCM

Vvirion

sto

exitthenu

cleus

Brincido

fovir

(new

treatm

ent,un

der

trial)

Oral

Intravenou

sinfusion

clinical

trialsareon

goingin

HSC

Tpatie

nts

Doselim

iting

toxicity

tothegastrointestinal

track.

No

detectable

myelotoxicity

Brincido

fovirisCido

fovirwith

alip

idside

chainthat

iscleavedintracellularly.

Thecleavage

redu

cesthedrug

circulationthus

mitigatin

gthehigh

renaltoxicity

ofthe

original

form

ulation.

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

3

approaches encompass the risk of developing drug resistance, especiallyamong patients that are exposed to low antiviral drug dosage for aprolonged time (more than 6 weeks of cumulative treatment)[1,7,37–41]. The CMV drug resistance prevalence ranges between5–12% in SOT patients, with peaks of 18 % in lung transplant patients[7,37,38] and between 0–4% in HSCT [37,40,41] with rates up to 14.5% among mismatch, haploidentical unrelated HSCT recipients [41].CMV-resistance to old [7] and new drugs [42–44] has been reported.The resistance may be caused by genetic mutations of the virus, or byrefractoriness to treatments due to reduced antiviral drug levels and/orhigh immunosuppressive regimens. Among North American transplantcenters, the overall prevalence of CMV resistance is 12.1 % in SOT withpeaks of 10–11.9% in lung recipients [38,39] and up to 7.9 % in HSCT[39,45] with peaks of 14.5 % in haploidentical recipients. Similarly, inEurope, rates of 10.7 % are reported in SOT and of 6.9 % in HSCT.However, only 5.9 % of SOT and 1.7 % of HSCT cases are associatedwith CMV mutations [37,46], whereas in Italy, single-center studiesreported rates of 3.8 % in allogeneic HSCT [40] and below 0.1 % in SOT[47].

In Italy, the pre-emptive approach is applied to HSCT patients andlow-risk SOT patients, mainly kidney transplants [8,20,48] whereasprophylaxis is administered almost routinely to SOT patients, especiallythose high-risk who are more prone to develop CMV disease and re-current opportunistic infections. Yet, the choice of the strategy is

carefully evaluated in consideration of the risks/benefits of exposingpatients to expensive and potentially toxic drugs (Fig. 2, Table 3).However, a recent retrospective study in paediatric liver transplantsdemonstrated equal effectiveness of pre-emptive and prophylaxis stra-tegies and suggested to reconsider pre-emptive approaches also forhigh-risk patients [49].

2.1. CMV DNAemia surveillance: the pillar of CMV management

Current guidelines recommend quantitative nucleic acid amplifica-tion [4,7,9,11] as the gold standard for monitoring of the CMV viralload in blood or plasma and for guiding therapy in the post-transplant.Such technique has replaced antigenemia, a method consisting of theimmunofluorescent detection of CMV pp65 antigen, in the nuclei ofinfected leukocytes. Though directly quantitating only infected cells,antigenemia has indeed limited sensitivity in severely neutropenic pa-tients, like HSCT recipients, and its kinetics does not strictly adherewith CMV replication [50]. Beyond such limitations, the laboriousprocedures with subjective interpretation of results also prevented thespreading of a consensus practice and the definition of an inter-la-boratory standardized antigenemia threshold for therapeutic interven-tion. Conversely, by targeting conserved regions of the viral genome ortranscripts, nucleic acid amplification assays are sensitive methods toquantify viral DNA or RNA in several biological matrices, regardless of

Fig. 2. Use of pre-emptive therapy and prophylaxis in relation to associated major risk factors.CMV disease onset is mainly influenced by the donor and recipient serostatus. In SOT D+R- patients represents the highest-risk group [55]. Additional factors suchas immunosuppression therapy, T-cell depletion, lymphopenia and HLA mismatch impact on the choice of CMV management strategy in relation to D/R status[1,34,35]. In HSCT, CMV disease is mainly observed in R+patients, especially when receiving graft from D- donor lacking CMV immunity. Steroid treatments GvDH,T-cell depletion, lymphopenia, cord blood transplantation and CMV specific T-cell response are additional risk factors for R+ and D+R- patients [19,22,32,33,36].Abbreviations: D: donor; HSCT: Hematopoietic stem cell transplantation; R: recipient; SOT: solid organ transplant.

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

4

the presence of cells [51,52]. CMV DNA is indicative of the virus pre-sence post-transplant. However, because of CMV persistence as a latentform in myeloid precursors, monitoring of the CMV DNAemia throughtime is needed for active viral infection identification. CMV RNA ispresent in the peripheral blood only in disease settings [53,54] and itsdetection is considered a direct marker of active viral infection.

Being a simpler method for the rapid quantification of CMV

DNAemia, qPCR has become the standard. In qPCR quantitation, CMVDNAemia correlates with the infection progression, reliably describesthe late-onset infection relapse in HSCT [55], predicts the CMV disease-associated morbidity, in particular in SOT seronegative recipients (R-)of organs from seropositive donors (D+) [56] and allows discrimina-tion of spontaneously resolving CMV infection in HSCT [32,57,58].Nevertheless, CMV DNA quantitation per se may provide insufficient

Table 4Major differences between pre-emptive and prophylaxis. Modified from Ramanan [26].

Parameters Pre-emptive therapy Universal anti-viral prophylaxis

DiagnosisCMV detection Yes, allows early detection of infection onset Not required in SOTVL cut-offs Some reference values per organ, but requires further research Non applicableQ-NAT monitoring Mandatory Desirable to monitor efficacy of antiviral treatment and

possible CMV resistance onset

Clinical effectiveness and implicationsRisk of CMV onset Low risk of CMV disease in short and long term Low risk CMV disease in short-termMissed detection of primary or recurrent CMV

infectionVery low, provided that frequent VL monitoring schedule isfollowed

High risk in case of suboptimal dosing of antiviral treatmentand without VL monitoring

Drug toxicities None, and limited to drug exposure Very likelyProtection against other Herpes viruses No To some degree, depending on drug administered.Development of CMV immunity Yes, development of CMV specific cellular-mediated immunity No long-term immunityRisk of late onset Very low, through continual surveillance Yes. Higher in SOT D+/R-

FeasibilityLogistics/resources Coordinated diagnostic-clinical network and availability of

technology for maximal effectivenessNon relevant

Surveillance for drug toxicity Not necessary Yes, close monitoring requiredPatient adherence Patient must strictly adhere to surveillance schedule Patient does not need to refer to centre as often

CostsDirect hospital expenses Higher lab costs Higher drug costs plus drug toxicity-related costsIndirect transplant complications Better overall cost/benefit Higher costs due to complications and transplantation failure

Table 5Virological diagnosis and immunological diagnosis relevant features [7,9].

Diagnostic approach Details Description Advantages

Viral load monitoring7,8 Method quantitative - NAT method is the gold standard method - allows viral monitoring independently from theimmunosuppression and neutropenia intensity

Patients viral load monitoring is applied to high-risk SOT and allHSCT patients

- rapid viral detection before symptom presentation- viral load quantitation correlates with the severity of theinfection and risk of disease- viral kinetic is predictive of the infection progression- limiting exposure to drug toxicity- indicative for CMV resistance if no impact on viral load isobserved under antiviral treatment- may predict resistance also under prophylaxis regimen

Post-transplantmonitoring frequency

- under pre-emptive therapy1-3 month → minimum once per week,4-6 month → minimum once every two weeks,7-24 months → minimum once a month,- under antiviral treatment once a week up to 1 week aftertherapy discontinuation.No recommendations for monitoring under prophylaxis

- viral load quantitation correlates with the severity of theinfection and risk of disease- viral kinetic is predictive of the infection progression- limiting exposure to drug toxicity- indicative for CMV resistance if no impact on viral load isobserved under antiviral treatment- may predict resistance also under prophylaxis regimen

Immunological monitoring8 Method CMV specific Immune monitoring assays describes thestate and activity of T cells or T cell subpopulation relevantfor reconstitution of the immune competencecomplementary to viral load quantitation

- predicts individuals at higher risk of post-transplant CMVdisease- measures T-cell activity and provides a direct measurementof the immune system response and recovery during the post-transplant treatment

Patients All patients in the pre- and post-transplant.All patients under pre-emptive therapy or antiviraltreatmentall patients at the end of prophylaxis all patients in the pre-and post-transplant all patients under pre-emptive therapyor antiviral treatmentall patients at the end of prophylaxis

- can predict CMV resistance CMV resistance

Post-transplantmonitoring frequency

1-3 month → once a month, 3-12 month → once every 3months*

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

5

information to guide pre-emptive therapy [57] and should be sub-stantiated by monitoring viral load variation through time (viral ki-netic), in blood or plasma. Especially when considering low viremiaload [7,9], viral kinetics is an accurate marker for discriminating be-tween self-resolving and non-self-resolving infections, even in presenceof critical viral load [7,59], and for predicting disease progression[4,57,60,61] and CMV-associated mortality [32]. Furthermore, in pa-tients presenting comparable levels of neutropenia, the CMV kineticsare similar, provide indirect information on the recipients’ immuneresponse regardless of the type of transplant [60,61] and allow com-paring results across laboratories, independently form methods, cali-bration, and measurement [62].

The rapid turnaround time of qPCR facilitates the implementation ofviral kinetics in informing the decisional processes in the pre-emptiveapproach [9,51]; however, the sensitivity of current qPCR assay mightbe a limit for reproducibly detecting low viral loads significant foridentification of self-resolving infections [7,57]. Finally, besides theaccess to the technology, pre-emptive regimes require also strict ad-herence to monitoring schedules and a logistical network to supportstreamlined tracking of CMV DNAemia to parallel immunologicalmonitoring [4,8,23].

2.2. Working towards standardization

In the pre-emptive strategy current guidelines endorse the stan-dardization of the viremia monitoring as major input for infectioncontrol and drug resistance detection.

Keeping up with SOT and HSCT international standards, the Italianscientific societies AMCLI (Italian Clinical Microbiologist Association),SITO (Organ Transplant Italian Society), GITMO (Italian Group forBone Marrow Transplant) recently published a consensus for CMV in-fection prevention in allogeneic hematopoietic stem cell transplant andin solid organ transplant [11], that defines a best practice for DNAemiamonitoring in HSCT and SOT recipients comprehensive of re-commendations with schedules, type of matrices, IU for result reportingand thresholds for initiating treatments [51].

The Italian group has identified three key and necessary elementsfor risk evaluation and post-transplant management: serostatus, im-mune response, and DNAemia [11,32]. The serostatus of donor andrecipient (i.e. presence of anti-CMV IgG) have so far constituted themainstay of transplant risk evaluation as a major factor for the allograftand patient survival after transplant. The highest risk of allograft re-jection is indeed for solid transplant CMV-seronegative recipients (R-)receiving organs from CMV-seropositive donors (D+) and seropositivepositive recipients (R+) receiving stem cells from a seronegative ne-gative donor (D-). Whereas D-/R- patients are at the lowest risk in bothSOT and HSCT (Fig. 2) [10,56,32]. The immune response is proposed asa mean for evaluating the state of reconstruction of the recipient’simmune system (Table 5). In providing quantitative and functionalinformation on CD4+ and CD8+ CMV-specific T-cells, as well as on theproduction of INF-gamma and other cytokines on an individual basis,the immunological monitoring (before and after transplant) allowscase-by-case the further grading of the risk of CMV infection and ad-justments of the post-transplant therapy [7,9,11,31]. Last but most re-levant, DNAemia is acknowledge as the first and direct marker for thevirus replication and the infection development and is the main triggerfor therapeutic intervention (Table 5).

The Italian AMCLI working group on infection in transplantation(GLaIT) recommends whole blood as more appropriate for DNAemiasurveillance during the pre-emptive therapy. Although the viral kineticis similar in both compartments, CMV is indeed detected earlier inwhole blood than in plasma and CMV DNAemia declines more rapidlyin whole blood, thus providing earlier an indication on the virusclearance [11,51] and safe discontinuation of treatments.

2.3. Systematic monitoring: the core of CMV disease management strategies

A variety of qPCR tests for CMV quantitation, commercial or home-made, are available nowadays for monitoring CMV DNAemia in thepost-transplant [4,20] and since the issue of WHO International Stan-dards on the use of IU, many laboratories have been working on vali-dating conversion factors [6,63–65]. Nonetheless, copies/mL ratherthan IU/mL are still broadly used in result reporting [3,66], and dif-ferences in CMV viral load quantitation in the range of 0.9 up to 3.35log have been reported worldwide in interlaboratory comparative stu-dies [4,67–69], where results were reported in copies/mL.

On the other hand, when reporting in IU, the laboratories oftenfollow their own developed protocols eventually applying incorrectconversion factors, even when using validated and certified commercialassays, which should be used according to manufacturer's instructions,application of conversion factors included. Furthermore, the resultcomparability remains suboptimal mainly because plasma and wholeblood are often equivalently used in monitoring the same patient, andthe clinical implementation of therapeutic intervention cut-off based onIU remains center dependent [57,70,71].

Considering that the commutability of the CMV WHO standard ismatrix and method-dependent [65,72], the development of a consensusprocedure for conversion factor calculation, may help to obtain morecomparable quantitation of viral loads in IU.

The GLaIT working group has recently taken a further step bypromoting a national study across transplant centers to identify center-specific conversion factors from copies/mL to IU/mL for the normal-ization of CMV DNA load, using a shared procedure of data collectionand analysis [73]. The study highlighted that a specific conversioncoefficient is needed for each matrix and the application of such acoefficient, when appropriately calculated, significantly improves theagreement of viral load values across laboratories. Still, differencespersist when grouping the results by diagnostic method, suggesting thata standardized procedure for calibrating methods against IU should alsobe applied during the validation of commercial assays.

This GLaIT study further strengths the recent Italian consensus andthe laboratory practice for the CMV disease management in the pre-emptive strategy, in which qPCR and whole blood are the pillars forCMV DNAemia monitoring [7,11]. The reduced variation in viral loadquantitation across centers allowed also to identify consensus cut-offsfor therapeutic intervention in pre-emptive therapy. In consideration ofthe rapid CMV replication rate, the GLaIT consensus encompasses alsoclear recommendations on the frequency of viral load monitoring. Aswell, the viral load values for initiating the antiviral treatment are in-dicated, for both HSCT recipients and SOT, always taking into con-sideration the difference in DNAemia in blood and plasma [11]. Whilein general, the treatment dismission is based on the finding of twoconsecutive negative viral load testing (Table 6) [1,7]. A comparison ofthe Italian consensus and current guidelines for pre-emptive strategy issummarised in Table 6.

As earlier suggested by Dioverti et al. [3], the Italian consensusundoubtedly endorses the application of molecular tests, on blood orplasma (as long as the same matrix is consistently used during thefollow-up), throughout prophylaxis for following fluctuations of CMVDNAemia as surrogate marker of subclinical viral replication and de-velopment of resistant CMV strains [74]. However, in prophylaxis, themonitoring frequency and viral cut-off for a therapeutic switch remain aconjecture so far. A comparison of the Italian consensus and currentguidelines for DNAemia monitoring in prophylaxis is summarised inTable 7.

3. Future perspectives

From the laboratory side in the next future, the implementation ofCMV disease management strategies might likely undergo evolutionthrough the identification of new indicators, the availability of CMV

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

6

Table6

CMVdiagno

sisandDNAem

iamon

itoring

associated

with

pre-em

ptivetherapy:

comparisonof

Italia

nCo

nsensus[11]

andInternationalg

uidelin

es.

Requ

irem

ent

Transplant

type

Italia

nconsensus

Internationalg

uidelin

eRe

f.

Eligiblepatients

SOT

alllow

risk

SOTpatie

nts

alllow

risk

SOTpatie

nts

[7,34]

HSC

Tall

all

[9]

Referencemethod

SOT,

HSC

Tqu

antitativeNATassay,

quantitativeNATassay

[7,9,34]

Methodrequirements

SOT,

HSC

Tsensitivity,m

easuring

rang

esensitivity

shou

ldbe

lower

than

103IU/m

L(3

logIU/m

L),

quantifi

catio

nprecisionshou

ldbe

with

in0.5logforq

uantities

equalo

rabo

ve10

3IU/m

L,and

with

in0.7logforqu

antitiesbelow

103IU/m

L

[7]

Resultreportingunit

SOT,

HSC

TIU/m

Lpreferable

-IU/m

Lpreferable

-Resultshallb

erepo

rted

inlogscale

[7,9]

Calculationofconversion

factortoIU

SOT,

HSC

Tdefin

edby

aconsensusprotocol

lack

ofindicatio

ns[7]

Samplematrix

SOT,

HSC

T-w

hole

bloo

drecommended

-plasm

aacceptable

-the

samematrixmustbe

used

whenmon

itoring

-lackof

indicatio

non

CMVdiseasediagno

sis

-eith

erwho

lebloo

dor

plasma.Th

esamematrixmustb

eused

whenmon

itoring

CMVrelapse

-other

tissues

shallb

eused

fordiagno

sisof

CMVdisease:

BALandtissueforCM

Vtissueinvasive

diseasevitreous

humou

rforCM

Vretin

itis

[7,9,34]

Monitored

parameters

SOT,

HSC

Tviralload,

viralk

inetic

viralload,

viralk

inetic

[7,9

]Cut-off

fortreatment

SOT

105copies/m

linbloo

d(5

logcopies/m

L),1

04copies/m

Lin

plasma(4

logcopies/m

L)each

transplant

centre

shou

lddefin

ethresholdvalues

basedon

arisk

policy

cut-o

ffshou

ldbe

adaptedaccordingto

themon

itoring

techniqu

eandthetransplant

metho

d[7,

9]HSC

T10

4copies

/mlinbloo

d(4

logcopies/m

L),1

03copies/m

Lin

plasma(3

logcopies/m

L)SignificantCM

VDNAvariations

SOT

CMVDNAvariations

exceeding0.5log(3

folds)

CMVDNAvariations

exceeding0.5log(3

folds)

[7]

HSC

TCM

VDNAvariations

exceeding0.5log(3

folds)

CMVDNAload

doub

lingin

less

than

2days

[9]

Post-transplantmonitoringminimal

frequency

HSC

T,SO

Ton

ceeveryweekin

thefirst3mon

ths,

once

every2weeks

from

mon

th4to

6,on

ceamon

thun

tilantiv

iral

therapydiscon

tinuatio

n

-inHSC

Tat

leaston

ceeveryweekin

thefirst3mon

ths

-inSO

Tevery1-2weeks

during

thefirst3mon

ths

[9]

[34]

Monitoringincaseofrelapse

HSC

T,SO

Tminim

alfrequencymustbe

maintained,

intensificatio

nof

mon

itoring

shou

ldbe

considered

morefrequent

testingto

beconsidered

inhigh

-riskSO

Tpatie

nts

[7]

Monitoringduration

SOT

upto

12mon

thspo

st-transplant

3-4mon

thsaftertransplant

[7]

HSC

Tup

to22

mon

thspo

st-transplant

-minim

um3mon

ths

-tobe

extend

edfor6-1

2mon

thsin

presence

ofchronicgraft-v

ersus-ho

stdisease(GvH

D)or

prolon

gedT-cellim

mun

odefi

ciency

[9]

Therapydiscontinuation

SOT

whentw

oconsecutivenegativ

eresults

areob

tained

-afte

r1resultislessthan

theLLoQ

,whentheqP

CRLLoQ

islower

than

200IU/m

L,or

lack

ofdetectionon

2consecutivetests

-twoconsecutivenegativ

edeterm

inations

with

one-weekinterval

betw

eenthem

[7,34]

HSC

Twhentw

oconsecutivenegativ

eresults

areob

tained

lack

ofindicatio

nsResistantCM

Vdiagnosis

SOT,

HSC

TLack

ofindicatio

ns-d

etectio

nof

persistent

orincreasedviralloadafter3weeks

expo

sure

toantiv

iral

adequate

treatm

ent

[34]

-detectio

nof

persistent

orrecurrentCM

VDNAem

iadu

ring

2weeks

ofon

goingexpo

sure

toantiv

iral

therapy

[7,9]

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

7

specific antiviral agents and combined diagnostic methodologies. In thepre-emptive approach, the CMV replication kinetic has been recognizedas predictive of disease progression. In both blood and plasma, newtechnologies for nucleic acid extraction will be needed to process alarger volume of plasma, and to guarantee more reproducible results atthose low viral concentrations that in whole blood mark the early onsetand clearance of the infection [51]. and in plasma represent a potentialpredictor for self-resolving infections and relapse.

Advantages might be also achieved in the universal prophylaxisregimen by implementing the frequency of DNAemia monitoring byqPCR to optimise treatment duration and drive treatment adjustment[26].

Currently, a combined approach pre-emptive/prophylaxis is fa-voured as beneficial for the patient’s life quality and cost-effectivenessof the CMV management strategy [44,74–76].

When assuming routine viral load quantitation as the driver in fu-ture treatment, detection of emergent drug-resistant strains [77,78] anddetection of deficiency in the immune response, yet a major effort willbe necessary for improving labs logistics, workflow, and availability ofeasy-to-use instrumentation, as well as in evaluating costs under ahealth–economic perspective.

On the treatment side, the development of new drugs might reopenthe discussion over matrix suitability and consensus cut-off for treat-ment. Letermovir is a promising and specific anti-CMV agent withoutapparent toxic effect [79–81]. However, it inhibits the maturation ofthe viral DNA but not its replication. This might imply the accumulationof immature CMV-DNA and delayed viral clearance in either wholeblood or plasma and reference cut-offs as defined nowadays might notbe adequate to describe the follow up under Letermovir [27–29,59]. Aswell, RNAemia may be reconsidered as a predictive marker of diseaseprogression. It is demonstrated that the immediate-early (IE) mRNAscorrelate with the infection progression [82] and reliably describe thedisease progression with peaks and kinetics that resemble those ofDNAemia [52,54]. If so, the development of new strategies for CMVmonitoring, based on IE mRNA quantitation, should be reconsidered.

Furthermore, at the Italian and international level there is a growingconsensus on monitoring of CMV-specific T-cell response for predictingindividuals at increased risk of CMV disease and guiding prophylaxisand pre-emptive decisions [7,9,11]. Recently a specific role was as-cribed to T-cell subtype (CD4+ and CD8+) in mounting the immuneresponse against CMV in the short [83] and long-term [25], the lack ofreconstitution of such protective immunity is pivotal in developingsymptomatic infections in the post-transplant period [70].

Therefore, methods for monitoring the T-cell activity should providequantitative and functional information on CMV-specific T cell sub-populations involved in the immuno-responses after transplant[7,9,11].

The available assays, Quantiferon and ELISpot, measure the IFN-γproduced by lymphocytes upon stimulation with CMV-pp65 protein-derived peptide, but only ELISpot differentiates and measures the CD4and CD8 activity [83,84]. Because ELISpot is highly influenced by thesample cellularity and might be of difficult resolution in severe neu-tropenic patients, a “normalised” ELISpot assay, that relates the cellactivity to the lymphocyte number [85] has been implemented. Thismay open the way for a standardized use of CMV-specific T-cell activityas a marker for CMV infection in the pre-transplant phase and of post-transplantation risk of CMV reactivation [84]. However, further evi-dence will be needed to demonstrate the efficacy and cost-effectivenessof such practice before its full endorsement within guidelines.

By applying the same cell-stimulation principle, adoptive T-cellimmunotherapy is seeking for therapeutic alternatives, aiming to re-constitute patient’s protective immunity [70]. CMV-pp65 peptides weredemonstrated to selectively trigger T-cell activity. These pre-committedcells are effective against autologous CMV infected targets in vitro [86]and show therapeutic effects in HSCT patients in vivo [87] after infu-sion. Clinical trials are ongoing to translate this approach into theclinics.

4. Conclusions

The continuous improvement of CMV disease management practicehas widely contributed to the success of organ transplantation. Theintroduction of molecular diagnostics has been pivotal in the processand the spreading of new molecular integrated, easy-to-use diagnosticinstruments will predictably result in a new acceleration in the stan-dardization of clinical and laboratory protocols. In the pre-emptiveapproach, the assignment of therapeutic clinical cut-off will strictlydepend on method equivalency.

The Italian scientific community has identified common key para-meters to describe the patient’s status and agreed on laboratory pro-cedures for efficient monitoring by qPCR in both SOT and HSCT andjointly worked on establishing conversion factors to IU. As a fact, aconsensus procedure for conversion factors calculation, including ex-perimental design and calculations, is crucial to support the use of theinternational standard and align results across methods and centers.The implementation of additional parameters such as DNAemia kineticsneeds also further efforts for standardization across centers. Designingmore stringent protocols for CMV monitoring after antiviral treatmentdiscontinuation remains a point for improvement in the prophylaxisapproach, to early detect and adequately treat late disease recurrenceand resistance development. The advent of the new drugs might changethe clinical approach and favour new combined strategies based on therevision of at-risk group stratification, the inclusion of technologiessuch as ELISpot, and molecular methods in a multidisciplinary

Table 7CMV DNAemia monitoring in prophylaxis: comparison of Italian Consensus and International guidelines.

Requirements Transplant type Italian consensus International guideline Ref.

Surveillance of CMVDNAemia

SOT not recommended - not recommended in adult SOT patients [7, 32]- applicable to paediatric SOT patients, weekly monitoring. should continuefor at least 3-4 months post-transplant or at least for 2-3 months afterantiviral discontinuation

HSCT recommended to identify emergency ofresistance

recommended in all patients under antiviral prophylaxis with Aciclovir andValganciclovir, Valaciclovir

[9]

Minimal duration oftreatment

SOT 3-6 months in all patients, starting fromday 10 after transplant

- 6-12 months in lung transplant and intestinal transplant- 3-6 months in kidney, heart and liver transplant

[32]

- 3 months of antiviral medication should be used for routine kidney,pancreas, liver, and heart transplant recipients

[7]

- 3-6 months in intestinal transplant recipients [7]minimum 6 months in lung recipients [7]

HSCT in all allo-transplant patients 100 days,starting from day 0 after transplant

lack of indication [9]

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

8

diagnostic. In this perspective teamworking between virologist andclinicians at Italian - AMCLI, SITO, GITMO - and international trans-plant centers, will be core to set aside the dualistic approach and re-define a strategy closer to the clinics.

Credit author contribution statement

All authors have equally contributed to the development and criticalappraisal of the paper.

Conflict of interest disclosure

The authors declare that they do not have anything to disclose re-garding funding or conflict of interest with respect to this manuscript.

Acknowledgements

The authors are grateful to ELITechGroup S.p.A. (Torino, Italy)forcovering the expenses of editorial support, to dr. Cristina Olivo(ELITechGroup S.p.A.) for the support and coordination, and to CarlaBenci and Manuella Walker for the drafting and editing of the paper.

References

[1] P.A. Andrews, V.C. Emery, C. Newstead, Summary of the british transplantationsociety guidelines for the prevention and management of CMV disease after solidorgan transplantation, Transplantation 92 (11) (2011) 1181–1187, https://doi.org/10.1097/TP.0b013e318235c7fc Review. PubMed PMID: 22002346.

[2] L. Azevedo, L. Pierotti, E. Abdala, et al., Cytomegalovirus infection in transplantrecipients, Clinics 70 (7) (2015) 515–523.

[3] M.V. Dioverti, R.R. Razonable, Clinical utility of cytomegalovirus viral load in solidorgan transplant recipients, Curr. Opin. Infect. Dis. 28 (4) (2015) 317–322, https://doi.org/10.1097/QCO.0000000000000173 Review. PubMed PMID: 26098497.

[4] D. Navarro, R. San-Juan, O. Manuel, et al., For the ESGICH CMV Survey StudyGroup, on behalf of the European Study Group of Infections in Compromised Hosts(ESGICH) from the Society of Clinical Microbiology and Infectious Diseases(ESCMID). Cytomegalovirus infection management in solid organ transplant re-cipients across European centers in the time of molecular diagnostics: an ESGICHsurvey, Transpl. Infect. Dis. 19 (Dec 6) (2017), https://doi.org/10.1111/tid.12773Epub 2017 Oct 25. PubMed PMID: 28859257.

[5] H.A. Freyer, R. Anderson, P.D. Minor, Collaborative study to evaluate the proposed1st WHO international standard for human cytomegalovirus for nucleic acid am-plification-based assays, WHO/BS/10.2138 (2010) 1–40.

[6] J.F. Fryer, A.B. Heath, Minor PD on behalf of the Collaborative Study Group. Acollaborative study to establish the 1st WHO International Standard for humancytomegalovirus for nucleic acid amplification technology, Biologicals 44 (4)(2016) 242–251, https://doi.org/10.1016/j.biologicals.2016.04.005 Epub 2016May 11. PubMed PMID: 27179913.

[7] C. Kotton, K. Deepal, A. Caliendo, et al., On behalf of the transplantation societyinternational CMV consensus group. The third international consensus guidelineson the management of cytomegalovirus in solid organ transplantation,Transplantation (2018) Publish Ahead of Print DOI: 10.1097/TP.0000000000002191.

[8] V. Emery, M. Zuckerman, G. Jackson, et al., On behalf of the British Committee forStandards in Haematology; British Society of Blood and Marrow Transplantation;UK Virology Network. Management of cytomegalovirus infection in haemopoieticstem cell transplantation, Br. J. Haematol. 162 (1) (2013) 25–39, https://doi.org/10.1111/bjh.12363 Epub 2013 May 6. PubMed PMID: 23647436.

[9] P. Ljungman, R. de la Camara, C. Robin, R. Crocchiolo, H. Einsele, J.A. Hill,K.N. Ward, Guidelines for the management of cytomegalovirus infection in patientswith haematological malignancies and after stem cell transplantation from the 2017European Conference on Infections in Leukaemia (ECIL 7), Lancet Infect. Dis.(2019), https://doi.org/10.1016/s1473-3099(19)30107-0.

[10] C. Cornella, M.C. Torazza, G.F. Strippoli, et al., On behalf of the Società Italiana diNefrologia. [Antiviral prophylaxis and pre-emptive therapy for the prevention ofCytomegalovirus infection in renal transplant recipients: guideline from the ItalianSociety of Nephrology], G. Ital. Nefrol. 24 (Suppl 37) (2007) S165–78 [Italian].PubMed PMID: 17347963. [In Italian].

[11] C. Girmenia, T. Lazzarotto, F. Bonifazi, F. Patriarca, G. Irrera, F. Ciceri, F. Aversa,F. Citterio, U. Cillo, E. Cozzi, E. Gringeri, F. Baldanti, R. Cavallo, P. Clerici,G. Barosi, P. Grossi, Assessment and prevention of cytomegalovirus infection inallogeneic hematopoietic stem cell transplant and in solid organ transplant: amultidisciplinary consensus conference by the Italian GITMO, SITO, and AMCLIsocieties, Clin. Transplant. 16 (July) (2019) e13666, , https://doi.org/10.1111/ctr.13666 [Epub ahead of print] Review. PubMed PMID: 31310687.

[12] Data from Gruppo Italiano per il Trapianto di Midollo Osseo (GITMO). Available athttp://www.gitmo.it. Last Accessed 12 August 2019. [In Italian].

[13] Trapianto di midollo osseo e di cellule staminali emopoietiche: l’impegno di Gitmo.

Intervista alla presidente Francesca Bonifazi_ https://www.quotidianosanita.it/scienza-e-farmaci/articolo.php?articolo_id=73518. Last Accessed 12 August 2019.[In Italian].

[14] Data from 2018 annual report of the National Transplant Centre of the ItalianNational Institute of Health (ISS). Available at http://www.trapianti.salute.gov.it/imgs/C_17_cntPubblicazioni_266_allegato.pdf. Last Accessed 12 August 2019. [InItalian].

[15] Fondazione Censis. IL VALORE DEL TRAPIANTO. Un’analisi empirica dei consumisanitari e dei costi dei trapiantati di rene in Italia 13192_2011 sintesi [In Italian].

[16] Data from Italian Ministry of Health. Valutazione di qualità dell’attività del tra-pianto di Fegato 2000-2016. Available at https://trapianti.sanita.it/statistiche/attivita/2019_D_QUALITA_ORGANI_FEGATO_00-16.pdf. Last Accessed 2 August2019. [In Italian].

[17] Data from Italian Ministry of Health. Valutazione di qualità dell’attività del tra-pianto di Rene 2000-2015. Available at https://trapianti.sanita.it/statistiche/attivita/2017_D_QUALITA_ORGANI_RENE_00-15.pdf. Last Accessed 2 August 2019.[In Italian].

[18] Data from Italian Ministry of Health. Valutazione di qualità dell’attività del tra-pianto di Cuore 2000-2014. Available at https://trapianti.sanita.it/statistiche/attivita/2017_D_QUALITA_ORGANI_CUORE_00-15.pdf. Last Accessed 2 August2019. [In Italian].

[19] D. Lilleri, G. Gerna, Strategies to control human cytomegalovirus infection in adulthematopoietic stem cell transplant recipients, Immunotherapy 8 (9) (2016)1135–1149, https://doi.org/10.2217/imt-2015-0028 Review. PubMed PMID:27485084.

[20] G. Gerna, D. Lilleri, A. Callegaro, et al., Prophylaxis followed by preemptive therapyversus preemptive therapy for prevention of human cytomegalovirus disease inpediatric patients undergoing liver transplantation, Transplantation 86 (Jul 1)(2008) 163–166, https://doi.org/10.1097/TP.0b013e31817889e4 PubMed PMID:18622294.

[21] J. Styczynski, G. Tridello, J.P. Donnelly, et al., Protective environment for hema-topoietic cell transplant (HSCT) recipients: the Infectious Diseases working PartyEBMT analysis of global recommendations on health-care facilities, Bone MarrowTransplant. (2018), https://doi.org/10.1038/s41409-018-0141-5.

[22] M. Stern, H. Hirsch, A. Cusini, et al., Cytomegalovirus serology and replicationremain associated with solid organ graft rejection and graft loss in the era of pro-phylactic treatment, Transplantation 98 (2014) 1013–1018.

[23] C.N. Kotton, D. Kumar, A.M. Caliendo, et al., on behalf of the TransplantationSociety International CMV Consensus Group. Updated international consensusguidelines on the management of cytomegalovirus in solid-organ transplantation,Transplantation 96 (4) (2013) 333–360, https://doi.org/10.1097/TP.0b013e31829df29d.

[24] L.M. McDevitt, Etiology and impact of cytomegalovirus disease on solid organtransplant recipients, Am. J. Health. Syst. Pharm. 63 (19 Suppl 5) (2006) S3–S9Review. PubMed PMID: 16990643.

[25] P. Ljungman, M. Hakki, M. Boeckh, Cytomegalovirus in hematopoietic stem celltransplant recipients, Hematol. Oncol. Clin. North Am. 25 (1) (2011) 151–169,https://doi.org/10.1016/j.hoc.2010.11.011.

[26] P. Ramanan, R. Razonable, Cytomegalovirus infections in solid organ transplanta-tion: a review, Infect. Chemother. 45 (3) (2013) 260–271.

[27] M. McIntosh, B. Hauschild, V. Miller, Human cytomegalovirus and transplantation:drug development and regulatory issues, J. Virus Erad. 2 (2016) 143–148.

[28] Y. Sankawa, Anwendung Und Dosierung Von CMV-Immunglobulinen NachTransplantation. 16 October, (2014) Mannheim, Germany. (Conference report).

[29] L. Bowman, J. Melaragno, D. Brennan, Letermovir for the management of cyto-megalovirus infection, Expert Opin. Investig. Drugs 26 (2) (2017) 235–241, https://doi.org/10.1080/13543784.2017.1274733.

[30] M. Cardillo, M. Scalamogna, C. Pizzi, et al., On behalf of the North Italy TransplantProgram. Kidney transplantation in the north Italy transplant program, Clin.Transpl. (2000) 371–373 PubMed PMID: 11512340.

[31] R. Razonable, A. Humar, Cytomegalovirus in solid organ transplant recipients-Guideline of the American Society of Transplantation Infectious DiseaseCommunity of Practice, Clin. Transplant. 28 (2019, Feb) e13512, , https://doi.org/10.1111/ctr.13512.

[32] J. Camargo, K. Komanduri, Emerging concepts in cytomegalovirus infection fol-lowing hematopoietic stem cell transplantation, Hematol. Stem Cell Ther. 10 (2017)233–238.

[33] A.J. Ullmann, M. Schmidt-Hieber, H. Bertz, et al., On behalf of the InfectiousDiseases Working Party of the German Society for Hematology and MedicalOncology (AGIHO/DGHO) and the DAG-KBT (German Working Group for Bloodand Marrow Transplantation). Infectious diseases in allogeneic haematopoieticstem cell transplantation: prevention and prophylaxis strategy guidelines 2016,Ann. Hematol. 95 (9) (2016) 1435–1455, https://doi.org/10.1007/s00277-016-2711-1.

[34] C. Lumbreras, O. Manuel, O. Len, et al., Cytomegalovirus infection in solid organtransplant recipients, Clin. Microbiol. Infect. 20 (Suppl. 7) (2014) 19–26.

[35] E. Jaskula, J. Bochenska, E. Kocwin, et al., CMV serostatus of donor-recipient pairsinfluences the risk of CMV infection/reactivation in HSCT patients, Bone MarrowRes. (2012) Article ID 375075.

[36] M. Boeckh, P. Ljungman, How we treat cytomegalovirus in hematopoietic celltransplant recipients, Blood 113 (23) (2009) 5711–5719.

[37] S. Hantz, F. Garnier-Geoffroy, M.C. Mazeron, et al., For the French CMV ResistanceSurvey Study Group. Drug-resistant cytomegalovirus in transplant recipients: aFrench cohort study, J. Antimicrob. Chemother. 65 (12) (2010) 2628–2640,https://doi.org/10.1093/jac/dkq368 Epub 2010 Oct 20.

[38] C.E. Fisher, J.L. Knudsen, E.D. Lease, et al., Risk factors and outcomes of

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

9

ganciclovir-resistant cytomegalovirus infection in solid organ transplant recipients,Clin. Infect. Dis. 65 (1) (2017) 57–63, https://doi.org/10.1093/cid/cix259.

[39] P.G. Young, J. Rubin, M. Angarone, J. Flaherty, et al., Ganciclovir-resistant cyto-megalovirus infection in solid organ transplant recipients: a single-center retro-spective cohort study, Transpl. Infect. Dis. 18 (3) (2016) 390–395, https://doi.org/10.1111/tid.12537 Epub 2016 Jun 9.

[40] T. Allice, A. Busca, F. Locatelli, et al., Valganciclovir as pre-emptive therapy forcytomegalovirus infection post-allogenic stem cell transplantation: implications forthe emergence of drug-resistant cytomegalovirus, J. Antimicrob. Chemother. 63(Mar 3) (2009) 600–608, https://doi.org/10.1093/jac/dkn521 Epub 2009 Jan 15.

[41] E. Shmueli, R. Or, M.Y. Shapira, I.B. Resnick, et al., High rate of cytomegalovirusdrug resistance among patients receiving preemptive antiviral treatment afterhaploidentical stem cell transplantation, J. Infect. Dis. 209 (4) (2014) 557–561,https://doi.org/10.1093/infdis/jit475 Epub 2013 Aug 27.

[42] S. Chou, Rapid in vitro evolution of human cytomegalovirus UL56 mutations thatconfer letermovir resistance, Antimicrob. Agents Chemother. 59 (10) (2015)6588–106593, https://doi.org/10.1128/AAC.01623-15 Epub 2015 Aug 10.

[43] K.E. Rolling, M.R. Jorgenson, J.L. Descourouez, et al., Ganciclovir-resistant cyto-megalovirus infection in abdominal solid OrganTransplant recipients: case seriesand review of the literature, Pharmacotherapy 37 (Oct 10) (2017) 1258–1271,https://doi.org/10.1002/phar.1987 Epub 2017 Sep 3. Review.

[44] C.J. Houldcroft, J.M. Bryant, D.P. Depledge, et al., Detection of low frequencymulti-drug resistance and novel putative maribavir resistance in im-munocompromised pediatric patients with cytomegalovirus, Front. Microbiol. 7(2016) 1317, https://doi.org/10.3389/fmicb.2016.01317 eCollection 2016.

[45] F. El Chaer, D.P. Shah, R.F. Chemaly, How I treat resistant cytomegalovirus infec-tion in hematopoietic cell transplantation recipients, Blood 128 (23) (2016)2624–2636 Epub 2016 Oct 19.

[46] RAVN 2017. Usage of antivirals and the Occurrence of Antiviral Resistance inNorway. Norwegian Institute of Public Health, Oslo 2018. ISBN: 978-82-8082-973-3.

[47] F. Baldanti, N. Lurain, G. Gerna, Clinical and biologic aspects of human cytome-galovirus resistance to antiviral drugs, Hum. Immunol. 65 (May 5) (2004) 403–409Review.

[48] G. Gerna, D. Lilleri, D. Caldera, et al., Validation of a DNAemia cutoff for pre-emptive therapy of cytomegalovirus infection in adult hematopoietic stem celltransplant recipients, Bone Marrow Transplant. 41 (May 10) (2008) 873–879,https://doi.org/10.1038/sj.bmt.1705986.

[49] E. Nicastro, S. Giovannozzi, P. Stroppa, et al., Effectiveness of preemptive therapyfor cytomegalovirus disease in pediatric liver transplantation, Transplantation 101(4) (2017) 804–810, https://doi.org/10.1097/TP.0000000000001531.

[50] G. Gerna, F. Baldanti, M. Torsellini, L. Minoli, M. Viganò, T. Oggionnis, T. Rampino,B. Castiglioni, A. Goglio, M. Colledan, C. Mammana, F. Nozza, L. Daniele, BergamoTransplant Group. Evaluation of cytomegalovirus DNAaemia versus pp65-anti-genaemia cutoff for guiding preemptive therapy in transplant recipients: a rando-mized study, Antivir. Ther. 12 (1) (2007) 63–72.

[51] T. Lazzarotto, A. Chiereghin, A. Piralla, et al., AMCLI-GLaIT working group.Cytomegalovirus and epstein-barr virus DNA kinetics in whole blood and plasma ofallogeneic hematopoietic stem cell transplantation recipients, Biol. Blood MarrowTransplant. 24 (8) (2018) 1699–1706, https://doi.org/10.1016/j.bbmt.2018.03.005 Epub 2018 Mar 12.

[52] G. Gerna, F. Baldanti, J. Middeldorp, D. Lilleri, Use of CMV transcripts for mon-itoring of CMV infections in transplant recipients, Int. J. Antimicrob. Agents 16 (Dec4) (2000) 455–460 PubMed PMID: 11118857.

[53] S.A. Ross, Z. Novak, S. Pati, S.B. Boppana, Overview of the diagnosis of cytome-galovirus infection, Infect. Disord. Drug Targets 11 (5) (2011) 466–474.

[54] G. Gerna, F. Baldanti, D. Lilleri, M. Parea, et al., Human cytomegalovirus im-mediate-early mRNA detection by nucleic acid sequence-based amplification as anew parameter for preemptive therapy in bone marrow transplant recipients, J.Clin. Microbiol. 38 (5) (2000) 1845–1853.

[55] N. Singh, Recent advances in the management of infections in liver transplant re-cipients, Curr. Infect. Dis. Rep. 3 (2) (2001) 123–130 PubMed PMID: 11286652.

[56] J.M. McBride, D. Sheinson, J. Jiang, N. Lewin-Koh, et al., Correlation of cytome-galovirus (CMV) disease severity and mortality with CMV viral burden in CMV-Seropositive donor and CMV-Seronegative solid organ transplant recipients, OpenForum Infect. Dis. 6 (2) (2019), https://doi.org/10.1093/ofid/ofz003ofz003eCollection 2019 Feb.

[57] S.K. Tan, J.J. Waggoner, B.A. Pinsky, Cytomegalovirus load at treatment initiationis predictive of time to resolution of viremia and duration of therapy in hemato-poietic cell transplant recipients, J. Clin. Virol. 69 (2015) 179–183, https://doi.org/10.1016/j.jcv.2015.06.006 Epub 2015 Jun 10.

[58] B. Muñoz-Cobo, C. Solano, E. Costa, D. Bravo, M. Clari, I. Benet, M.J. Remigia,J. Montoro, D. Navarro, Dynamics of cytomegalovirus (CMV) plasma DNAemia ininitial and recurrent episodes of active CMV infection in the allogeneic stem celltransplantation setting: implications for designing preemptive antiviral therapystrategies, Biol. Blood Marrow Transplant. 17 (11) (2011) 1602–1611, https://doi.org/10.1016/j.bbmt.2011.08.014 Epub 2011 Aug 24.

[59] Griffiths PD, Rothwell E, Raza M, et al. Randomized Controlled Trials to DefineViral Load Thresholds for Cytomegalovirus Pre-Emptive Therapy. PLoS ONE. 2016;11(9): e0163722. 10.1371/journal.pone.0163722.

[60] S.F. Atabani, C. Smith, C. Atkinson, R.W. Aldridge, M. Rodriguez-Perálvarez,N. Rolando, M. Harber, G. Jones, A. O’Riordan, A.K. Burroughs, D. Thorburn,J. O’Beirne, R.S. Milne, V.C. Emery, P.D. Griffiths, Cytomegalovirus replicationkinetics in solid organ transplant recipients managed by preemptive therapy, Am. J.Transplant. 12 (9) (2012) 2457–2464, https://doi.org/10.1111/j.1600-6143.2012.04087.x Epub 2012 May 17.

[61] H. Hebart, T. Rudolph, J. Loeffler, et al., Evaluation of the NucliSens CMV pp67assay for detection and monitoring of human cytomegalovirus infection after allo-geneic stem cell transplantation, Bone Marrow Transplant. 30 (3) (2002) 181–187.

[62] M. Furione, V. Rognoni, E. Cabano, F. Baldanti, Kinetics of human cytomegalovirus(HCMV) DNAemia in transplanted patients expressed in international units as de-termined with the Abbott RealTime CMV assay and an in-house assay, J. Clin. Virol.55 (4) (2012) 317–322 doi: 10.16/j.jcv.2012.08.017. Epub 2012 Sep 16.

[63] C. Kraft, W. Armstrong, A. Caliendo, Interpreting Quantitative CytomegalovirusDNA Testing: Understanding the Laboratory Perspective, Clinical InfectiousDiseases 54 (12) (2012) 1793–1797.

[64] H.H. Hirsch, I. Lautenschlager, B.A. Pinsky, et al., An international multicenterperformance analysis of cytomegalovirus load tests, Clin. Infect. Dis. 56 (3) (2013)367–373, https://doi.org/10.1093/cid/cis900 Epub 2012 Oct 24.

[65] S. Jones, E.M. Webb, C.P. Barry, et al., Commutability of cytomegalovirus WHOinternational standard in different matrices, J. Clin. Microbiol. 54 (6) (2016)1512–1519, https://doi.org/10.1128/JCM.03292-15 Epub 2016 Mar 30. PubMedPMID: 27030491; PubMed Central PMCID: PMC4879292.

[66] R.R. Razonable, A. Asberg, H. Rollag, et al., Virologic suppression measured by acytomegalovirus (CMV) DNA test calibrated to the world health organization in-ternational standard is predictive of CMV disease resolution in transplant recipients,Clin. Infect. Dis. 56 (2013) 1546–1553.

[67] I. Abbate, A. Piralla, A. Calvario, A. Callegaro, et al., On behalf of the AMCLI −Infections in Transplant Working Group GLaIT. Nation-wide measure of variabilityin HCMV, EBV and BKV DNA quantification among centers involved in monitoringtransplanted patients, J. Clin. Virol. 82 (2016) 76–83, https://doi.org/10.1016/j.jcv.2016.07.001 Epub 2016 Jul 2. PubMed PMID:27467016.

[68] J. Rychert, L. Danziger-Isakov, B. Yen-Lieberman, et al., Multicenter comparison oflaboratory performance in Cytomegalovirus and Epstein-Barr virus viral loadtesting using international standards, Clin. Transplant. 28 (12) (2014) 1416–1423,https://doi.org/10.1111/ctr.12473 Epub 2014 Nov 13.

[69] R.T. Hayden, Y. Sun, L. Tang, et al., Progress in quantitative viral load testing:variability and impact of the WHO quantitative international standards, J. Clin.Microbiol. 55 (Feb 2) (2017) 423–430, https://doi.org/10.1128/JCM.02044-16Epub 2016 Nov 16.

[70] M. Boeckh, W.J. Murphy, K.S. Peggs, Reprint of: recent advances in cytomegalo-virus: an update on pharmacologic and cellular therapies, Biol. Blood MarrowTransplant. 21 (2 Suppl) (2015) S19–S24, https://doi.org/10.1016/j.bbmt.2014.12.034.

[71] J.F. Camargo, E. Kimble, R. Rosa, et al., Impact of cytomegalovirus viral load onprobability of spontaneous clearance and response to preemptive therapy in allo-geneic stem cell transplantation recipients, Biol. Blood Marrow Transplant. 24 (4)(2018) 806–814, https://doi.org/10.1016/j.bbmt.2017.11.038 Epub 2017 Dec 5.

[72] R.T. Hayden, J. Preiksaitis, Y. Tong, X. Pang, Y. Sun, L. Tang, L. Cook, S. Pounds,J. Fryer, A.M. Caliendo, Commutability of the first world health organization in-ternational standard for human cytomegalovirus, J. Clin. Microbiol. 53 (10) (2015)3325–3333, https://doi.org/10.1128/JCM.01495-15 Epub 2015 Aug 12.

[73] F. Sidoti, A. Piralla, C. Costa, M.L. Scarasciulli, A. Calvario, P.G. Conaldi, P. Paba,C.F. Perno, A. Gaeta, G. Antonelli, G. Sodano, R. Santangelo, M. Sanguinetti,M.L. Vatteroni, L. Barzon, G. Palù, I. Abbate, M.R. Capobianchi, G. Piccirilli,T. Lazzarotto, F. Baldanti, R. Cavallo, Collaborative national multicenter for theidentification of conversion factors from copies/mL to international units/mL forthe normalization of HCMV DNA load, Diagn. Microbiol. Infect. Dis. (2019),https://doi.org/10.1016/j.diagmicrobio.2019.05.012 pii: S0732-8893(18)30703-X.doi.

[74] J.W. McGillicuddy, N.A. Weimert, D.J. Taber, et al., Can preemptive cytomegalo-virus monitoring be as effective as universal prophylaxis when implemented as thestandard of care in patients at moderate risk? Transplantation 89 (10) (2010)1218–1223, https://doi.org/10.1097/TP.0b013e3181d54ba6.

[75] R. Hellemans, P. Beutels, M. Ieven, et al., Cost analysis in favor of a combinedapproach for cytomegalovirus after kidney transplantation: a single-center experi-ence, Transpl. Infect. Dis. 15 (1) (2013) 70–78, https://doi.org/10.1111/tid.12023Epub 2012 Nov 23.

[76] L. Annemans, K. Moeremans, D. Mutimer, et al., Modeling costs and cost-effec-tiveness of different CMV management strategies in liver transplant recipients as asupport for current and future decision making, Value Health 5 (4) (2002) 347–358.

[77] S. Chou, Approach to drug-resistant cytomegalovirus in transplant recipients, Curr.Opin. Infect. Dis. 28 (4) (2015) 293–299, https://doi.org/10.1097/QCO.0000000000000170.

[78] P. Lischka, G. Hewlett, T. Wunberg, et al., In vitro and in vivo activities of the novelanticytomegalovirus compound AIC246, Antimicrob. Agents Chemother. 54 (3)(2010) 1290–1297, https://doi.org/10.1128/AAC.01596-09.

[79] F. Marty, P. Ljungman, R. Chemalay, et al., Letermovir prophylaxis for cytomega-lovirus in hematopoietic-cell transplantation, N. Engl. J. Med. 377 (2017)2433–2444, https://doi.org/10.1056/NEJMoa1706640.

[80] R. Chemalay, A. Ulmann, S. Stoelben, et al., Letermovir for cytomegalovirus pro-phylaxis in hematopoietic-cell transplantation, N. Engl. J. Med. 370 (2014)1781–1789, https://doi.org/10.1056/NEJMoa1309533.

[81] S. Stoelben, A. Wolfang, L. Renders, et al., Preemptive treatment ofCytomegalovirus infection in kidney transplant recipients with letermovir: results ofa Phase 2a study, Transpl. Int. 27 (1) (2014) 77–86, https://doi.org/10.1111/tri.12225 Erratum in: Transpl Int. 2014 Apr;27(4):416. PubMed PMID: 24164420.

[82] A.E. Greijer, H.M. Adriaanse, C.A. Dekkers, J.M. Middeldorp, Multiplex real-timeNASBAfor monitoring expression dynamics of human cytomegalovirus encoded IE1and pp67 RNA, J. Clin. Virol. 24 (Feb 1-2) (2002) 57–66.

[83] S. Calarota, J. Aberle, E. Puchhammer-Stöckl, F. Baldanti, Approaches for mon-itoring of non virus-specific and virus-specific T-cell response in solid organ

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

10

transplantation and their clinical applications, J. Clin. Virol. 70 (2015) 109–119.[84] M. Rittà, C. Costa, F. Sidoti, et al., Pre-transplant assessment of CMV-specific im-

mune response by Elispot assay in kidney transplant recipients, New Microbiol. 38(2015) 329–335.

[85] I. Cassaniti, F. Sidoti, K.M.G. Adzasehoun, et al., A bi-centric longitudinal analysisof HCMV-specific T-cell immune response in kidney tranplsant recipients (KTRs)-abstract, European Symposium of Clinical Virology ESCV (2017).

[86] D. Trivedi, R.Y. Williams, R.J. O’Reilly, G. Koehne, Generation of CMV-specific T

lymphocytes using protein-spanning pools of pp65-derived overlapping pentade-capeptides for adoptive immunotherapy, Blood 105 (7) (2005) 2793–2801, https://doi.org/10.1182/blood-2003-05-1433.

[87] G. Khoene, A. Hasan, E. Doubrovina, S. Prockop, E. Tyler, G. Wasilewski,R.J. O’Reilly, Immunotherapy with donor t cells sensitized with overlapping pen-tadecapeptides for treatment of persistent cytomegalovirus infection or viremia,Biol. Blood Marrow Transplant. 21 (9) (2015) 1663–1678, https://doi.org/10.1016/j.bbmt.2015.05.015.

P.A. Grossi, et al. Journal of Clinical Virology 123 (2020) 104211

11

This e-print is distributed with the support of MSD