Molecular Microbiology in

Transplantation 28
Adnan A. Alatoom and Robin Patel

Contents 28.1 Common Infections in

28.1 Common Infections in Transplant Transplant Recipients
Recipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
28.2 Cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
• Solid organ and stem cell transplantations are
standard therapeutic options for selected
28.3 Epstein–Barr Virus (Posttransplantation
diseases
Lymphoproliferative Disease) . . . . . . . . . . . . 801
• Allograft rejection and opportunistic infection
28.4 BK Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802 are major causes of morbidity and mortality in
28.5 JC Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804 transplant recipients
28.6 Hepatitis C Virus . . . . . . . . . . . . . . . . . . . . . . . . . . 804 • Infection in organ transplant recipients occurs
in three phases posttransplantation, related to
28.7 Herpes Simplex Virus . . . . . . . . . . . . . . . . . . . . . 807
surgical factors, level of immunosuppression,
28.8 Varicella Zoster Virus . . . . . . . . . . . . . . . . . . . . . 807 and environmental exposures (Fig. 28.1)
28.9 Human Herpes Virus 6 . . . . . . . . . . . . . . . . . . . . 808 – First month posttransplantation
28.10 Parvovirus (Erythrovirus) B19 . . . . . . . . . . . 808
• Infection relating to surgical complica-
tions (e.g., wound infection, pneumonia,
28.11 Adenovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 809 urinary tract infection, line sepsis, infec-
28.12 Hepatitis B Virus . . . . . . . . . . . . . . . . . . . . . . . . . . 809 tion of drainage catheters)
28.13 Mycobacterium tuberculosis . . . . . . . . . . . . . . . 809 • Reactivated herpes simplex virus (HSV)
infection (individuals seropositive for
28.14 Toxoplasma gondii . . . . . . . . . . . . . . . . . . . . . . . . . 809
HSV pretransplantation not receiving
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 810 antivirals active against HSV)
– 1–6 months posttransplantation
• Opportunistic pathogens
– Cytomegalovirus (CMV), Pneum-
ocystis jiroveci, Aspergillus species,
Nocardia species, and Epstein–Barr
virus (EBV)-related posttrans-
A.A. Alatoom, MD, PhD, ABMM, FCAP (*) plantation lymphoproliferative disease
Department of Pathology, University of Texas
(PTLD)
Southwestern Medical Center, Dallas, TX, USA
• Reactivation of infection present
R. Patel, MD(CM), FRCP(C), D(ABMM), FIDSA, FACP,
in recipient before transplantation
F(AAM)
Divisions of Clinical Microbiology and Infectious
Diseases, Mayo Clinic, Rochester, MN, USA

L. Cheng, D.Y. Zhang, J.N. Eble (eds.), Molecular Genetic Pathology, 795
DOI 10.1007/978-1-4614-4800-6_28, # Springer Science+Business Media New York 2013
796 A.A. Alatoom and R. Patel

Fig. 28.1 Timeline of Timeline of Infections in Organ Transplant Recipients

infections in organ Infection type
transplant recipients
Parasitic Toxoplasma gondii

Epstein-Barr virus, Adenovirus

Varicella zoster virus, influenza, BK virus
Viral
Reactivated herpes simplex virus
Cytomegalovirus

Fungal Pneumocystis jiroveci,

Aspergillus species
Bacterial and Cryptococcus neoformans
candidal
infections relating Nontuberculosis mycobacteria
to surgical
Bacterial complications: Mycobacterium tuberculosis
wound infection,
line sepsis Nocardia species

Pneumonia, urinary tract infection

1 1 month 6 months
Transplant
(liver, kidney, pancreas, lung) 3158115B-1

(e.g., Mycobacterium tuberculosis, viral • Reactivated HSV infection (individuals

hepatitis, Histoplasma capsulatum,
Coccidioides immitis)
• Presentation of donor-transmitted infec-
tion [e.g., human immunodeficiency
virus, hepatitis B virus (HBV), hepatitis
C virus (HCV), fungal and mycobacterial
infection]
– >6 months posttransplantation
• Infection types typical of general popu-
lation (e.g., influenza, urinary tract infec-
tion, pneumococcal pneumonia)
• Reactivated varicella zoster virus
(VZV) infection (herpes zoster)
• In stem cell transplant (SCT) recipients, infec-
tion also occurs in three phases related to the
level and type of immunosuppression (i.e.,
neutropenia, cell-mediated and humoral
immune deficiency; Fig. 28.2)
– Preengraftment (typically first 30 days after
SCT, infection associated with neutrope-
nia, and breaks in mucosal
barriers – autologous and allogeneic SCT
recipients)
• Oral, gastrointestinal, and skin flora
(including Candida species)
• Aspergillus species
seropositive for HSV pretransplantation
not receiving antivirals active against
HSV)
– Postengraftment (30–100 days after
SCT – allogeneic SCT recipients)
• Opportunistic pathogens
– CMV pneumonia, hepatitis, and colitis
– P. jiroveci and Aspergillus species
– EBV-related PTLD
– Late (>100 days after SCT – allogeneic
SCT recipients, especially with graft versus
host disease)
• CMV
• Infection types typical of general popu-
lation (e.g., influenza, community-
acquired respiratory viruses)
• Infection with encapsulated bacteria
(e.g., Streptococcus pneumoniae,
Haemophilus influenzae)
• Reactivated VZV infection (herpes zoster)
• Molecular methods used in diagnosis and
management of infections in transplant
populations.
– Application of molecular methods in com-
mon posttransplant infections is presented
below.
28 Molecular Microbiology in Transplantation 797

Fig. 28.2 Timeline of Timeline of Infections in Stem Cell Transplant Recipients

infections in stem cell
Infection type
transplant recipients
Reactivated herpes simplex virus
Cytomegalovirus

Viral
Epstein Barr virus,
Respiratory viruses
(influenza).
Varicella zoster virus

Fungal Pneumocystis jiroveci

Aspergillus species (and others)

Streptococcus
Oral, skin, pneumoniae,
Bacterial gastrointestinal
flora (bacteria, Haemophilus
influenzae
Candida species)

↑ 30 days 100 days

Stem cell
infusion 3158115B-2

• CMV serostatus of recipient and donor

28.2 Cytomegalovirus
• Enveloped, double-stranded DNA virus,
Herpesviridae family
•
50–80% US adults infected
• Asymptomatically, intermittently shed (e.g.,
urine, saliva, semen, tears)
• Transmission
– Primary infection, secondary infection,
superinfection
• Primary infection develops when
CMV-seronegative transplant recipient
becomes infected
– Most often transmitted from donor
• Secondary infection or reactivation infec-
tion develops when endogenous latent
virus is reactivated in CMV-seropositive
transplant recipient
• Superinfection or reinfection occurs
when seropositive recipient receives
latently infected cells from seropositive
donor and virus that reactivates
posttransplantation is of donor origin
– Occurrence, severity of disease in trans-
plant recipients related to
– Organ transplant recipients – CMV-
seronegative recipients of organs from
CMV-seropositive donors highest risk
– SCT recipients – allogeneic
CMV-seropositive recipients of
stem cells from CMV-seronegative
donors highest risk
• Type of organ transplanted
• Immunosuppression
– Degree, type (e.g., antilymphocyte
antibodies, mycophenolate mofetil)
– Other means of transmission – close
contact with individual excreting CMV in
saliva or other body fluids (especially
young children) or blood transfusion
• Clinical presentation
– Wide range of clinical manifestations –
asymptomatic infection to severe, lethal,
CMV disease
– Mostly mild to moderate severity and
rarely fatal
– Fever and malaise alone is common;
leukopenia with or without thrombocytope-
nia may be present. Myalgias, arthralgias,
and occasionally frank arthritis may occur
798
– Solid organ transplant recipients
• Organ involvement correlates with
organ transplanted
– Hepatitis most frequent in liver
transplant recipients
– Pancreatitis most frequent in pancreas
transplant recipients
– Pneumonia most frequent in lung
transplant recipients
– Gastroenteritis
• Affects any segment of gastrointestinal
tract, including esophagus, stomach,
and small and large intestines
• Symptoms – dysphagia, odynophagia,
nausea, vomiting, abdominal discomfort,
gastrointestinal hemorrhage, and/or
diarrhea
• Potential intestinal perforation
• Endoscopic findings
– Erythema and diffuse, shallow erosions
or localized ulcerations (nonspecific)
– Biopsy – definitive diagnosis
• Particularly common among CMV-
seronegative recipients of allografts from
CMV-positive organ donors following
completion of prophylaxis – delayed
primary CMV disease
– CMV pneumonia
• Fever, dyspnea, cough, and/or
hypoxemia
• Radiography – bilateral interstitial,
unilateral lobar, nodular infiltrates
– CMV retinitis
• Asymptomatic or blurred vision, scoto-
mata, and/or decreased visual acuity
• Diagnosis by fundoscopy
• Usually presents >6 months
posttransplantation
– Other sites of involvement
• Liver, gallbladder, pancreas, epididy-
mis, biliary tract, skin, endometrium,
central nervous system, etc.
– CMV infection/disease in organ transplant
recipients associations
• Acute rejection
• Graft failure [vasculopathy (cardiac trans-
plant), bronchiolitis obliterans (lung trans-
plant), cirrhosis (liver transplantation)]
A.A. Alatoom and R. Patel
• More aggressive relapse of HCV
• Immunosuppression (other opportunis-
tic, especially fungal, infections)
• Other viral infections (EBV PTLD,
HHV6)
• Prevention
– CMV-seronegative, filtered or
leukoreduced blood products
– Selection of CMV-seronegative donors for
CMV-seronegative recipients (not typi-
cally done)
– Passive immunoprophylaxis (CMV
immunoglobulin)
– Pharmacologic prophylaxis (e.g., oral
valganciclovir, oral ganciclovir)
– Preemptive therapy
• Asymptomatic at-risk patients tested for
CMV DNA (or antigen) in blood, and if
positive preemptive therapy given
before symptoms develop
– May be preferred over prophylaxis
for patients at low or intermediate
risk for CMV disease
– Common in allogeneic SCT recipi-
ents due to risk of ganciclovir- or
valganciclovir-associated neutropenia
• Treatment
– Intravenous ganciclovir (oral
valganciclovir)
• Most widely used drug
• Undergoes initial phosphorylation by
thymidine kinase encoded by CMV
UL97 (phosphotransferase). Cellular
enzymes add two more phosphates to
make active form (analog of guanosine
triphosphate) which is incorporated
into replicating DNA by CMV DNA
polymerase (encoded by UL54 [DNA
polymerase]) resulting in termination
of replication
• Side effect: leukopenia
– Foscarnet
• Inhibits DNA replication
• Usually administered with intolerance
of or failure to respond to ganciclovir
• Side effects: nephrotoxicity, electrolyte
disturbances
28 Molecular Microbiology in Transplantation 799

Table 28.1 Nonmolecular tests for cytomegalovirus

Assay Principle Advantages Disadvantages Comments
Histology, Intranuclear inclusions Specific and Insensitive and requires invasive Useful for
cytology in tissue, definitive procedure gastrointestinal disease
immunohistochemistry, diagnosis which may occur
in situ hybridization without detectable
DNAemia
Culture Focal cytopathic effect Low sensitivity for CMV Gold standard for CMV
in cell lines [e.g., human viremia, prolonged turnaround detection
embryo lung fibroblasts time
(MRC5)]
Shell vial Immunofluorescent- More sensitive Low sensitivity for CMV
assay labeled antibodies and faster than viremia
against CMV antigens conventional
culture (1–2 days)
Serology lgG produced during lgM not useful in transplant Used to assess immune
primary infection, recipients status prior to
persists lifelong transplantation and for
donor testing
CMV Detects structural Rapid turnaround Labor-intensive; requires
antigenemia protein pp65 on surface time processing within 6–8 h of
of infected lymphocytes specimen collection:
compromised by low leukocyte
count

– Cidofovir – Treatment of clinical CMV disease

• Inhibits DNA replication
– CMV immunoglobulin
• Diagnosis
– Nonmolecular assays for CMV
diagnosis – Table 28.1
– Clinical utility of molecular CMV testing
in transplant recipients
• Diagnosis of active CMV disease
– CMV causes latent infection and may
be detected without symptoms; latent
infection distinguished from active
disease by
• Cutoff values – CMV disease
associated with higher viral loads
asymptomatic infection (cutoffs
may vary between assays)
• Targeting mRNA (present in
active infection) instead of DNA
(present in active and latent
infection)
• Measurement of viral load in
plasma versus whole blood or
leukocytes
• Monitoring response to antiviral
therapy
usually requires 2 weeks full-dose
intravenous ganciclovir
• Document clearance of viremia
before intravenous therapy is
stopped
– Clearance time inversely correlates
with pretreatment viral load
– Persistent on therapy elevated viral
load suggests ganciclovir resistance
• Molecular assays for detection and quantifica-
tion of CMV nucleic acid
– Conventional or real-time PCR [qualita-
tive, quantitative (viral load)]
• Performance varies due to differences
in acceptable specimen type,
nucleic acid extraction, target genes,
primer sequences, quantitation stan-
dards, amplified product detection
methods, etc.
– CMV viral load by PCR – plasma or whole
blood
• Indications
– Preemptive therapy marker (thresh-
olds vary between SCT and solid
organ transplant recipients)
800
– Diagnosis of CMV-associated signs
and symptoms
– Monitoring response to antiviral
therapy
• World Health Organization Interna-
tional Standard for CMV available
from National Institute for Biological
Standards and Control (NIBSC product
code: 09/162) for standardization
– Without this standard, results not
always comparable between assays
use same assay for monitoring indi-
vidual patients
• PCR advantages versus pp65 antigenemia
– CMV DNA stable in whole blood/
plasma for prolonged periods
– PCR may be performed with low leu-
kocyte count
– Qualitative PCR
• Aid to diagnose CMV disease
– Various clinical specimens (e.g.,
cerebrospinal fluid, urine, various
tissues, respiratory specimens, body
fluids)
– Formalin-fixed, paraffin-embedded tissues
• CMV antigens demonstrated by
immunohistochemistry
• DNA demonstrated by in situ
hybridization
– FDA-cleared non-PCR molecular diagnos-
tic test example
• CMV pp67 mRNA (bioMérieux, Dur-
ham, NC)
– Nucleic acid sequence-based ampli-
fication (NASBA)
– Qualitative
– Detects pp67 mRNA in whole blood
– High levels pp67 expressed in active
disease (not latent infection)
• Testing for antiviral resistance
– De novo resistance rare – resistance usually
occurs after prolonged therapy
– Risk factors
• CMV donor seropositive/recipient sero-
negative organ transplant
• High viral load
A.A. Alatoom and R. Patel
• Profound immunosuppression
• Suboptimal antiviral therapy
• Lung or kidney–pancreas transplant
recipient
– Indication for antiviral resistance testing
• Inadequate antiviral treatment response
– Stable or rising viral load or persis-
tence of symptoms after 2–3 weeks
full-dose intravenous therapy
– Assay types for detection of antiviral
resistance
• Phenotypic detection of CMV antiviral
resistance
– Growth of CMV in presence of
varying concentrations of antivirals
– Plaque reduction assay – concentration
that reduces number of plaques by
50% relative to control wells without
drug is 50% inhibitory concentration
(IC50)
– Time-consuming (4–6 weeks),
labor-intensive, subjective, not
standardized
– Genotypic
• Genotypic detection of CMV antiviral
resistance – UL97 and/or UL54 ampli-
fied using PCR and PCR products
sequenced
• From cultured virus or directly from
clinical specimen
• Ganciclovir resistance most commonly
due to point mutations/deletions in
UL97 (foscarnet and cidofovir not
affected)
– Decreased levels of ganciclovir
triphosphate in CMV-infected cells
– Mutations at codons 460, 594, and
595 most common
• UL54 point mutations/deletions
– Less frequent than UL97 mutations
– If selected by ganciclovir or
cidofovir, confers cross-resistance to
one another but usually not foscarnet
– If selected by foscarnet, usually
does not confer cross-resistance to
ganciclovir or cidofovir
28 Molecular Microbiology in Transplantation 801

Table 28.2 Risk factors and
poor prognostic factors of
posttransplant
lymphoproliferative disease
(PTLD)
Risk factors Poor prognostic factors
Early PTLD Poor performance status
Primary EBV infection Multisite disease
Young recipient age Central nervous system disease
Type of organ transplanted Monoclonal disease
CMV disease T cell or NK cell PTLD
Antilymphocyte antibody receipt EBV-negative PTLD
Late PTLD Hepatitis B or C virus coinfection
Recipient (versus donor) origin disease
Duration/amount of immunosuppression
Proto-oncogene or tumor suppressor gene
Type of organ transplanted
mutation
Older recipient age

• Risk factors
28.3 Epstein–Barr Virus – EBV seronegativity prior to organ
(Posttransplantation transplantation
Lymphoproliferative Disease) – CMV disease
– Type and intensity of immunosuppression
• Enveloped, double-stranded DNA virus, (especially administration of
Herpesviridae family antilymphocyte therapy for rejection)
• >90% adults infected – High EBV viral load
• Transmission • Clinical presentation
– Donor-transmitted infection (organ donor – Median time to onset
seropositive/recipient seronegative) • Organ transplant recipients – 6 months
– Blood transfusion, exposure to saliva of • SCT recipients – 3 months
infected asymptomatic person – SCT recipients – more severe,
– Reactivation disseminated versus organ transplant
• Disease associations recipients
– Posttransplantation lymphoproliferative – 1–15% of transplant patients
disease (PTLD) • Small intestinal transplant recipients,
• Rarely non-PTLD disease up to 20%
• Infectious mononucleosis • Pancreas, heart, lung, and liver trans-
– Sore throat, malaise, fever, headache plants recipients, 3–12%
• Hemophagocytic syndrome • Renal transplant and SCT recipients,
• Posttransplantation Lymphoproliferative 1–2%
Disease Pathogenesis – Mortality, 40–70%
– EBV replication, without lymphocytes that – More frequent in children than adults
normally control expression of EBV- (children more likely seronegative)
infected, transformed B cells (due to – Risk factors and prognostic factors; see
antilymphocyte therapy) Table 28.2
• Allogeneic SCT recipient, EBV- – EBV genome in majority (>90%) of early
infected B cells usually donor-derived (within first year after organ transplant)
• Organ transplant recipient, EBV is B cell PTLD
typically released from transplanted – 21–38% of late PTLD EBV-negative,
organ and infects recipient B cells non-B cell
802
– Clinical presentations include fever
(including “fever of unknown origin”),
weight loss, adenopathy, abdominal pain,
anorexia, jaundice, gastrointestinal bleed-
ing, intestinal perforation, renal dysfunc-
tion, liver dysfunction, pneumothorax, and
pulmonary infiltrates or nodules
– Often multicentric, may involve central
nervous system, eyes, gastrointestinal
tract, liver, spleen, lymph nodes, lungs,
allograft, oropharynx, and other organs
• Diagnosis
– Serology
• Determines pretransplant donor and
recipient EBV serostatus (assess PTLD
risk)
• Unreliable for diagnosis of PTLD
– PTLD diagnosis
• Definitive diagnosis – biopsy
• Monoclonal, oligoclonal, and polyclonal
• B cell or T cell lymphoma
• Diagnosis of EBV-associated
PTLD – EBV DNA, RNA, or protein in
biopsy tissue
– Gold standard: in situ hybridization
targeting EBER1 and/or EBER2
• More sensitive than targeting viral
DNA because EBER is expressed
at high levels in infected cells
• More sensitive than
immunohistochemistry
• Example commercial systems
(Ventana [Tucson, AZ], Leica
[Bannockburn, IL], Dako
[Glostrup, Denmark], Invitrogen
[Carlsbad, CA], Biogenex [San
Ramon, CA])
– Conventional or real-time PCR [qualitative
or quantitative (viral load)]
• Workup of transplant recipient with
symptoms suggestive of PTLD
– High EBV load triggers search for
mass lesions with computerized
tomography of chest, abdomen, and
pelvis, or organ dysfunction,
pinpointing potential site(s) for biopsy
• Increase in EBV viral load in peripheral
blood (whole EDTA blood, peripheral
A.A. Alatoom and R. Patel
blood lymphocytes, plasma) may be
detected before development of EBV-
associated PTLD; levels typically
decrease with effective therapy
– Levels of EBV DNA above threshold
trigger evaluation for PTLD (see
above); even if PTLD not found
may trigger preemptive lessening of
immunosuppression, administration
of murine humanized chimeric
anti-CD20 monoclonal antibody
(Rituximab), or adoptive
immunotherapy
– Cutoff values to trigger evaluation
unclear
• Some pediatric liver and heart
transplant recipients have chronic
high viral loads
• Low level elevated EBV viral
load frequent; may resolve with-
out intervention
• No standardization of optimal assay
technique, sample type (i.e., whole
blood, lymphocytes, plasma), or sam-
pling schedule
• Treatment
– Early detection; reduction in
immunosuppression – response high in
low risk patients
– Murine humanized chimeric anti-CD20
monoclonal antibody (Rituximab)
– Adoptive immunotherapy using donor-
derived cloned EBV-specific cytotoxic
T cells
– Chemotherapy
– Surgical resection
– Radiotherapy
– Antiviral drugs (limited role)
28.4 BK Virus
• Nonenveloped, double-stranded DNA virus,
Polyomaviridae family
– 75% sequence homology with JC virus
– Four genotypes (1–4)
• Primary infection in childhood; 60–90%
adults infected
28 Molecular Microbiology in Transplantation
– Following primary infection, remains
latent in urogenital tract, B lymphocytes,
or other tissues (e.g., spleen, brain)
– May reactivate with urinary shedding of
infected urothelial cells following
immunosuppression
– Up to 5% of normal hosts – asymptomatic
viruria
• Viruria after transplantation
– Kidney transplantation, 27%
– Heart transplantation, 26%
– Liver transplantation, 8%
• Viremia after transplantation
– Kidney transplantation, 12%
– Heart transplantation, 7%
• Clinical presentation
– Polyomavirus-associated nephropathy
(PVAN) of allograft – kidney transplant
recipients
• Incidence varies, up to 8%
• Histologic progression – initial cyto-
pathic stage, followed by cytopathic
inflammatory stage and late stage tubu-
lar atrophy and fibrosis
• Mostly in first posttransplant year
• Graft loss 15–80%
• Definitive diagnosis requires renal
allograft biopsy with in situ hybridiza-
tion for BK virus
– Hemorrhagic cystitis (HC) – SCT
recipients
• Risk greater for allogeneic than autolo-
gous SCT recipients (rare in organ trans-
plant recipients)
• Typically 1–18 weeks after
transplantation
• Symptoms similar to bacterial urinary
tract infection
• Resolves spontaneously
• Diagnosis and monitoring
– Urine cytology
• BK virus-infected – “decoy”
cells – enlarged round nuclei with
ground glass appearance and marginal
chromatin
– Useful for early diagnosis PVAN
with 100% sensitivity but low posi-
tive predictive value (<30%)
803
– Not specific for BK virus – may be
found with JCV, adenovirus
– Renal biopsy
• Viral cytopathic changes in renal tubu-
lar epithelium
• Early stage PVAN may be focal (false
negative biopsy)
– Molecular assays
• Active replication of BK virus in
urothelial cells, with tissue damage,
releases BK virus into urine and blood
• Early diagnosis of BK virus replication
before renal function deteriorates
• Guide management of immunosuppres-
sive therapy
• Monitor response to intervention
• Real-time PCR – method of choice for
BK virus viral load
• Assays differ in equitable detection of
genotypes, limit of quantitation and
dynamic ranges, sample type (urine vs.
urine sediment vs. unextracted urine;
plasma vs. serum vs. whole blood),
DNA extraction and purification
method, primer and probe sequences,
and PCR amplification conditions
– Plasma preferred over serum, whole
blood
– Whole urine preferred over urine
sediment
• Clinical utility of molecular testing in kidney
transplant recipients
– Diagnosis of PVAN with allograft dysfunc-
tion or preemptively (Fig. 28.3)
– Urine testing
• Cytology may be used as screening test,
followed by BK viral load testing, if
positive
• PCR more sensitive than cytology; BK
virus DNA may be present in urine ear-
lier than decoy cells
– Plasma testing
• BK viremia occurs only when virus is
detected in urine and is prerequisite for
progression to PVAN
– Rare transient BK viremia without
urinary excretion may reflect
reactivation of BK virus in
804 A.A. Alatoom and R. Patel

Fig. 28.3 BK virus

Screen every 3 months for 2 years; then annually for 3 years
monitoring algorithm
example EITHER BK viruria - decoy cells detected or urine viral load > 7 log10 geq/ml → assess plasma viral load
OR plasma viral load
IF plasma viral load >4 log10 geq/ml for >3 weeks

Increased creatinine Normal creatinine

Allograft biopsy
OR
Allograft biopsy
Reduce immunosuppression,
and monitor plasma viral load

circulating leukocytes, PCR inhibi- • Etiologic agent of progressive multifocal

tors in urine, or sequence differ-
ences between urine and plasma
BK virus
• Median interval from onset of viruria to
onset of viremia – 1–3 months
• Viremia precedes PVAN – median 1–12
weeks
• PVAN higher BK viral load than no
PVAN
• Plasma viral load >104 genome equiva-
lents/ml >3 weeks – presumptive PVAN
• Monitoring of therapy
• Treatment
– Reduction in immunosuppression
• Enables immune system to recover and
control infection
• Risk of acute rejection
– Cidofovir, leflunomide, or intravenous
immunoglobulin
– Viremia resolves before viruria
• Clearance t1/2 6 h to 17 days
– Monitor viral load every 2–4 weeks
28.5 JC Virus
• Nonenveloped, double-stranded DNA virus,
Polyomaviridae family
– 75% sequence homology with BK virus
leukoencephalopathy (PML)
– Fatal central nervous system demyelinating
disease
– Brain biopsy
• Characteristic pathologic changes local-
ized mainly in oligodendrocytes and
astrocytes
• In situ hybridization for JC virus on
brain tissue
– Detection of JC virus DNA by PCR in
cerebrospinal fluid has largely replaced tis-
sue biopsy for diagnosis
28.6 Hepatitis C Virus
• Enveloped, positive sense, single-stranded
RNA virus, Flaviviridae family
– Genome encodes single open reading
frame coding structural proteins (one core
and two envelope proteins) and
nonstructural proteins (NS1–NS5)
• Envelope region hypervariable – high
mutation rate
– Six genotypes based on sequence homol-
ogy of 50 NTR
• Genotypes 1–6
– <70% homology in nucleotide
sequence
28 Molecular Microbiology in Transplantation 805

Acute infecion
Most asymptomatic

Clearance of HCA RNA Fulminant hepatitis

(15-25%) (Rare)

Extrahepatic manifestations
Chronic infection
(glomerulonephritis, arthritis
(75-85%)
cryoglobulinemia)

Chronic active hepatitis

Cirrhosis
(10-20% in 20 years)

Decompensated cirrhosis
(ascites, upper gastrointestinal Hepatocellular carcinoma
bleeding, hepatic encephalopathy) (1-4% per year)
(5-year survival rate, 50-60%)

Liver transplantation

Fig. 28.4 Clinical presentation of hepatitis C virus infection

– Types la and lb
60% of infections – Early infection (window period, 7–8

– United States –
70–75% genotype weeks)
1, most remaining types 2 or 3 – Some immunocompromised patients
•
1.6% adults infected; most common indica- (e.g., dialysis, agammaglobulinemia)
tion for liver transplantation – Delayed in some intravenous drug
• Transmission users
– Injection drug use, transfusion of blood • False positive (up to 30% in low preva-
products (before 1992), transplantation, lence populations)
dialysis, needle stick injury, vertical • Confirmatory test – recombinant immu-
(infected mother to child), sexual noblot assay (RIBA)
• Clinical presentation – Most helpful in low signal-to-cutoff
– Clinical presentation of HCV presented in ratio samples
Fig. 28.4 – Samples with high signal-to-cutoff
• Diagnosis of HCV infection ratio are rarely RIBA negative
– Serologic assays – Molecular assays
• Immunoassay • Qualitative assays
– Enzyme-linked immunosorbent – Uses
assay (ELISA) • Confirm infection
– Chemiluminescent assay • Document viral clearance
• Supplemental or reflex testing distin- during therapy, end of therapy,
guishes new from past infection and following therapy
• Antibody production absent completion
806
– Technologies
• Reverse transcription–polymerase
chain reaction
• Transcription-mediated amplifi-
cation (TMA)
– Ideally detect 50 HCV RNA IU/ml
or less and have equitable sensitivity
for detection of genotypes
• Quantitative assays
– Uses
• Quantify baseline RNA
• Measure viral decline during
therapy
– Technologies
• Reverse transcription–polymerase chain
reaction
• Branched DNA
• TMA
– HCV RNA levels above upper limit of
quantification of assay underestimated
• Samples retested after 1/10 to 1/100
dilution for accurate quantification
• Genotype determination
– Cure rates with therapy higher with geno-
types 2 and 3
• Allows shorter duration of therapy
(compared to genotype 1)
• Interleukin – 28B genotype
– Single nucleotide polymorphisms on chro-
mosome 19 in or near the interleukin-28B
gene predict successful antiviral treatment
response
• C (vs. T) allele advantages for single
nucleotide polymorphism rs129789860
• T (vs. G) allele advantages for single
nucleotide polymorphism rs8099917
• Clinical utility of molecular testing in trans-
plant recipients
– Organs from donors with HCV antibody
and detectable HCV RNA more likely to
transmit HCV infection than those from
donors with HCV antibody but without
detectable HCV RNA
– HCV infection independent risk factor for
mortality following liver transplantation
– Liver transplantation for HCV cirrhosis
• Recurrent HCV disease in most with
viremia at transplant
A.A. Alatoom and R. Patel
• Spontaneous viral clearance rare
• May lead to graft failure, cirrhosis
• Histologic progression accelerated com-
pared to general population
– Acute hepatitis associated with recurrent
HCV infection >50%, typically in first
6 months following liver transplant –
increased viral load, rise in serum
aminotransferase levels, and/or histologic
evidence of acute HCV infection
– Risk factors for severe recurrent HCV and
poor allograft survival after liver
transplantation
• Fibrosing cholestatic HCV syndrome
(infrequent severe disease with high
viral load occurring in first 6 months
after transplant)
• Early recurrence or severe histologic
inflammatory activity within 1 year
• Increased donor age (>50 years)
• High serum HCV RNA levels before or
after transplantation
• Certain immunosuppressive agents
(e.g., high dose steroids)
• Other infections (CMV, human immu-
nodeficiency virus)
– Serum viral RNA
• May reach pretransplant levels within
first few days postoperatively
• Serum RNA peaks 1–3 months
posttransplant, reaching levels many
fold higher than in pretransplant period
– Liver biopsy for evaluation of
posttransplant recurrent HCV infection
– Treatment
• Preemptive approach (treat immediately
following transplantation)
• Recurrence-based approach (patients
with histologic liver disease selected
for treatment)
• Recurrent HCV infection
– Liver transplant recipients usually
treated
– Other types of organ transplant
recipients usually not treated
(graft rejection/loss secondary to
interferon’s immunomodulatory
effects)
28 Molecular Microbiology in Transplantation
– Kidney transplantation of HCV positive
recipients – no increased risk of graft loss
or death following transplantation
• RNA levels and liver biopsy assist in
management of HCV infection before
transplantation
• Treat before transplantation
– HCV infection associated with transient
hepatitis and veno-occlusive disease in
posttransplant period in SCT recipients
• Persistent hepatitis associated with
increased risk of earlier cirrhosis
compared with HCV-infected
nontransplanted patients
28.7 Herpes Simplex Virus
• Enveloped, double-stranded DNA virus,
Herpesviridae family
• Following primary infection, virus remains
latent in sensory nerve ganglia
• Anti-HSV IgG antibodies
75% adults
• Most commonly presents as reactivation
infection
– Usually in first month after transplantation
– Oral or genital mucocutaneous lesions
– Most orolabial infections are mild,
although severe ulceration and discom-
fort, complicated by bacterial superinfec-
tion or esophageal involvement, are
possible
– Anogenital infection usually presents as
ulceration and may or may not have typical
vesicular appearance
– Rare zosteriform lesions on buttocks, or
mucocutaneous nodules or plaques
– Prevented by acyclovir, valacyclovir, gan-
ciclovir, or valganciclovir
• Occasional primary infection, transmitted
by contact from person to person or via
allograft
• Reactivation or primary HSV infection
occasionally causes:
– Pneumonitis, tracheobronchitis
• HSV pneumonitis usually secondary
process in intubated patients with pneu-
monia of other etiology
807
• Due to oropharyngeal shedding, detec-
tion of HSV in respiratory secretions
does not definitively imply HSV
pneumonitis
– Lung biopsy needed for definitive
diagnosis of pneumonitis
– Esophagitis
• Dysphagia
• Mimics candidal and CMV esophagitis
– Hepatitis
– Disseminated infection
– Ocular infection
– Central nervous system infection
• Diagnosis
– PCR more sensitive and faster than culture,
more sensitive and specific than Tzanck,
and more sensitive than direct fluorescent
antibody testing
• Mucosal or skin lesion – vigorously rub
culture transport swab over suspect lesion
– Assay should detect and differentiate
HSV types 1 and 2
28.8 Varicella Zoster Virus
• Enveloped, double-stranded DNA virus,
Herpesviridae family
•
90% of adult organ transplant recipients
seropositive before transplantation – at risk
for reactivation (zoster) typically after first 6
months of transplantation
– 11% renal transplant recipients within 4
years of transplant
– 37% SCT recipients within 3 years of
transplant
– Dermatomal distribution (may involve
multiple adjoining dermatomes), occasion-
ally with a few or many sites of cutaneous
dissemination at distant sites, or more
widespread dissemination with visceral
and/or central nervous system involvement
• 10% adult solid organ transplant recipients
seronegative
– Risk primary infection after transplantation
• Occurs at any time
• Acquired via contact with infected indi-
vidual via respiratory route
808
– Primary infection
• Chickenpox syndrome, hepatitis, or
fatal disseminated infection
– Screen transplant candidates for antibody
to VZV prior to transplantation
• Vaccinate organ transplant candidates
before transplantation if no history of
prior VZV disease and VZV seronegative
• Postexposure prophylaxis in nonimmune
transplant recipients
• Diagnosis
– PCR more sensitive and faster than culture
• Culture transport swab vigorously
rubbed over suspect lesion
• Cerebrospinal fluid tested as an aid to
diagnosis of VZV central nervous sys-
tem infection
• Treatment
– Localized dermatomal zoster – acyclovir,
famciclovir, or valacyclovir
– Primary infection – intravenous acyclovir
+/ varicella zoster immune globulin
28.9 Human Herpes Virus 6
• Enveloped, double-stranded DNA virus,
Herpesviridae family
• Primary infection
– Transmitted via infected donor organs,
cells, or cellular blood products
– Nontransplant childhood disease – roseola
infantum, asymptomatic infection
• Reactivation infection (common)
• Clinical presentation
– Asymptomatic
– Symptomatic
• Febrile illness
• Rash
• Pneumonitis
• Myelosuppression
• Hepatitis
• Encephalitis
– CMV reactivation
• Diagnosis
– PCR
A.A. Alatoom and R. Patel
• Plasma, serum, whole blood,
peripheral blood mononuclear cells,
whole blood, biopsy, and tissue
specimens
• Ideally detects and differentiates vari-
ants A and B
• As with CMV, does not differentiate
replicating from latent virus
– Differentiate latent from active
infection
• High/increasing viral load or
reverse transcription PCR to
detect RNA
• Serum rather than whole blood
– In situ hybridization of formalin-fixed,
paraffin-embedded tissues
– Culture from peripheral blood mononu-
clear cells (other clinical specimens)
• Labor-intensive
• Takes up to 21 days (1–3 days with shell
vial assay)
• Treatment – ganciclovir, foscarnet
28.10 Parvovirus (Erythrovirus) B19
• Nonenveloped, single-stranded DNA virus,
Parvoviridae family
• 60–90% adults seropositive
• Infects erythroid progenitor cells by binding
P antigen
• Clinical presentation
– Nontransplant patients
• Children – erythema infectiosum
• Adults – occasional arthropathy
• Pregnancy – hydrops fetalis
• Hemolytic disorders – severe anemia
– Transplant patients
• Usually presents in first 3 months after
transplantation
– Anemia is most common (weakness,
dyspnea, orthostasis)
– Rare – hepatitis, myocarditis, pneumo-
nitis, glomerulopathy, graft dysfunc-
tion, leukopenia, thrombocytopenia,
fever and flulike manifestations, rash,
arthralgia, carditis, or hepatitis
28 Molecular Microbiology in Transplantation
• Diagnosis
– PCR (plasma or serum) most sensitive non-
invasive technique for diagnosis of parvo-
virus B19-related anemia
• May remain positive for extended periods
– Bone marrow examination
• Giant pronormoblasts (not always
detected) suggest parvovirus B19
infection
– Serology – IgM lacks sensitivity
• Treatment
– Reduction in immunosuppression
– Intravenous immune globulin (contains
parvovirus B19-specific antibodies)
28.11 Adenovirus
• Nonenveloped, double-stranded DNA virus,
Adenoviridae family – 52 serotypes
• Respiratory transmission (also water, fomites)
• Normal host – respiratory tract infection, gas-
troenteritis, or conjunctivitis
• Transplant patients – respiratory tract infec-
tion (pneumonia, upper respiratory tract infec-
tion), disseminated infection, gastroenteritis,
hemorrhagic cystitis, meningoencephalitis, or
hepatitis
• Diagnosis – viral culture (long turnaround
time), PCR (qualitative, quantitative), or
histopathology
• Treatment – symptomatic, cidofovir, or
ribavirin
• Preemptive therapy – based on viral load, used
in some pediatric SCT populations
28.12 Hepatitis B Virus
• Enveloped, DNA virus, Hepadnaviridae family
• Recurrent HBV 80–90% patients following
liver transplantation
– Detected by appearance of hepatitis
B surface antigen 2–6 months after trans-
plantation, followed by hepatocellular injury
• Molecular methods used for diagnosis and
resistance testing
809
• Primary HBV, acquired from donor liver or by
blood product transfusion, usually mild
• Cirrhosis of liver allograft uncommon in
patients transplanted for fulminant HBV
28.13 Mycobacterium tuberculosis
• Transplant recipients increased risk for
primary and reactivation infection
• Disseminated disease more common
in transplant recipients than in other
populations
• Rarely transmitted by allograft
• Clinical presentation
– Cavitary and noncavitary pulmonary
disease
– Extrapulmonary – intestinal, skeletal, cuta-
neous, central nervous system, or dissemi-
nated disease
• Molecular methods used to diagnose
M. tuberculosis from positive cultures,
and directly from clinical specimens (e.g.,
sputum, tissues), and for drug resistance
testing
– FDA-approved test example
• AMPLIFIED™ Mycobacterium tuber-
culosis Direct Test (MTD) [Gen-Probe,
San Diego, California] – target-
amplified nucleic acid probe test for
detection of M. tuberculosis complex
ribosomal RNA in sputum,
bronchoalveolar lavage fluid, or bron-
chial or tracheal aspirates
28.14 Toxoplasma gondii
• Toxoplasmosis generally develops within the
first six months after transplantation with the
highest incidence in the second and third
months
• Usually results from reactivation of latent
disease in the seropositive donor heart when
transplanted into seronegative recipient but
may occur in any type of organ transplant
recipient
810
• Prophylaxis given to seronegative heart trans-
plant recipients who receive allografts from
seropositive donor
• Meningoencephalitis, brain abscess, pneumo-
nia, myocarditis, pericarditis, hepatitis, or
retinochoroiditis
• PCR can be performed on blood, cerebrospi-
nal fluid, respiratory secretions, or tissues
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811
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