Viral MoViral MoDiagnoDiagno

TransplapSeyed Alireza Nadji Ph DSeyed Alireza Nadji, Ph.D.Associate Professor of Medical VirolVirology Research CentergyShahid Beheshti University of Medic

olecularolecular osis inosis in antation

logy

cal Sciences

Which techniques qClinical Diagnosis

C it iCriteria;• Fast, Sensitive, Specific, Simple to co

Viral detection techniquesViral detection techniques• Electron microscopy

• Insensitive & laborious• Cell culture

• Time consuming, relative insensitive, lagrown

• Immunoassays (Serology) • Indirect & depends on host immune sys

• Direct detection (Antigen & Nucleic ADirect detection (Antigen & Nucleic A• NAT; universally applicable, revolutiona

should be used in s of Viral infection

nduct

rge part of clinically relevant viruses can not be 

stemAcid)Acid)ry technology 

HumanHuman CytomegaloCytomegaloovirusovirus

HCCMV continues to be an important

omplication after transplantationinical complications of HCMV• Gastrointestinal disease;

• can escape blood-based surveillance by PCR and the pp65 antigenemia assay in approximately 25% of patients

HCMV i i• HCMV pneumonia; most serious complication

• Rare manifestations include retinitisRare manifestations include retinitis and encephalitis

• invasive bacterial and fungal disease ll GVHDas well as GVHD

MV• Diagnosis of HCMV infection and

disease• Histopathology• Histopathology

• confirms the presence of tissue-invasive CMVdisease

• invasive procedure but recommended• another concomitant pathology (e.g. graft

rejection) or copathogens or negative HCMV testing in the blood

• repeated histopathology not clinically necessarynecessary

• Culture; specific but modest sensitivity and slow turn-around time

• Serology; limited utility for diagnosis• delayed or impaired ability to mount an

antibody response• antigenemia pp65 • molec lar assa s• molecular assays

• Q-NAT• Late mRNA pp67

HCMV DiAntigenemia assay pp65

Semiquantitative assay in HCMV-infected peripheral blood leukocytesinfected peripheral blood leukocytesHigher sensitivity than culture & and is comparable to NAT (PCR)Useful to guide

• preemptive therapy, for rapid and sensitive diagnosis of HCMV disease, g ,

• treatment responses

Main disadvantage• S bj ti th d• Subjective method• need to process the clinical sample within

few hours• limited utility in leukopenic patients• limited utility in leukopenic patients

iagnosisl lMolecular tests

• Detection of HCMV DNA (QNAT) or Late mRNA (pp67) are the preferred methods for the diagnosis of HCMVdiagnosis of HCMV

• HCMV RNA is indicative of active HCMV replication

• In contrast, detection of HCMV DNA may or may not reflect HCMV replicationmay not reflect HCMV replication

• a highly sensitive NAT may amplify latent viral DNA

• Hence, QNAT assays can differentiate• Active viral replication (typically associated with• Active viral replication (typically associated with

high viral load) from latent virus (low-level HCMV DNAemia if using highly sensitive tests)

• Higher HCMV load values are generally associated with tissue-invasive disease, while lower values are seen with asymptomatic HCMV infection andseen with asymptomatic HCMV infection, and intermediate-range viral loads are seen with HCMV syndrome

• however, there is wide overlap between these categories

• late mRNA-based anti-HCMV therapy showlate mRNA based anti HCMV therapy show comparable safety and efficacy with PCR-based therapy in patients after Transplantation

HCMV Dh tno consensus on how to use mo

diagnose HCMV pneumonia

PCR i th f t t d dPCR is therefore not accepted as depneumonia or gastrointestinal disea

• no data on what level of HCMV DNAi h HCMV diwith HCMV disease• negative predictive value of PCR is high

There are occasional patients with plate-onset gastrointestinal CMV disundetectable viral load in the blood

• these cases may be due to CMV disethese cases may be due to CMV diseless sensitive QNAT assays.

In HCMV encephalitis, PCR is a us

iagnosisl l th d t l i lolecular methods to conclusively and gastrointestinal disease

fi iti f f HCMVefinitive proof of HCMV aseA in BAL fluid or tissue correlates best

h, so it can be used to rule out disease

tissue-invasive disease (especially ( p ysease and retinitis) with very low to dase compartmentalization or the use ofase compartmentalization, or the use of

seful diagnostic tool

Threshold Quanrecomme

PAST, the biggest drawback of HCMV AT was the variability of test results oss laboratories due to the lack of ay standardizationdifferences in commercial detection reagents, calibration, nucleic acid extraction methods and the selection of primers and probes targetingthe selection of primers and probes targeting different genesup to a 3 log10 difference in viral load values across different laboratoriesSignificant variations in viral load may be due to SAMPLE TYPE

• WHOLE BLOOD samples is more sensitive and yields higher viral load and earlier time to viral detection compared to PLASMA samples

• In a study which compared plasma versus whole blood for monitoring of CMV levels during treatment of CMV disease, there was a higher rate of detectable virus at day 21 in the whole blood samples when compared to the21 in the whole blood samples when compared to the plasma samples (70% versus 52%)

ntities; no clear endation

• In 2011, the WHO released the firInternational Reference Standard fthe quantification of CMV nucleicacid

• CMV QNAT assays should now be calibrated to this standard

• may ensure uniformity in viral load reporting, thereby facilitating to defiviral thresholds for various clinical applicationsapplications

• preemptive therapy, disease prognostication, therapeutic monitoringg

• Threshold Quantities• A study; HCMV load of 250 IU/mL or greater

plasma / HSCT• A study; a viral load of 3,983 IU/mL in whole

as cut-off for starting pre-emptive therapy in Rpatients / SOT

Recommendatdiagndiagn

Viral culture of blood and urine has lidiagnosis and management of CMV Serologic assays to detect HCMV-Ig

d f th di i f CMV dig y g

used for the diagnosis of CMV diseasHCMV QNAT or pp65 antigenemia shHCMV diseaseHCMV QNAT or pp65 antigenemia smonitoring the response of CMV diseHCMV QNAT or pp65 antigenemia spredict risk of HCMV disease if preepredict risk of HCMV disease, if preepreventionHCMV QNAT assays should be calibReference StandardReference Standard

• Studies should report HCMV load in IU/mcalibrated to the WHO International Refe

Patients suspected to have tissue-inQ

pnegative QNAT or pp65 antigenemiahistopathology to confirm the clinic

tions for HCMV nosisnosismited clinical utility for prediction, disease in adult patients

gM and IgG antibodies should not be g gsehould be used for rapid diagnosis of

should be performed once weekly for ease to antiviral treatment.should be performed once weekly to mptive therapy is used for CMVmptive therapy is used for CMV

brated based on the WHO Internationa

mL using QNAT assays that have been erence Standardnvasive HCMV disease but with should have tissue biopsy and

cal suspicion of CMV disease

Herpes SimHerpes Simmplex virusmplex virus

Herpes SimTransplant recipients shed virufrequent and severe clinical mab l d hbe slower to respond to therapyMost symptomatic HSV diseasresults from reactivation of pre

• particularly early after transplansetting of antirejection therapsetting of antirejection therapy

• Hepatitis & Pneumonitis are the• Keratitis (infection of the corneKeratitis (infection of the corne

manifestation of HSV in the eyeHSV in immunocompromisedHSV in immunocompromised laboratory confirmation may b

mplex Virusus more frequently, have more anifestations of HSV, and may y.se in adult transplant recipients eviously acquired virus, ntation (in the 2nd weeks) and in the

e major manifestationsa) is the most commona) is the most common ehosts may be atypical, thus,hosts may be atypical, thus,

be helpful.

HSV laborato• Tissue cultureTissue culture

• most isolates are identified within 5 day• Timing of sampling is important; 

• sampling of genital lesions >5 days old h• sampling of genital lesions >5 days old h• DFA testing; more of mucocutaneouscan provide rapid resultsPCR• PCR assays 

• up to four fold more sensitive than tissuculture as the preferred diagnostic test

• diagnostic test of choice for HSV enceph• Tissue histopathology with immunocytoconfirm a PCR positive diagnosis the samcontaminated from oropharynx)contaminated from oropharynx) 

• Serologic testing is rarely useful for d• useful to acquire pre‐transplant for appr

• Diagnosis of HSV keratitis remains pri

ry Diagnosisys

had a yield of less than 35%had a yield of less than 35%

s lesions, BAL and other clinical samples, 

ue culture for diagnosing and have replaced viral 

halitisochemistry is helpful and is recommended to mples contaminated from another site (e.g. BAL 

iagnosing acute infectionsropriate post‐transplant risk stratificationimarily a clinical diagnosis

AdenovirusAdenovirus

Clinical Manifestations Vary with the sites affected and th

• Liver transplant recipients; commonldand urinary tract

• lung transplant recipients; acute flu‐likpneumonia and chronic changes such as brobronchiectasisbronchiectasis

• Heart transplant recipients; detection specimens might be predictive of coronary v

• In transplants involving the small bowIn transplants involving the small bowproportion of these patients develop dissem

• renal transplant recipients; Hemorrhagmore often in adult than pediatric

• Bone marrow transplant recipients; dbeen found to be predictive of invasive dise

• similar data are not available for organ 

e type of transplanted organly hepatitis, gastrointestinal tract, respiratory 

ke illness, diffuse alveolar damage or necrotizing onchiolitis obliterans, interstitial fibrosis or 

of adenoviral genome in myocardial biopsy vasculopathy and graft losswel, enteritis is common and a significant wel, e te t s s co o a d a s g ca tminated adenovirus disease gic cystitis and graft dysfunction are described 

detection of adenovirus at two or more sites has ase transplant recipients

ADENOVVIRUS …

ADENOVVIRUS …

BK polyomaBK polyomaa virusa virus

BK polyoma virus DBKV infection manifestation

• BK viruria, BK viremia and BKVN• BK viruria precedes BK viremia by a median• BK viruria precedes BK viremia by a median 

Diagnostic techniques • Urine cytology using Papanicolaou stainsy gy g p

• detection of ‘decoy’ cells in the urine

• BK viral load• In urine to monitor BKV infectionIn urine to monitor BKV infection

• Low levels of viruria may reflect asymp• Increasing viral load is indicative of act• Variables; fluctuations in urine content and;

contribute to interassay variations• BK viral copy number depending on supern

Diagnosis

of 4 weeks and BKVN by a median of 12weeksof 4 weeks and BKVN by a median of 12weeks

s or on phase contrast microscopyp py

ptomatic sheddingive BKV replicationd the method of sample processing and shipment, can p p g p ,

atants, cell pellets or resuspended urine are used for DNA preparation

… BKV diagnosis tests for BK viruria

• amplification of viral VP1 mRNA in urit t ti BKV li tiit represents active BKV replication

• Using a cutoff value of 6.5 × 105 BKV VP• a 94% sensitivity and specificity for BKV

• a larger cohort study reported urinary Baccurately diagnose BKVN

• a non‐invasive test for prediction of BK

BK viremia; can be measured quantitatively• BKVN

5000 i / L i i i f 100% /• 5000 copies/mL; sensitivity of 100% / a• >7700 copies/mL• 1000 copies/mL

• a threshold value that can accurately diagnos

rine may be a better test for BK viruria as

1 mRNA copies per nanogram total RNAVNBKV VP1 mRNA expression continued to 

KVN

y by PCR in plasma

f l i i di i i 15 2% false‐positive diagnosis in 15.2%

se BKVN without the need for a biopsy does not exist

RNA RespirRNA Respirvirusesviruses

ratoryratory