infections

Kyong Ran Peck Division of Infectious Diseases Samsung Medical Center Sungkyunkwan University School of Medicine Herpesviruses

-Herpesviruses  (HSV-1 and HSV-2) Varicella-zoster virus (VZV, HHV-3) -Herpesviruses  (CMV, HHV-5) Human herpesvirus-6 and -7 (HHV-6, HHV-7) -Herpesviruses Epstein-Barr virus (EBV, HHV-4) HHV-8 (Kaposi’s sarcoma–associated herpesvirus) Fishman JA. Am J Transpl. 2017;17:856-879 Characteristics of herpesviruses

 Herpesvirus infections usually occur during childhood or adolescence.  Life-long latency is established.  Seroprevalence rates are high in adults except HHV-8

 Latent herpesviruses reactivate later especially when host immune status is compromised. SOT recipients are at risk of reactivation and severe infections Effects of Viral Infections

 Direct clinical manifestations - invasive disease  Pneumonitis, nephritis, , neutropenia  Allograft injury often greater than systemic manifestations  Immunomodulatory effects  Systemic immune suppression  other infections  “Indirect” or cellular effects  graft rejection  Oncogenesis, cell proliferation  Epstein-Barr virus: B-cell lymphoma (PTLD)  HHV-8 (KSHV): Kaposi’s sarcoma, effusion lymphoma  CMV: accelerated atherogenesis

Rubin RH. Curr Clin Top Infect Dis. 2002;22:125; Ljungman P. J Infect Dis. 2002;186:S99 Boeckh M Herpes. 2003;10:12 Herpesviruses in Transplantation Risk factors for the herpesviruses  Lack of pathogen-specific immunity (seronegative)  Recurrent reactivation/persistence for life  MHC-linked immunity may be defective in graft environment  “Crosstalk”— infections with one stimulate activation of others (eg, CMV ↔ HHV6, CMV → EBV-PTLD ) resulting in “herpesvirus syndrome” (multiple )

Dockrell DH Rev Med Virol. 2001;11:23 DesJardin JA. Clin Infect Dis. 2001;33:1358 Babel N. Transplantation. 2001;72:736 Diagnosis of herpesviruses

Methods Serology Difficult to diagnose reactivation Culture Gold standard method Highly specific Limited by its modest sensitivity and slow turn-around time Antigenemia Higher sensitivity than culture, and is comparable to NAT

Nucleic acid test Preferred methods for confirmative diagnosis (NAT) Quantitative NAT may differentiate active viral replication from latent virus Histopathology Specific diagnosis with immunohistochemical staining Confirms the presence of tissue-invasive disease May be needed when blood tests are negative Antiviral agents for herpesviruses

Agents Formulations Clinical uses Acyclovir IV, PO HSV, VZV PO HSV, VZV PO HSV, VZV

Ganciclovir IV CMV, EBV, HHV-6, 7

Valganciclovir PO CMV

Foscarnet IV Resistant HSV, CMV

Cidofovir IV CMV

Over 90% of adult SOT recipients are seropositive for HSV-1 Most HSV-1 and -2 diseases are from reactivation, particularly early after transplantation Clinical infections Mucocutneous herpes is the most common Orolabial herpes  Herpes encephalitis Visceral herpes in immunocompromised hosts , hepatitis, pneumonitis Diagnosis of HSV

 Clinical diagnosis is possible based on typical manifestations  Clinical presentation may be atypical in immunocompromised hosts

 Laboratory confirmation is needed, especially in atypical presentation or visceral involvement  Serology: not helpful for diagnosis  Tzanck smear: rapid but nonspecific (HSV or VZV)  Direct fluorescent antibody (DFA) staining: rapid and specific  Culture: confirmative but slow  Polymerase chain reaction (PCR): sensitive  Tissue histopathology: confirmative but difficult to acquire samples < Case 1>

M/51

Hepatocellular carcinoma d/t HBV s/p multiple TACE

2017.8.31 ABO incompatible living donor LT done (preLT HSV-1,2 IgG: positive, CMV IgG : positive)

POD #10 , globus pharyngeus developed POD #18 Odynophagia aggravation POD #18 EGD

Ulcers in oral cavity, pyriform sinus

Ulcers in , IT 23 cm and 40 cm < Case 1 >

Esophageal Bx. showed IHC positive for HSV

1. Esophagus, #1x1 : IT 40 cm, : Herpes esophagitis

2. Esophagus, #2x2 : IT 23 cm, biopsy : Herpes esophagitis Treatment of HSV

 Rapid initiation of acyclovir therapy is associated with improved outcome.  Reduction in immunosuppression should be considered for life-threatening HSV disease. (III)  Therapy should be continued for minimum of 5–7 days or until complete healing.  Therapy in severe disease (e.g. encephalitis) should be continued for a minimum of 14 days. (III)

Am J Transplant 2013; 13: 121-127 Treatment of HSV

Indication Treatment Severe, visceral, Acyclovir 30mg/kg IV in 3 divided doses (II-1) Disseminated, CNS disease Mucocutaneous Acyclovir 400mg PO three times daily (I) disease Valacyclovir 1g PO twice daily (I) Famciclovir 500mg twice daily (I) Acyclovir 15mg/kg IV in 3 divided doses if unable to take PO Acyclovir-resistant Foscarnet 80-120mg/kg IV in 2-3 divided doses (I) HSV Cidofovir (II-3)

Am J Transplant 2013; 13: 121-127 POD #18 Acyclovir 10mg/kg IV q8h was started  Symptoms improved

POD #24 Patient wanted to discharge Acyclovir was changed to Valacyclovir 1g TID PO

POD #41 f/u EGD after 3 weeks of treatment

Antiviral treatment was discontinued

 Primary varicella or herpes zoster

 Over 90% of adult SOT recipients are seropositive for VZV  Primary varicella: rare  Herpes zoster: 8-11% during the first 4 years after SOT

 Risk factors  Old age  Types of organ: heart, lung transplantation  MMF Diagnosis of VZV

 Clinical diagnosis based on typical manifestations  Herpes zoster: painful vesicles localized in dermatomal area of involvement  Disseminated in immunocompromised hosts

 Definitive laboratory tests  Useful for atypical cases  Disseminated, visceral disease or central nervous system

 Diagnosis  Serology: not helpful for diagnosis, use in risk stratification before transplant  Direct fluorescent antibody (DFA) staining: reliable , rapid  Culture: specific, but less sensitive for PCR and slow  PCR of vesicles is most sensitive test for VZV < Case 2 >

F/32

Acute fulminant /t 2008.07.14 Living donor LT done (preLT HSV-1,2 IgG: positive, VZV IgG: positive)

No specific event during routine follow-up

2017.7.5 Rt. Arm pain developed 2017.7.7 Multiple vesicles developed on Rt. Arm, back 2017.7.12 visited ED 2017.7.12 Erythematous papules with grouped vesicles were identified on Rt. Arm (C5) and multiple scattered vesicles on back and face Treatment of VZV infections Disease Treatment Acute primary varicella Acyclovir 30mg/kg IV in 3 divided doses (I) Herpes zoster Acyclovir 30mg/kg IV in 3 divided doses (I) Disseminated or invasive disease or zoster ophthalmicus or Ramsay-Hunt syndrome/ Herpes zoster oticus Herpes zoster Acyclovir 800mg PO five times daily Localized or Valacyclovir 1g PO three times daily or Famciclovir 500mg PO three times daily (II-1)

Am J Transplant 2013; 13: 138-146 2017.7.12 Previous varicella history was identified Acyclovir 10mg/kg IV q8h was started with diagnosis of disseminated herpes zoster

2017.7.13 Vesicle fluid PCR: positive for VZV 7/21

HD #11 she was discharged with additional famciclovir 500mg PO three times daily for 4 days because some lesions were not crusted Cytomegalovirus

 The seroprevalence rates of CMV ranges from 30–97%  Without a prevention strategy, CMV disease typically occurs during the first 3 months after SOT  Clinical CMV infections  CMV infection: the presence of CMV replication regardless of symptoms  CMV syndrome: symptomatic reactivation of CMV  CMV organ disease: CMV infection accompanied by clinical signs and symptoms of organ involvement < Case 3 >

F/37 Underlying alcoholic liver

2017.7.15 esophageal variceal bleeding  open gastric varix ligation, S-B tube insertion

2017.7.26 Deceased donor split LT Primary repair of stomach perforation done (preLT HSV-1,2 IgG: positive, CMV IgG : positive) Postop. #13 EGD was done for N-J tube insertion

 ulcer was observed on the high body of stomach, and biopsy was taken. Diagnosis of CMV Methods Serology Unreliable diagnostic tool in immunocompromised patients due to delayed or absent humoral responses Antigenemia Higher sensitivity than culture, and is comparable to NAT by PCR Useful to guide preemptive therapy, for rapid and sensitive diagnosis of CMV disease, and to guide treatment responses Nucleic acid test Preferred methods (NAT) Quantitative NAT may differentiate active viral replication from latent virus Histopathology Confirms the presence of tissue-invasive CMV disease Recommended in cases where another concomitant pathology (e.g. graft rejection) or copathogens are suspected, especially when patients do not respond to anti-CMV treatment May be needed when CMV disease is suspected but CMV testing in the blood is negative Culture Highly specific Limited by its modest sensitivity and slow turn-around time Postop. #13 EGD was done for N-J tube insertion CMV antigenemia: 26 / 200,000 WBC Ganciclovir 5mg/kg IV q12h was started

Stomach, #1x3 : LC of HB, biopsy : . Chronic , active, with granulation tissue and some CMV-positive cells, consistent with cytomegalovirus gastritis . No H. pylori identified. Treatment of CMV

 Ganciclovir and valganciclovir: the first-line agents for CMV treatment Drug Treatment Maintenance/prophylaxis Leukopenia: major ganciclovir 5mg/kg IV every 12h 5mg/kg IV once daily toxicity Ease of administration ValGCV 900mg PO twice daily 900mg PO once daily Leukopenia: major toxicity  Foscarnet, cidofovir: the second, third-line agents Drug Treatment Prophylaxis 60mg/kg IV every 8h Second-line agent Foscarnet NOT recommended 90mg/kg IV every 12h Highly nephrotoxic 5mg/kg once weekly x 2 Third-line agent Cidofovir then every 2 weeks NOT recommended Highly nephrotoxic thereafter Ganciclovir resistant CMV

 Ganciclovir-resistant (ganR) cytomegalovirus (CMV) infection in solid organ transplant (SOT) recipients has been increasingly reported  Suspicion of resistance in  failure to achieve ≥1 log reduction in CMV viral load or  failure to have a significant improvement in clinical symptoms despite ≥2 weeks of appropriate full-dose ganciclovir or valganciclovir treatment  Resistance mechanisms: UL97 and/or UL54 mutations  ganR-CMV is associated with  type of organ transplanted,  CMV serostatus of the recipient and the donor (CMV D+R−)  lower doses or longer duration of ganciclovir prophylaxis  high CMV viral loads  more intensive immunosuppression

Fisher CE et al. Clin Infect Dis. 2017;65:57-63 Ganciclovir resistant CMV

 Associated with  longer hospitalization  increased mortality

 Treatment  Reducing immunosuppression  High dose of ganciclovir (for UL97 mutations)  Foscarnet, cidofovir  Other agents: leflunomide, rapamycin  New antivirals: maribavir, brincidofovir, letermovir, cyclopropavir

Fisher CE et al. Clin Infect Dis. 2017;65:57-63 Epstein-Barr Virus

 EBV syndromes  Infectious mononucleosis in primary infections  Organ specific diseases: hepatitis, pneumonitis, gastrointestinal symptoms and hematological manifestations (leucopenia, thrombocytopenia, hemolytic anemia and hemophagocytosis)  Post-transplant lymhoproliferative disease (PTLD) in SOT recipients  Risk factors for PTLD  Early PTLD: primary EBV infection, type of organ, OKT3 and polyclonal antilymphocyte antibodies, young recipient age, CMV mismatch or CMV disease  Late PTLD: duration of immunosuppression, type of organ, older recipient age Incidence of PTLD in SOT recipients

Green M and Michaels MG. Am J Transpl. 2013;13:41-54 < Case 4 >

M/52

Decompensated LC d/t HBV 2005. Deceased donor LT (preLT HSV-1,2 IgG: positive, CMV IgG: positive, EB-VCA IgG: positive, EBV-EA: negative, EBNA: positive)

CKD d/t chronic glomerulonephritis 2016.3.14 Hemodialysis started via Lt. AVF 2016.5.8 visited ED with EGD: no specific lesion Sigmoidoscopy: active bleeding

HD #2 Colonoscopy: multiple shallow ulcers

Descending colon, hepatic flexure, 65,40 cm from anal verge, biopsy: . Chronic active inflammation with some cytomegalic inclusion . Consistent with CMV associated . HSV IHC: Negative, CMV IHC: Positive HD #2 Ganciclovir was started under the presumptive Dx. of CMV (1.25mg/kg 3 times weekly after HD)

HD #18 f/u colonoscopy showed remaining ulcers and patient was transferred to another hospital for continuation of ganciclovir CMV activates EBV 4 days after transfer, visited ER d/t RUQ pain 2016.5.30 Emergent extended right hemicolectomy was done Post-transplant lymphoproliferative disorder, diffuse large B cell lymphoma Diagnosis of EBV Methods Unreliable diagnostic tool in immunocompromised patients due to delayed or absent humoral responses EBV serology Important in the determination of pretransplant donor and recipient EBV serostatus for PTLD risk assessment Serial monitoring of high risk seronegative populations is NAT recommended (II-2) Depends largely on the location of suspected lesions Radiographic Brain CT or MRI be included as part of the initial work-up, as the imaging presence of central nervous system lesions will significantly for influence treatment and outcome Localization PET–CT: useful test in the evaluation of PTLD and monitoring response to therapy The gold standard for PTLD diagnosis (IHC) Histopathology Excisional biopsy is preferred Treatment of EBV and PTLD

Reduction of immunosuppression: initial approach Preemptive antiviral agents (acyclovir, ganciclovir)/passive antibody (IVIG): may lower risks of PTLD Monoclonal PTLD treatment Insufficient data on the efficacy of antivirals Surgical resection/local irradiation Monoclonal B cell antibody therapy (Anti-CD20): rituximab Treatment of CD20+ B cell PTLD Cytotoxic Quantitative Epstein–Barr virus shedding and its correlation with the risk of post-transplant lymphoproliferative disorder

 Over a 7.5-yr period, PTLD occurred in 66 (5.8%)1131 patients (700 SOT patients and 431 HCT patients)  SOT recipients developed PTLD significantly later than HCT recipients (median, 2.8 yr vs. 121 d; p < 0.001).

 The occurrence of PTLD was significantly related to the quantity of EBV (p < 0.001)

(copies/mL, whole blood)

Holman CJ, et al. Clin Transplant 2012;26;741-747 Preemptive strategies for EBV

High risk populations (D+/R-)  EBV viral load monitoring  Weekly or biweekly for the first year after transplant  Optimal methods are not determined: whole blood is adequate  Quantitative PCR –threshold not established  Treatment  Reduce immunosuppression  Preemptive antiviral agents (acyclovir, ganciclovir)/passive antibody (IVIG)  Rituximab  May lower risks of PTLD HHV-6 & HHV-7

 Cause of infantum ( subitum)  Latency in T lymphocytes  Seropositivity 90 % by age 3 HHV-6 & HHV-7

Reactivation : 20-40% in SOT Clinical manifestations `Direct' clinical manifestations: febrile syndromes, pneumonitis, hepatitis, encephalopathy and marrow suppression (analogous to the CMV viral syndrome and CMV organ specific disease) `Indirect' effects Immune modulating virus triggering of immunological phenomena Cofactor for CMV, HHV-7 transactivation of other herpesviruses Diagnosis of HHV-6 & HHV-7

Methods Serology Limited because of high seroprevalence rate Culture Laborious and not routinely used Antigenemia Rapid, relatively easy to perform, and may discriminate between active and latent infection, no clear cut-off level

Immunohistochemistry More informative than viremia in cases where tissue invasive HHV-6 disease is suspected

Nucleic acid Can distinguish between HHV-6A, HHV-6B and HHV-7, amplification assays but they may not differentiate active from latent infection Qualitative or quantitative HHV-6 PCR of the cerebrospinal fluid is useful to diagnose HHV-6 encephalitis in patients with the appropriate clinical signs Treatment of HHV-6 & HHV-7

 Treatment of asymptomatic viral reactivation: not recommended (II-2)  Treatment of HHV-6 encephalitis and other clinical syndromes: should be considered (III)  Reduction of pharmacologic immunosuppression may be complemented in the moderate or severe disease. (III)

 Antiviral therapy is not well established  in vitro susceptible to cidofovir, with ganciclovir or foscarnet preferred because of lower nephrotoxicity

 Routine monitoring is not indicated in SOT recipients

Le et al . Am J Transplant 2013; 13: 128-137 HHV-8

 Prevalence: widely variable according to the geographic region  North America, northern Europe, Asia: 0~5%  Mediterranean and Middle East: 5~20%  Parts of Africa: >50% Seroprevalence of specific IgG to herpesviruses in Korea Group Prevalence of tested antibody % (95% CI) HHV-8 EBV VZV CMV Blood donor 0 64.0 97.0 92.0 (n=200) (56.9-70.6) (93.6-98.9) (87.3-95.4) Visitors for STI 0 74.5 92.1 99.0 (n=220) (67.9-80.3) (87.6-95.4) (96.5-99.9)

HIV 7.0 88.5 95.5 98.5 seropositive (4.0-11.3) (83.2-92.6) (91.6-97.9) (95.7-99.7) (n=214)

Kim O. J Bacteriol Virol. 2001; 31: 275 HHV-8 (KAHV)

 Association Kaposi sarcoma (KS) Castleman disease Primary effusion lymphoma (PEL) in immunocompromised individuals from endemic regions Some cases of EBV-negative PTLD  Linked with Hemophagocytic syndrome Combination of febrile hepatosplenomegaly, pancytopenia, hypofibrinemia, and liver dysfunction Diagnosis of HHV-8

Methods Serology limited in the diagnosis of acute HHV-8 infection (III) Use in risk stratification before transplant in endemic regions (II-2) Immunohistoc Immunohistochemistry using monoclonal hemistry antibodies against HHV-8 antigens is useful for the pathological diagnosis of KS and other angiogenic proliferative diseases (II-2) Nucleic acid Used for the diagnosis of active HHV-8 infection testing Surveillance of HHV-8 infection in high risk patients (D+/R-) (II-2) Treatment of HHV-8

 Reducing the immunosuppressive regimen  Antiviral agents for the treatment of HHV-8 disease  The benefits are not defined  Ganciclovir, foscarnet, and cidofovir have activity against HHV-8 in vitro  Kaposi sarcoma  A switch to sirolimus from existing CNI should also be considered (II-3)  Local or isolated lesions: intralesional chemotherapy, surgical excision, or radiation therapy (II-2)  Visceral or severe disease: systemic chemotherapy using liposomal doxorubicin, paclitaxel, or other agents (II-2)

Am J Transplant 2013; 13: 128-137 J Infect Dis. 2008 Jul 1;198(1):23-30 Summary

 Life-long latency is established after primary infections of herpesviruses which usually occur during childhood or adolescence.  Reactivation of latent herpesviruses are common in SOT recipients.  Monitoring of reactivation using antigenemia or PCR followed by reduction on immune suppression and/or preemptive antiviral therapy is helpful for prevention of severe diseases in high risk patients.(D+/R- for CMV, EBV)  Clinical suspicion and early laboratory diagnosis are important.  Tissue pathology with immunohistochemical staining  Reduction of immunosuppression is needed for treatment  Antiviral agents (acyclovir, ganciclovir, foscarnet, and cidofovir) can be used according to virus species.  Ganciclovir resistance of CMV should be considered in clinical failure cases Thank you for your attention ! Determination, validation and standardization of a CMV DNA cut-off value in plasma for preemptive treatment of CMV infection in solid organ transplant recipients at lower risk for CMV infection.

BACKGROUND: Valganciclovir preemptive therapy guided by the viral load is the current strategy recommended for preventing CMV disease in CMV-seropositive Solid Organ Transplant Recipients (SOTR) at lower risk for developing CMV infection. However, universal viral load cut-off has not been established for initiating therapy. OBJECTIVES: Our goal was to define and validate a standardized cut-off determined in plasma by real-time PCR assay for initiating preemptive therapy in this population. STUDY DESIGN: A prospective cohort study of consecutive cases of CMV-seropositive SOTR was carried out. The cut-off value was determined in a derivation cohort and was validated in the validation cohort. Viral loads were determined using the Quant CMV LightCycler 2.0 real-time PCR System (Roche Applied Science) and results were standardized using the WHO International Standard for human CMV. RESULTS: A viral load of 3983 IU/ml (2600 copies/ml) was established as the optimal cut-off for initiating preemptive therapy in a cohort of 141 patients with 982 tests and validated in a cohort of 252 recipients with a total of 2022 test. This cut-off had a 99.6% NPV indicating that the great majority of patients at lower risk will not develop CMV disease without specific antiviral therapy. The high sensitivity and specificity (89.9% and 88.9%, respectively) and the relatively small numbers of patients with CMV disease confirm that real-time PCR was optimal. CONCLUSIONS: We have established a cut-off viral load for starting preemptive therapy for CMV-seropositive SOT recipients. Our results emphasized the importance of a mandatory follow-up protocol for CMV-seropositive patients receiving preemptive treatment.

Spanish Network for Research in Infect Dis & Marin-Gandul C, et al. J Clin Virol. 2013;56:13-8 Epstein-Barr Viral Load and Disease Prediction in a Large Cohort of Allogeneic Stem Cell Transplant Recipients

Background. to determine the clinical significance and predictability of EBV infections among a large cohort of recipients of allogeneic, unselected stem cell transplants. Methods. During 1988–1999, a total of 5479 consecutive serum samples obtained during 406 transplantations performed in Helsinki, Finland, were retrospectively analyzed by quantitative polymerase chain reaction for the presence of EBV DNA. Results. Overall, EBV DNA was noted in at least 1 serum sample for 57 patients (14.0%), of whom 22 (5.4%) were found to have progressively increasing and ultimately high (150,000 copies/mL) EBV DNA levels (median level, 179,000 copies/mL). In addition, 16 patients (4.0%) had low EBV DNA levels (median level, 3260 copies/mL) in isolated sera before death. Among the transplant recipients who survived, transient EBV DNAemia (median level, 3110 copies/mL), which apparently corresponded to asymptomatic EBV infection, was noted in 19 patients (4.7%). Conclusions. Low-level EBV DNA positivity in serum occurs relatively frequently after stem cell transplantation and may subside without specific treatment. However, high EBV DNA levels (i.e., 150,000 copies/mL) are strong predictors for the development of PTLD, are not spontaneously reversible, and should be treated immediately. If the EBV DNA level is 50,000 copies/mL, the patient can be classified as having life- threatening EBV infection. Alto SM et al, Clin Infecti Dis 2007; 45:1305-9 The Role of Antiviral Prophylaxis for the Prevention of Epstein–Barr Virus– Associated Posttransplant Lymphoproliferative Disease in Solid Organ Transplant Recipients: A Systematic Review

therapy for 56-100 days f/u for 8-23 mo

AlDabbagh MA, et al. Am J Transplant 2017; 17: 770–781 The Role of Antiviral Prophylaxis for the Prevention of Epstein–Barr Virus– Associated Posttransplant Lymphoproliferative Disease in Solid Organ Transplant Recipients: A Systematic Review

Treatment with HSV active agents in both studies.

AlDabbagh MA, et al. Am J Transplant 2017; 17: 770–781 Current preventive strategies and management of EBV related PTLD in SOT in Europe. Results of the ESGICH Questionnaire- based Cross-sectional Survey

Cumulative % 78.2 43.7 36.4 16.4

San-Juan R, et al. Clin Microbiol Infect 2015; 21: 604.e1–e9 Kaposi’s sarcoma in SOT (in Korea)

Ahn SJ. Korean J Dermatol 2005; 43; 1468 PTLD Categories of posttransplant lymphoproliferative disorder Early lesions Plasmacytic hyperplasia Infectious mononucleosis-like lesion Polymorphic PTLD Monomorphic PTLD (classify according to the lymphoma they resemble) B cell neoplasms Diffuse large B cell lymphoma Burkitt lymphoma Plasma cell myeloma Plasmacytoma-like lesion Other T cell neoplasms Peripheral T cell lymphoma, NOS Hepatosplenic T cell lymphoma Classical Hodgkin lymphoma-type PTLD Fishman JA. Am J Transpl. 2017;17:856-879 Viral infections in LDLT recipients

 A transplantation center (CMC) in Korea  208 subjects who had undergone LDLT during a 9-year period  351 episodes of infections

* Number of CMV end organ diseases

Kim YJ et al. Transpl Infect Dis. 2008;10:316-324