1. DR. ATUL HUMAR INFECTIOUS DISEASES / MULTI-ORGAN TRANSPLANTATION INFECTIONS IN SOLID ORGAN TRANSPLANT RECIPIENTS

2. OBJECTIVES To review the concept of compromised hostTo review the concept of compromised host To gain an understanding of the common infections after transplantTo gain an understanding of the common infections after transplant To gain further understanding into herpesvirus infections after transplantTo gain further understanding into herpesvirus infections after transplant –CMV –EBV

3. Definition Compromised hostCompromised host –Patient lacks resistance to infection due to a deficiency in defense mechanisms against microbial invasion and/or disease –Inherited or acquired

4. PATHOGENESIS DISEASE DETERMINANTS MicrobeHost Inoculum or OrganismsVirulenceLatency DefenseMechanisms

5. HOST DEFENSE MECHANISMS Intact skin and mucous membranesIntact skin and mucous membranes –Disrupted due to trauma, burns, ulceration, IV catheters, surgery Types of infectionTypes of infection –Wound infections, burn sepsis, diabetic foot infection, line sepsis Usual organismsUsual organisms –Bacteria – environmental, endogenous –Fungi – environmental, nosocomial

7. HOST DEFENSE MECHANISMS Physical removal / clearance of micro-organismsPhysical removal / clearance of micro-organisms –Respiratory muco-ciliary clearance –Peristalsis and dynamics of hollow viscus (gut, bile ducts, ureter, fallopian tube) –Maybe abnormal due to underlying disease, surgery, smoking etc. Intact sphincters/valvesIntact sphincters/valves Types of infectionTypes of infection –Pneumonia, urosepsis, biliary sepsis Usual organismsUsual organisms –Bacteria – environmental, endogenous

10. HOST DEFENSE MECHANISMS Endogenous microfloraEndogenous microflora –Oropharyngeal, gut, skin, vagina –Important for preventing colonization with disease causing organisms (competitive) –Antibiotics remove natural flora –E.g. C. difficile colitis Chemical antimicrobial agentsChemical antimicrobial agents –Gastric acidity, cutaneous fatty acids

11. HOST DEFENSE MECHANISMS Inflammatory responseInflammatory response –Number (mass) and function of circulating and tissue phagocytic cells –Neutrophils, monocytes, macrophages, spleen Humoral MediatorsHumoral Mediators –Complement, fibronectin

12. HOST DEFENSE MECHANISMS Specific Immune responseSpecific Immune response –T-lymphocytes CD4+, CD*+ (helper, cytotoxic)CD4+, CD*+ (helper, cytotoxic) Number, functionNumber, function –B-lymphocytes Make antibodiesMake antibodies IgG, IgAIgG, IgA

13. Common problems Host Defect: Inflammatory response Common microbes Neutropenia (<0.5) Splenectomy Gram negative bacilli, Staph, Candida, Aspergillus S. Pneumonia, H. influenza, N. Meningitis

14. Common problems Host Defect: Complement Common microbes Early (C3, C5) Late (C6,7,8) S. Aureus, S. Pneumonia, gram negative bacilli Neisseria species

15. Common problems Host Defect: Immune response Common microbes T- Lymphocyte e.g. HIV, organ transplant B-Lymphocyte Numerous microbes S. Pneumonia, H. influenza, Giardia

16. INFECTION: BASIC PRINCIPLES Inflammatory response attenuated by immunosup.Inflammatory response attenuated by immunosup. may abolish typical signs/symptomsmay abolish typical signs/symptoms decreased sensitivity of serological, radiological testsdecreased sensitivity of serological, radiological tests Efffects of established infection may be devastatingEfffects of established infection may be devastating Treatment may have more toxicitiesTreatment may have more toxicities Rifampin - decrease CsARifampin - decrease CsA Erythromycin, azoles increase CsAErythromycin, azoles increase CsA Synergistic nephrotoxicity - aminoglycosides, AmB, septra, cipro, vancomycin, pentamidineSynergistic nephrotoxicity - aminoglycosides, AmB, septra, cipro, vancomycin, pentamidine

17. INFECTIONS IN TRANSPLANTATION Three main determinants of the risk of infection in transplant recipients Infections related to technical / surgical problemsInfections related to technical / surgical problems

18. TECHNICAL COMPLICATIONS Liver - biliary tree - leaks, stricturesLiver - biliary tree - leaks, strictures Lung - bronchial anastomosis necrosis, dehiscence ; mediastinal fluid collectionLung - bronchial anastomosis necrosis, dehiscence ; mediastinal fluid collection Kidney - uroterocystostomy - leak, urinomaKidney - uroterocystostomy - leak, urinoma Pancreas - duodenum-bladder; duodenum-bowel: anastomotic leaks, abscessPancreas - duodenum-bladder; duodenum-bowel: anastomotic leaks, abscess

20. INFECTIONS IN TRANSPLANTATION Major determinants of the risk of infection The net state of ImmunosuppressionEpidemiologicalexposures

21. NET STATE OF IMMUNOSUPPRESSION Immunosuppressive therapy: dose, duration, temporal sequence - ‘area under the curve’Immunosuppressive therapy: dose, duration, temporal sequence - ‘area under the curve’ Underlying immune deficiencyUnderlying immune deficiency Mucocutaneous barrier integrity: intubation, drains, catheters, central linesMucocutaneous barrier integrity: intubation, drains, catheters, central lines Devitalized tissue, fluid collectionDevitalized tissue, fluid collection Neutropenia, lymphopeniaNeutropenia, lymphopenia

22. NET STATE OF IMMUNOSUPPRESSION Metabolic conditionsMetabolic conditions UremiaUremia MalnutritionMalnutrition DiabetesDiabetes Viral infection: Immune modulationViral infection: Immune modulation CytomegalovirusCytomegalovirus Epstein-Barr virusEpstein-Barr virus Hepatitis B, C, HIVHepatitis B, C, HIV

23. EPIDEMIOLOGICAL EXPOSURES Community –Community acquired pneumonia pathogens –Environmental fungi –Enteric bacterial pathogens (salmonella) –TB, zoonosis, HIV, hepatitis viruses Nosocomial –MRSA,VRE –Pseudomonas, MDR gram negatives –Aspergillus

24. CASE PRESENTATION 61 y.o. male heart transplant 199161 y.o. male heart transplant 1991 Stable immunosuppression x yearsStable immunosuppression x years –cylosporin, prednisone 3 week history of progressive leg cellulitis, fever unresponsive to antibiotics3 week history of progressive leg cellulitis, fever unresponsive to antibiotics Intermittent confusionIntermittent confusion

28. TIMETABLE: 0-1 MONTH Infections usual to post-op patientsInfections usual to post-op patients –nosocomial pneumonia, wound, line sepsis, UTI Key factors: nature of the operation, technical skillKey factors: nature of the operation, technical skill Lung, heart, liver at highest riskLung, heart, liver at highest risk –longer intubation, ICU stay, lines, catheters Most OI’s (eg. PCP) absent in the first monthMost OI’s (eg. PCP) absent in the first month –Exceptions – HSV, HHV6, Candida, Aspergillus

29. TIMETABLE: 0-1 MONTH Also may seeAlso may see –Infection transmitted with the allograft: eg. lung transplant with pneumonia or a donor bacteremia which seeds the vascular anastamosis –Pre-existing infection within the recipient made worse by the transplant

30. TIMETABLE OF INFECTION One to 6 months post-Tx –Maximal period of immunosuppression –Effect of sustained immunosuppression or ‘area under the curve’ –Opportunistic infections in the absence of excessive epidemiological hazard

31. TIMETABLE - 1 TO 6 MONTHS VIRAL –CMV, EBV, VZV, HHV-6, Adenovirus, Influenza, RSV BACTERIAL –Nocardia, Legionella, Listeria, TB FUNGAL –PCP, Aspergillus, Cryptococcus, endemic mycosis PARASITIC –Toxoplasma, Strongyloides

32. TIMETABLE - > 6 MONTHS GROUP 1: Good graft function, minimal immunosuppression –Community acquired pneumonia, UTI, OI based on intense exposure GROUP 2: Recurrent or chronic rejection, high level immunosuppression, chronic viral replication –Continued risk of opportunistic infections

33. CASE PRESENTATION 55 year old female OLTx for PSC55 year old female OLTx for PSC Acute rejection: Steroid resistant requiring OKT3 for 10 days (pre-emptive ganciclovir)Acute rejection: Steroid resistant requiring OKT3 for 10 days (pre-emptive ganciclovir) Neoral, prednisone, MMFNeoral, prednisone, MMF 2 months later presents with fever, malaise, elevated transaminases2 months later presents with fever, malaise, elevated transaminases

34. CMV HEPATITIS

35. 0255075100125 0 1 2 3 4 5 6 CMV hepatitis Days Post-transplant Log viral load CMV VIRAL LOAD

36. CYTOMEGALOVIRUS BetaherpesvirusBetaherpesvirus –DS DNA –Icoshedral capsid –Lipid envelope Establishes latencyEstablishes latency –“Once infected always infected”

37. CLINCAL MANIFESTATIONS DIRECT EFFECTS –Asymptomatic viral shedding –Acute viral syndrome –Pneumonitis: BMT, Lung Transplant –Infection of allograft: hepatitis, pneumonitis, nephritis, myocarditis, pancreatitis –Infection of native tissue: GI, CNS, retina

38. CMV INFECTION/DISEASE Cellular effects: Antigen/cytokineexpression PTLD-EBVImmunosuppression Bacterial and fungal infections AllograftInjuryAllograftRejection OB, VBD, graft vasculopathy chronicacuteacute INDIRECT EFFECTS

39. INFLAMMATION (CYTOKINES, NF-  B) LATENT CMV INFECTION ANTILYMPHOCYTEANTIBODIES OTHER HERPES VIRUSES SEPSIS/SURGERYREJECTION

40. CMV: THE ROLE OF CYTOKINES TNF-  has been shown to stimulate the CMV-IE gene enhancer/promotor region in a dose-dependent manner leading to CMV reactivationTNF-  has been shown to stimulate the CMV-IE gene enhancer/promotor region in a dose-dependent manner leading to CMV reactivation CMV has direct effects on cytokines: CMV-IE gene products shown to increase IL-6 and IL-8 gene expressionCMV has direct effects on cytokines: CMV-IE gene products shown to increase IL-6 and IL-8 gene expression This has been shown to enhance neutrophil trans-endothelial migrationThis has been shown to enhance neutrophil trans-endothelial migration Cytokine mediated PMN recruitment may enhance CMV disseminationCytokine mediated PMN recruitment may enhance CMV dissemination

41. CYTOKINE LEVELS AND CMV DISEASE Humar et al. J Infect Dis 1999; 179: 484

42. CMV INFECTION CMV DISEASE Steroids CsA MMFIncreasing viral load

43. MULTIVARIATE ANALYSIS OF RISK FACTORS FOR CMV DISEASE Humar et al. Transplantation 2000

44. HHV-6 AND TRANSPLANTATION Cytopathic lymphotrophic virus : roseola infantumCytopathic lymphotrophic virus : roseola infantum Seroprevalence almost universal by age 2-3Seroprevalence almost universal by age 2-3 Post-transplant: implicated as a cause of febrile illness, hepatitis, pneumonitis and other infections.Post-transplant: implicated as a cause of febrile illness, hepatitis, pneumonitis and other infections. Rates of reactivation estimated from 14 - 82 %Rates of reactivation estimated from 14 - 82 % Its main effect post-transplant may be immunomodulatory including an interaction with CMVIts main effect post-transplant may be immunomodulatory including an interaction with CMV

45. HHV-6 AND TRANSPLANTATION Infection of T-cells results in down-regulation of IL-2 mRNA and protein synthesis, and a reduction in mitogen-driven proliferative responses resulting in a cell mediated immune defect HHV-6 infection results in cytokine dysregulation; induction of TNF-  and other immunomodulatory cytokines Interactions among herpesviruses may be more direct, including specific binding via glycoproteins resulting in cellular co-infections and facilitating viral spread

46. 0255075100125 0 1 2 3 4 5 6 CMV hepatitis Days Post-transplant Log viral load VIRAL CULTURE MEDIA

47. HERPESVIRUS INTERACTIONS Serial Quantitative HHV-6 in 200 liver transplant recipientsSerial Quantitative HHV-6 in 200 liver transplant recipients Serial Quantitative CMV PCRSerial Quantitative CMV PCR Direct effects and Indirect effects of viral replication on development of graft rejection and opportunistic infection were assessedDirect effects and Indirect effects of viral replication on development of graft rejection and opportunistic infection were assessed

48. HHV-6 RESULTS HHV-6 infection occurred in 28% (56/200) patients (defined as VL > 2 logs)HHV-6 infection occurred in 28% (56/200) patients (defined as VL > 2 logs) peak VL occurred at a median of 35 days (mean 44.1 days; range 8-177)peak VL occurred at a median of 35 days (mean 44.1 days; range 8-177) Symptomatic disease occurred in only 2/200 patients (1%) and presented as fever and pancytopeniaSymptomatic disease occurred in only 2/200 patients (1%) and presented as fever and pancytopenia

49. OutcomeMultivariate modelOR (95% CI)P-value CMV disease n=32 HHV-6 infection Antilymphocyte globulin Steroid boluses Immunosuppression 3.27 (1.43-7.51) 3.13 (1.31-7.55) 1.00 (0.84-1.19) 2.15 (0.85-7.50) 0.005 0.01 0.99 0.094 HHV-6 AND CMV

50. CMV PREVENTION Universal prophylaxis: anti-viral therapy to all ‘at- risk’ patientsUniversal prophylaxis: anti-viral therapy to all ‘at- risk’ patients Pre-emptive therapy: anti-viral therapy to subgroups of ‘at-risk’ patients usually based on further diagnostic tests aimed at identifying early viral reactivationPre-emptive therapy: anti-viral therapy to subgroups of ‘at-risk’ patients usually based on further diagnostic tests aimed at identifying early viral reactivation

51. PRE-EMPTIVE THERAPY + _ ++++ ___ ++ _ 048 12 weeks Could have initiated pre-emptive therapy CMV disease TEST

52. CMV IN LIVER TRANSPLANT RECIPIENTS PRE-TRANSPLANT: Donor and recipient CMV serology POST-TRANSPLANT: D+/R+, D-/R+D+/R+, D-/R+ –Week 2-12: E very clinic visit :  CMV antigenemia  CMV quantitative PCR D+/R-:D+/R-: –G anciclovir prophylaxis 12 weeks  Bloodwork at week 12, 14, 16, 18.  CMV antigenemia and quantitative PCR testing

53. STUDY PROTOCOL: CMV IN LIVER TRANSPLANT RECIPIENTS OUTCOME: CMV disease defined according to biopsy evidence; viral syndrome based on specific clinical criteriaCMV disease defined according to biopsy evidence; viral syndrome based on specific clinical criteriaANALYSIS: Predictive value for antigenemia and PCRPredictive value for antigenemia and PCR –Positive: >0 cells/slide; >400 copies/ml –Sensitivity, specificity, PPV and NPV for different cut-off points –Multivariate logistic regression for predictors of CMV disease

54. RESULTS CMV disease: 21/97 ( 21.7%) patients; mean 60 days post- transplantCMV disease: 21/97 ( 21.7%) patients; mean 60 days post- transplant PCR: sensitivity of 100%, specificity 47.4%, PPV 34.4 % and NPV 100% for prediction of CMV diseasePCR: sensitivity of 100%, specificity 47.4%, PPV 34.4 % and NPV 100% for prediction of CMV disease Antigenemia: 95.2%, 55.3%, 37.0% and 97.7 %.Antigenemia: 95.2%, 55.3%, 37.0% and 97.7 %. The optimal cut-off for PCR in the range of 2000-5000 copies/ml (sensitivity 85.7%, specificity 86.8%, PPV 64.3%, NPV 95.7)The optimal cut-off for PCR in the range of 2000-5000 copies/ml (sensitivity 85.7%, specificity 86.8%, PPV 64.3%, NPV 95.7) The optimal cut-off for antigenemia was in the range of 6 positive cells/slide.The optimal cut-off for antigenemia was in the range of 6 positive cells/slide.

55. 0.000.250.500.751.00 0.00 0.25 0.50 0.75 1.00 >0 >1000 >2000 >5000 >7000 >12000 >15000 >20000 1- Specificity Sensitivity ROC CURVE FOR CMV QUANTITATIVE PCR

56. PREEMPTIVE THERAPY CMV antigenemia or CMV quantitative PCR useful for predicting the development of CMV diseaseCMV antigenemia or CMV quantitative PCR useful for predicting the development of CMV disease Either of these tests could be employed in a pre- emptive strategy using optimal cut-offsEither of these tests could be employed in a pre- emptive strategy using optimal cut-offs The CMV viral load is the most important determinant for the development of CMV diseaseThe CMV viral load is the most important determinant for the development of CMV disease

57. RESPONSE TO THERAPY 0255075100125 0 1 2 3 4 5 6 CMV hepatitis Days Post-transplant Log viral load 8 weeks after treatment relapsed with fever, Recurrent CMV disease

58. RESPONSE TO THERAPY Virologic response to therapy assessed in 52 patients with CMV disease treated with ganciclovirVirologic response to therapy assessed in 52 patients with CMV disease treated with ganciclovir Viral loads done at regular intervals after starting treatmentViral loads done at regular intervals after starting treatment Genotypic resistance testingGenotypic resistance testing Clinical response to treatment : Relapsing disease occurred in 24% of patientsClinical response to treatment : Relapsing disease occurred in 24% of patients Humar et al. JID 2002

59. y=y 0 e ax VIRAL LOAD KINETICS

60. 0 1 2 3 4 5 6 010203040 Time (days) Patient 3 T 1/2 = 4.5d Viral load (log 10 copies/ml) 0 1 2 3 4 5 6 0102030405060 Time (days) Patient 4 T 1/2 = 6.2d Viral load (log 10 copies/ml) FIGURE 1 Patient 2 Viral load (log 10 copies/ml) 0 1 2 3 4 5 0510152025 Time (days) T 1/2 = 1.6d Patient 1 0 1 2 3 4 5 6 0510152025 Time (days) T 1/2 = 1.3d Viral load (log 10 copies/ml) Humar et al. JID 2002

62. CONCLUSIONS Different people have different rates of response to antiviral therapyDifferent people have different rates of response to antiviral therapy Early phase kinetics are predictive of relapsing disease.Early phase kinetics are predictive of relapsing disease. Differential response likely combination ofDifferential response likely combination of –Host factors - CTL, immunosuppression –Viral Factors – genotype, immune evasion genes

63. EBV AND PTLD DEFINITION An abnormal proliferation of B-cells driven by EBVAn abnormal proliferation of B-cells driven by EBV –May be polyclonal or monoclonal –(occasional tumors are T-cell, NK cell)

64. PTLD Viral Infection Tumor

65. EPSTEIN-BARR VIRUS Lytic infectionLytic infection – ~100 genes expressed, lysis of B-cell Latent infectionLatent infection –< 10 genes expressed –LMP 1,2, EBNA 1,2,3, EBER, BCRF, BHRF, BARF –Evades host immune response –Latent gene products drive B-cell proliferation

66. Growthadvantage Monoclonal Cytogeneticabnormality Malignanttransformation Polyclonal IL-1,6,10 LyticBCRF-1LMP-1 latent

67. CASE PRESENTATION 34 y.o. male 2 years post-kidney transplant34 y.o. male 2 years post-kidney transplant On Neoral, Prednisone and ImmuranOn Neoral, Prednisone and Immuran Fever, sore throat, and multiple subcutaneous nodulesFever, sore throat, and multiple subcutaneous nodules

68. INVESTIGATIONS EBV Viral load > 1000 copies / 10 6 PBLEBV Viral load > 1000 copies / 10 6 PBL Biopsy – Aggressive, undifferentiated monoclonal PTLD, EBV positiveBiopsy – Aggressive, undifferentiated monoclonal PTLD, EBV positive Withdrawal of MMF, treatment with IV ganciclovirWithdrawal of MMF, treatment with IV ganciclovir

69. 0 1 2 3 4 0 1 2 3 4 5 6 TIME (months) Log viral load ( -o- ) Ganciclovir

70. RISK FACTORS FOR PTLD EBV D+/R-EBV D+/R- –1-5% incidence in R+ vs. 20-30% in R- Intensity of ImmunosuppressionIntensity of Immunosuppression Type of transplantType of transplant –Small bowel > lung > heart > liver, kidney Herpesvirus interactionsHerpesvirus interactions

71. EBV SEMIQUANTATIVE PCR Timing of TestPTLDNo PTLDP-value (log/10 6 )(log/10 6 ) Prior to PTLD2.9(1.5)1.4(1.5) 0.005 PTLD Diagnosis3.1(1.2)1.4(1.5)< 0.001 Peak value 3.4(0.5)1.8(1.5)< 0.001 (12 months post-transplant)

72. EBV PROPHYLAXIS STUDY Multicentre RCT in EBV D+/R- transplant recipientsMulticentre RCT in EBV D+/R- transplant recipients –Group 1: Ganciclovir + CMVIG –Group 2: Ganciclovir + placebo EBV viral loads taken at regular intervals post-transplantEBV viral loads taken at regular intervals post-transplant

73. EBV PROPHYLAXIS STUDY Viral load data was analyzed from 28 (20 pediatric and 8 adult) patients (15 cytogam and 13 placebo).Viral load data was analyzed from 28 (20 pediatric and 8 adult) patients (15 cytogam and 13 placebo). Transplant types were liver (n=11), kidney (n=10), lung (n=6), and pancreas (n=1).Transplant types were liver (n=11), kidney (n=10), lung (n=6), and pancreas (n=1). During the first 6 months post-transplant, detectable viremia occurred in 9/13 (69.2%) placebo patients and 10/15 (66.7%) cytogam patientsDuring the first 6 months post-transplant, detectable viremia occurred in 9/13 (69.2%) placebo patients and 10/15 (66.7%) cytogam patients

75. SUMMARY Reactivation of herpesviruses post-transplant are due to a complex interaction of multiple factorsReactivation of herpesviruses post-transplant are due to a complex interaction of multiple factors Viral infections likely produce multiple direct and indirect effects on the post-transplant course of these patientsViral infections likely produce multiple direct and indirect effects on the post-transplant course of these patients Efforts to minimize the impact of these infections should lead to improvement in graft outcomesEfforts to minimize the impact of these infections should lead to improvement in graft outcomes