**3. Cytomegalovirus (CMV)**

control both primary infection ant periodic reactivation that occur in all EBV-seropositive persons [1, 3, 4]. The EBV causes nasopharyngeal carcinoma, Burkitt lymphoma, and other lymphoepithelic tumors (non-Hodgkin's lymphoma, B- and T-cellular lymphomas) [5]. Development of these diseases is based on some cellular factors, as well as 14th chromosome translocation. Once infected with EBV, the virus persists latently in a person for life, in B cell lymphocytes, and chronically replicating in the cells of the oropharynx [5, 6]. In patients with HIV and transplanted ones, EBV becomes a main problem because of the inability of the immune system to control B cell proliferation and immortalization. EBV infection is registered in nearly 75% of transplanted recipients as the source usually is the donor. Contagion can also occur after blood transfusion. In the course of the immunosuppression, the latent EBV infection can be reactivated. Clinical disease represent mononuclear syndrome with temperature, lymphadenopathy, hepatosplenomegaly and monocytosis. The central nervous system is rarely involved with symptoms of serous meningitis, encephalitis, Guillen Barre syndrome.

The immunosuppression required to prevent graft rejection post-transplantation impairs T cell immunity, potentially allowing for uncontrolled proliferation of EBV-infected B cells, which may result in a spectrum of B cell proliferations that range from hyperplasia to true lymphoma [7, 8]. In the initial stages of PTLD, prolypheration is polyclonal. With mutation and selective growth, the lesion becomes oligoclonal and later, monoclonal. Lymphocytes from patients treated with cyclosporine do not exhibit an appropriate T cell response to EBV-infected B cells in vitro. The activity of natural killer cells is reduced for several months following

PTLD is a well-recognized complication of both solid organ transplantation and allogeneic hematopoietic stem cell transplantation (HSCT). It is one of the most common post-transplant malignancies. In most cases, it is associated with EBV infection of B cells, either as a conse‐ quence of post-transplant reactivation of the virus or from primary EBV infection. The median onset of disease in solid organ transplant population is 6 months and in hematopoietic stem cell recipients 70–90 days [11, 12] after transplantation. The frequency of PTLD depends largely on the type of transplant received and the immunosuppression that the particular transplant requires [6, 11, 12]. Primary EBV infection may develop, such as in an EBV seronegative recipient who received an allograft from an EBV-seropositive donor. This is recognized as probably the most significant risk factor for developing PTLD and be higher in pediatric transplant recipients [12]. The incidence ranged from 0.6%–2.1% in adult kidney recipients to 4.4%–6.9% in pediatric kidney recipients [12, 13] at different time after transplantation. Lung and heart transplantation in adult population is associated with a relatively high rate of PTLD with an incidence of approximately 5% or more [14]. After liver transplantation, reported rate of incidence is approximately 1% in adult recipients and pediatric recipients [15]. In the setting of allogeneic hematopoietic stem cell transplantation, PTLD rates vary greatly depending on the conditioning regimen and the amount of T cell depletion. In pediatric recipients, PTLD occurs in less than 1% of non-T-cell-depleted grafts from matched siblings, compared with as high as 30% of patients with unrelated or HLA-mismatched donors when extensive T cell depletion of the donor bone marrow is performed. Treatment of graft versus host disease with antitimocyte globulin or anti-T-cell monoclonal antibodies is another risk factor for PTLD [16].

transplantation [9, 10].

104 Immunopathology and Immunomodulation

CMV is a ubiquitous herpesvirus that infects majority of humans and is transmitted via saliva, body fluids, cell, and tissue. Primary infection in immunocompetent individuals manifests as an asymptomatic or self-limited febrile illness or as mononucleosa-like syndrome in childhood and older age. The seroprevalence depends on the socioeconomic status and ranges from 30%– 97% in Europe and North America [2, 21]. Following primary viral replication in seronegative individuals, CMV establishes non-replicative infection for life, named latency, in CD34+ myeloid progenitor cells as a major site [22] and in lymphoid organs and tissues as well (23). Various latently infected cells serve as reservoirs for reactivation and as carriers of infection to susceptible individuals [24]. After reactivation, CMV multiplies inside. In immunocompro‐ mised patients and especially after transplantation, CMV is one of the main clinical problems in almost all types of allograft recipients. Basic risk factor in the development CMV replication and disease is transmission via transplanted organs or tissues including the heart, kidney, lung, liver, and hematopoietic stem cells [25, 26]. CMV disease risk is highest when primary infection occurs in seronegative transplant recipients by the transplanted organ from the seropositive donor (27). On the other hand, secondary infection presumably occurs following the reactiva‐ tion of the recipient's endogenous latent infection and is more common than primary infection. The frequency depends on the specific immunosuppression utilized. The third type of infection can be correlated with a presumed superinfection that is reinfection of the previously sero‐ positive recipients by donor virus present in allograft [28].

The initial infection is dangerous for all immunosuppressed patients, because of numerous CMV indirect effects, due to the ability to modulate the immune system, and is an important contributor to active and chronic allograft injury [26, 29]. CMV can cause dysfunction of the transplanted organ or can participate in its rejection from the organism, which is often seen in recipients of liver, heart, and lungs. Infections and diseases with CMV are also typical for recipients of kidneys and bone marrow, as mortality is in the rate of 32–70%. Other risk factors are the overall state of immunosuppression as determined by the immunosuppressive protocol (e.g. type of drug, dose, timing, and duration), host factors (e.g. age, comorbidity, leucopenia and lymphopenia, genetic factors), and others [30]. The degree of immunosuppression correlates with the severity of the clinical symptoms of CMV infection. According to the data, conventional immunosuppressive therapy is increasing the gravity of the disease.

Source of primary infection and reinfection are also blood and blood products, which have not been checked for the presence of latent CMV virus in lymphocytes. A CMV seronegative recipient who received donor organ of a seronegative individual has the lowest risk of CMV disease when receiving CMV-negative blood or leuco-depleted blood products. The use of mTOR inhibitors (everolimus, sirolimus) is associated with a lower risk of CMV disease [31]. Transplant recipients who receive treatment with lymphocyte-depleted drugs, especially if given for the treatment of rejection, should be considered at high risk for CMV disease [32].

It is considered that in almost 100% of immunocompromised patients, the latent CMV infection will become reactivated. This reactivation refers, especially, to recipients from seropositive donors, although clinical manifestation is developed in 20–25 % of them [28, 33].

To assess the risk for CMV-related disease, serology testing of all donors and transplant candidates prior to transplantation can be performed. The clinical symptoms of active CMV infection are often nonspecific, also known as CMV syndrome (prolonged fever, weakness, hematological abnormalities such as thrombocytopenia, atypical lymphocytosis and leukope‐ nia, and abnormalities of hepatic function). The symptoms occur 1–4 months after transplan‐ tation, in some cases, even later and sometimes it is difficult to differentiate them from those of organ rejection. The greatest risk for this condition is at the first 30 days after the immuno‐ suppression. Tissue-invasive CMV disease is when it implicates the gastrointestinal tract, pneumonitis, hepatitis, nephritis, myocarditis, pancreatitis, retinitis, etc. [34]. In patients with transplanted liver, CMV hepatitis occurs in 17% of the cases. The "vanishing bile duct syndrome" (VBS) is related with CMV infection and organ rejection. Heart and lung recipients usually develop interstitial pneumonia, as those with bone marrow transplantation. Mortality is from 33–100% in a half of the patients. Atherosclerosis of coronary vessels develops three times faster in patients with active CMV infection in heart recipients [35–42].

mised patients and especially after transplantation, CMV is one of the main clinical problems in almost all types of allograft recipients. Basic risk factor in the development CMV replication and disease is transmission via transplanted organs or tissues including the heart, kidney, lung, liver, and hematopoietic stem cells [25, 26]. CMV disease risk is highest when primary infection occurs in seronegative transplant recipients by the transplanted organ from the seropositive donor (27). On the other hand, secondary infection presumably occurs following the reactiva‐ tion of the recipient's endogenous latent infection and is more common than primary infection. The frequency depends on the specific immunosuppression utilized. The third type of infection can be correlated with a presumed superinfection that is reinfection of the previously sero‐

The initial infection is dangerous for all immunosuppressed patients, because of numerous CMV indirect effects, due to the ability to modulate the immune system, and is an important contributor to active and chronic allograft injury [26, 29]. CMV can cause dysfunction of the transplanted organ or can participate in its rejection from the organism, which is often seen in recipients of liver, heart, and lungs. Infections and diseases with CMV are also typical for recipients of kidneys and bone marrow, as mortality is in the rate of 32–70%. Other risk factors are the overall state of immunosuppression as determined by the immunosuppressive protocol (e.g. type of drug, dose, timing, and duration), host factors (e.g. age, comorbidity, leucopenia and lymphopenia, genetic factors), and others [30]. The degree of immunosuppression correlates with the severity of the clinical symptoms of CMV infection. According to the data,

conventional immunosuppressive therapy is increasing the gravity of the disease.

donors, although clinical manifestation is developed in 20–25 % of them [28, 33].

Source of primary infection and reinfection are also blood and blood products, which have not been checked for the presence of latent CMV virus in lymphocytes. A CMV seronegative recipient who received donor organ of a seronegative individual has the lowest risk of CMV disease when receiving CMV-negative blood or leuco-depleted blood products. The use of mTOR inhibitors (everolimus, sirolimus) is associated with a lower risk of CMV disease [31]. Transplant recipients who receive treatment with lymphocyte-depleted drugs, especially if given for the treatment of rejection, should be considered at high risk for CMV disease [32]. It is considered that in almost 100% of immunocompromised patients, the latent CMV infection will become reactivated. This reactivation refers, especially, to recipients from seropositive

To assess the risk for CMV-related disease, serology testing of all donors and transplant candidates prior to transplantation can be performed. The clinical symptoms of active CMV infection are often nonspecific, also known as CMV syndrome (prolonged fever, weakness, hematological abnormalities such as thrombocytopenia, atypical lymphocytosis and leukope‐ nia, and abnormalities of hepatic function). The symptoms occur 1–4 months after transplan‐ tation, in some cases, even later and sometimes it is difficult to differentiate them from those of organ rejection. The greatest risk for this condition is at the first 30 days after the immuno‐ suppression. Tissue-invasive CMV disease is when it implicates the gastrointestinal tract, pneumonitis, hepatitis, nephritis, myocarditis, pancreatitis, retinitis, etc. [34]. In patients with transplanted liver, CMV hepatitis occurs in 17% of the cases. The "vanishing bile duct syndrome" (VBS) is related with CMV infection and organ rejection. Heart and lung recipients

positive recipients by donor virus present in allograft [28].

106 Immunopathology and Immunomodulation

Laboratory diagnosis of CMV infection and CMV disease can be accomplished with various methods. Preliminarily, before starting with the immunosuppression or transplantation, the serological status of the donor and recipient is defined. Generally, the method used for this purpose is ELISA, which detects specific IgG Ab in the serum of the patient. CMV infection after transplantation represents the presence of the virus and viral replication in body fluids or tissue samples regardless of clinical symptoms. CMV disease after transplantation repre‐ sents the presence of any clinical symptoms in patients with CMV infection [43]. The laboratory methods to confirm CMV infections are histology, culture, serology, antigenemia (pp65 antigenemia), and molecular assay that detect and quantify CMV nucleic acid (NAT) [35]. Serology to detect CMV-IgM and IgG has limited use for diagnosis of CMV disease after transplantation (44). Molecular tests that detect CMV DNA or RNA are the preferred methods. Detection of CMV RNA is indicative of CMV replication. Detection of CMV DNA may or may not reflect CMV replication since a highly sensitive NAT may amplify latent viral DNA. Quantitative NAT (QNAT) assay have been developed to potentially differentiate active viral replication typically associated with high viral load from latent virus with low level CMV DNAemia [35, 45]. QNAT is useful for guiding preemptive therapy, for rapid and sensitive diagnosis of CMV infection, and to guide treatment responses [45]. Patients suspected to have tissue-invasive CMV disease but with negative QNAT or pp65 antgenemia should undergo tissue biopsy and histopathology to confirm the clinical suspicion of CMV disease [35].

The approaches to CMV prevention in recipients vary among different transplant population and risk profile. The two major strategies for CMV prevention are: antiviral prophylaxis and preemptive therapy. Antiviral prophylaxis is the administration of antiviral drug to "at-risk" patients for a defined period after transplantation. Preemptive therapy is the administration of antiviral drug only to asymptomatic patients with evidence of early CMV replication in order to prevent disease. Recipients are monitored at regular intervals (usually once weekly) using a laboratory assay such as CMV QNAT or pp65 antgenemia.

Antiviral prophylaxis has the advantage of preventing reactivation of other herpesviruses, and has been associated with lower incidence of indirect CMV effects [46]. Antiviral prophylaxis can be administered to any at-risk recipients. The duration varies depending on the CMV donor and recipient serostatus and the transplant types, extended between 100 days and 12 months in different group [35]. Valgancyclovir is the preferred drug. Alternative options are intravenous gancyclovir, oral gancyclovir, and for kidney recipients only valacyclovir. Unselected intravenous immunoglobulin (IVIG) may also be used but only as an adjunct to antiviral therapy in lung, heart, and intestinal transplant recipients. In general, antiviral prophylaxis should be started as early as possible and within the first 10 days after transplan‐ tation [35]. However, antiviral prophylaxis is associated with late-onset CMV disease partic‐ ularly among CMV D+/R- patients, probably due to development of drug resistance [47]. The potential options for prevention and management of late-onset CMV disease are careful clinical follow up with early treatment of CMV disease when symptoms occur, CMV QNAT or pp65 antgenemia monitoring after completion of antiviral prophylaxis, and prolonged antiviral prophylaxis.

Preemptive therapy requires weekly patient monitoring for evidence of early CMV replication, which is then treated with valgancyclovir or intravenous gancyclovir. The recommended doses are valgancyclovir (900 mg twice daily) or intravenous ganciclovir (5 mg/kg every 12 h). Many authors prefer antiviral prophylaxis for D+/R- and lung transplant recipients while recognizing the clinical utility of preemptive therapy in CMV R+ kidney, liver, pancreas, and heart recipients [21, 35]. The same laboratory test for monitoring is recommended, with frequency of once weekly for 12 weeks after transplantation.

Indications of use of ganciclovir also include severe local (often eye damages) and life threat‐ ening conditions in patients with HIV, organ transplantations, and neoplasms. The use of lymphocyte-depleting therapy is a major risk factor for CMV disease when used for rejection treatment. The optimal duration of antiviral prophylaxis is given for 1–3 months with valgan‐ cyclovir (900 mg once daily, oral gancyclovir 1 g p.o. thrice daily) or intravenous gancyclovir (5 mg/kg every 24 h) [35].

Patients who develop CMV disease after prolonged courses of gancyclovir or vagancyclovir administration, and those failing to respond to standard gancyclovir treatment, should be suspected of having gancyclovir resistant virus. In these conditions, genotype testing should be performed. Immunosupression should be cautiously reduced. Therapeutic options for gancyclovir resistant CMV are limited. Foscarnet is often the first line for the treatment of UL97 mutant gancyclovir-resistent CMV (48). Switching to sirolimus-containing regimen may be an option for patients receiving mTOR inhibitors. Other therapeutic options are administration of cidofovir or its new oral formulation that may be available for compassionate release brinsidofovir (CMX001), compassionate release letermovir (AIC246), compassionate release maribavir, off-label leflunomid and off-label artesunate [49, 50]. Due to the virus, ability to evade host defenses of primary infection with CMV has not been shown to confer immunity from subsequent infections. Notwithstanding this, there are efforts to develop a CMV vaccine for prevention and therapy [51]. Due to some toxic effects of ganciclovir, patients need preliminary tests for renal function and blood count. Renal function is defined with the means of creatinine clearance, which has to be more than 70 ml/min. In blood, the number of neutro‐ philes has to be more than 1000 cells/mm3 , platelets –above 25000 cells/mm3 . During the treatment process these indicators are monitored every week and if they begin to decrease drastically, therapy is ceased. CMV therapy is not recommended in pregnant women, children under 12 years old and people more than 65 years old.

## **4. Varicella Zoster Virus (VZV)**

VZV is a human herpesvirus that spreads through direct contact with skin lesions or through air from respiratory droplets. Primary exposure, usually in childhood, leads to varicella, typically presents with fever, constitutional symptoms, and widely disseminated vesicular rush that primary involves the trunk and face [52]. Symptoms usually resolve within 7–10 days in immunocompetent children and young adults. More than 90% of adults acquire the infection in childhood and will be seropositive for VZV [2]. After initial infection, VZV establishes lifelong latency in the cranial nerve and dorsal root ganglia, and can reactivate years to decades later as herpes zoster in some individuals [53]. In children with primary and secondary immunodeficiency because of immunosuppressive therapy (leukemia, lymphoma, solid tumors), after transplantation VZV causes progressive varicella characterized by the continu‐ ous development of vesicular rash because of high viral replication and inadequate immune response [54, 55]. The high mortality among these children and adult organ recipients is because of systematic infection with multiple organ involvement, especially in the lungs, liver, pancreas, and central nervous system and, in some cases, disseminated intravascular coagul‐ opathy. Relapses are often seen. More recent reports have shown that pediatric renal and liver transplant recipients are at lower risk (4%–6.2%) for complication when given immediate antiviral therapy [56–60].

antgenemia monitoring after completion of antiviral prophylaxis, and prolonged antiviral

Preemptive therapy requires weekly patient monitoring for evidence of early CMV replication, which is then treated with valgancyclovir or intravenous gancyclovir. The recommended doses are valgancyclovir (900 mg twice daily) or intravenous ganciclovir (5 mg/kg every 12 h). Many authors prefer antiviral prophylaxis for D+/R- and lung transplant recipients while recognizing the clinical utility of preemptive therapy in CMV R+ kidney, liver, pancreas, and heart recipients [21, 35]. The same laboratory test for monitoring is recommended, with frequency

Indications of use of ganciclovir also include severe local (often eye damages) and life threat‐ ening conditions in patients with HIV, organ transplantations, and neoplasms. The use of lymphocyte-depleting therapy is a major risk factor for CMV disease when used for rejection treatment. The optimal duration of antiviral prophylaxis is given for 1–3 months with valgan‐ cyclovir (900 mg once daily, oral gancyclovir 1 g p.o. thrice daily) or intravenous gancyclovir

Patients who develop CMV disease after prolonged courses of gancyclovir or vagancyclovir administration, and those failing to respond to standard gancyclovir treatment, should be suspected of having gancyclovir resistant virus. In these conditions, genotype testing should be performed. Immunosupression should be cautiously reduced. Therapeutic options for gancyclovir resistant CMV are limited. Foscarnet is often the first line for the treatment of UL97 mutant gancyclovir-resistent CMV (48). Switching to sirolimus-containing regimen may be an option for patients receiving mTOR inhibitors. Other therapeutic options are administration of cidofovir or its new oral formulation that may be available for compassionate release brinsidofovir (CMX001), compassionate release letermovir (AIC246), compassionate release maribavir, off-label leflunomid and off-label artesunate [49, 50]. Due to the virus, ability to evade host defenses of primary infection with CMV has not been shown to confer immunity from subsequent infections. Notwithstanding this, there are efforts to develop a CMV vaccine for prevention and therapy [51]. Due to some toxic effects of ganciclovir, patients need preliminary tests for renal function and blood count. Renal function is defined with the means of creatinine clearance, which has to be more than 70 ml/min. In blood, the number of neutro‐

treatment process these indicators are monitored every week and if they begin to decrease drastically, therapy is ceased. CMV therapy is not recommended in pregnant women, children

VZV is a human herpesvirus that spreads through direct contact with skin lesions or through air from respiratory droplets. Primary exposure, usually in childhood, leads to varicella, typically presents with fever, constitutional symptoms, and widely disseminated vesicular rush that primary involves the trunk and face [52]. Symptoms usually resolve within 7–10 days

, platelets –above 25000 cells/mm3

. During the

prophylaxis.

108 Immunopathology and Immunomodulation

(5 mg/kg every 24 h) [35].

of once weekly for 12 weeks after transplantation.

philes has to be more than 1000 cells/mm3

**4. Varicella Zoster Virus (VZV)**

under 12 years old and people more than 65 years old.

Herpes zoster is characterized by vesicular rash units all over the corresponding nerve and estimated to occur in up to 20% of the immunocompetent individuals during their lifetime. In immunosuppressed and transplanted patients, herpes zoster is a frequent infectious compli‐ cation during the first four years after the transplantation [61, 62]. About half of the cases in the first year after the transplantation, a disseminated infection with mortality about 9% is observed, especially in the cases of organ rejection. Allogeneic stem cell transplantation is another procedure that greatly heightens the risk of herpes zoster. The incidence of VZV reactivation is 20.7%. VZV-related complications occur in 29% of patients with reactivation, most common of which is disseminated disease and postherpetic neuralgia. Radiotherapy can also become a reason for herpes zoster in about 15%–34 %. There is dissemination of the rash units outside the affected dermatome. In about 1% of all cases, encephalitis develops. This is typical, a second relapse that manifests, involving other body parts. In children with leukemia, herpes zoster or varicella develops more than one episode of clinical manifestation. Older transplant recipients are at greater risk for the development of herpes zoster and postherpetic neuralgia as secondary complication [62–65].

To determine the risks of VZV primary infection or reactivation after immunosupression and transplantation, all patients being considered for these procedures should undergo serologic testing (ELISA anti VZV IgG) to document prior exposure to VZV. Patients who are seroneg‐ ative are at high risk for the development of primary VZV, and seropositive patients are at high risk for developing herpes zoster. In general, both primary varicella and herpes zoster have typical clinical presentations. Definitive laboratory testing can be used for atypical cases and should be used for suspected disseminated, visceral disease, or central nervous system disease. Rapid diagnostic methods, including polymerase chain reaction (PCR) and direct immunofluorescent assay, are the methods of choice. PCR can be used for detecting VZV in vesicle fluid, serum, spinal fluid, and other tissues. Viral culture is specific and can help distinguish VZV from other herpesvirus pathogens (herpes simplex virus - HSV) [66].

Post-transplant and immunosuppressive patients who develop primary varicella should be treated with intravenous (IV) acyclovir early in the course of the illness, especially within 24 hours of rash onset. Reduction of immunosuppressive therapy should be considered. How‐ ever, IVIG or VZV immunoglobulin (VZIG) have been used in those with severe infection. Patients with disseminated or organ invasive herpes zoster should be treated with IV acyclovir. Localized nonsevere dermatomal herpes zoster can be treated with oral acyclovir, valacyclovir or famcyclovir [65].

Oral acyclovir and its pro-drugs have been shown to prevent VZV reactivation in immuno‐ suppressed population. During the early post-transplant period, many current regimens used for CMV prevention will likely prevent VZV reactivation. In patients who do not receive CMV prophylaxis, short-term antivirals given for HSV prophylaxis may also be effective against VZV during the period immediately post-transplant [65]. Other authors recommended one year prophylactic with acyclovir, which has been shown to effectively prevent VZV-reactiva‐ tion after allogeneic hematopoietic stem cell transplantation [61].

In the U.S., potential transplant recipients who are susceptible to VZV should be given varicella vaccination (one or two doses) with live attenuated Oka vaccine (Varivax, Merck & Co., Inc., Whitehouse Station, NJ, USA). There is currently a herpes zoster vaccine (Zostavax, Merck & Co., Inc.) that has not been studied in patients with end-organ disease awaiting transplantation. The Oka varicella vaccines have been shown to be safe in select children undergoing chemo‐ therapy, and studies have shown that they can be given safely to posttransplant recipients receiving immunosupression. Inactivated VZV vaccines, which are in development, may eventually provide another option for this high-risk population [65–68].
