ZF Jiang and KL Shen

Introduction

CMV is a ubiquitous agent. It is a DNA virus and is a member of herpes group of viruses. CMV is not eliminated from the body after primary infection but persists in the form of low-grade chronic infection or remains in a latent state. It causes widespread infection but under normal conditions rarely kills its host. When the host's immune system is immature or compromised, infection can be associated with diseases, and the target organs include liver, brain, eye, lung, gastro-intestinal tract, bone marrow. CMV pneumonia is a major cause of morbidity and mortality in immunocompromised patients. This review will be restricted to CMV pneumonia in non-immune children.

CMV pneumonia might occur in congenital CMV infection as a part of cytomegalic inclusion disease (CID). It often infects neonates and small infants because of their relatively immunocompromised state. In a study of 205 infants younger than 3 months of age who were hospitalized for pneumonitis, CMV is a common etiologic agent (20%).¹

CMV pneumonia occurs mainly in immunocompromised individuals including infants and children with impaired cell-mediated immunity such as primary immune deficiency (PID), secondary immune deficiency (SID) and acquired immune deficiency syndrome (AIDS), transplant recipients, patients with connective tissue diseases and malignancies treated with immunosuppressive drugs.^2,3

In solid organ and marrow transplant recipients, serious CMV disease most often involves the lungs, producing an interstitial pneumonitis that is fatal in up to 30% of cases.^4,5

The frequency and morbidity of CMV infection in patients with malignancies is not as high as after marrow and organ transplantation. However, the use of chemotherapy, especially in leukemia, is associated with significant CMV disease, especially pneumonitis.^6,7

In children with AIDS, persistent pneumonia has been a major and significant problem. In a study of 307 pediatric cases of AIDS, disseminated CMV being the most common cause of persistent pneumonia (57 patients 19%).^8,9 In a clinical and pathologic study on the causes of death in 75 AIDS patients with emphasis on the role of pulmonary diseases CMV was the most prevalent pathogen, and was isolated from pulmonary tissue in 44 patients and caused significant disease in 21 patients. Five patients died of CMV pneumonia.¹⁰

Pathogenesis

Infections with CMV can be latent and nonproductive, productive yet asymptomatic or productive and symptomatic, depending on the host defense of CMV. The poor maturity of immunity and reduced cell-mediated immune responses due to diseases or immunosuppressive therapy appear to be the major factors associated with development of serious CMV diseases.

The pathogenesis of CMV pneumonia, is not yet well understood. There are several hypotheses:

1. Direct consequence of cytopathogenic effects of HCMV. Aukrust reported a case of fatal pulmonary disease in patient infected with HIV, where CMV was the only causative agent identified in the lungs at autopsy. The most prominent histopathological features were numerous interalveolar cells containing CMV inclusion bodies combined with scanty signs of inflammation. They propose that the lung damage caused by CMV in AIDS patients is a direct consequence of cytopathogenic effects of the virus related to the extent of active virus replication.¹¹

2. Reactivation of infectious virus from the latent viral genomes. Animal experiment showed that lungs are an authentic site of CMV latency and a high risk organ for CMV recurrence. When the host becomes immuno-suppressed, CMV is likely to become reactivated and this may explain in part why interstitial pneumonia is a frequent manifestation of recurrent CMV infection.¹²

3. CMV pneumonia - an immunopathologic condition. Squire described severe CMV pneumonitis in two HIV infected patients with higher than average CD 4 counts. Both patients with severe pneumonitis had relatively well preserved immune function, which support the hypothesis that severe CMV pneumonitis is an immunopathological condition.¹³

CMV pneumonitis in bone marrow transplant recipients has been hypothesized to be resulted from host cell-mediated events produced in response to chronic viral replication.¹⁴ The severe tissue destruction seen in CMV pneumonia may be, in part, an autoimmune phenomenon.

The difference in histopathological features between AIDS patients and transplant recipients may well reflect different pathogenetic mechanisms. It has been suggested that CMV pneumonitis in allogeneic transplant recipients is caused by immune mechanisms mediated by a T-cell response to virally induced antigens expressed in the lungs, and severe necrotizing pneumonitis may occur in spite of suppression of virus replication during ganciclovir therapy.¹⁵

Analysis of the cells in the lungs obtained by bronchoalveolar lavage (BAL) from mice with interstitial pneumonitis showed an influx of Thy 1.2-positive cells of donor origin. Depletion of Thy 1.2-positive cells from the donor inoculum prevented the development of interstitial pneumonitis. Thus, it was proposed that some immune response, triggered by MCMV and mediated by donor lymphocytes, that was directed against recipient cells in the lung was involved in the pathogenesis of the pneumonitis.^16,17 CMV late viral proteins may still be expressed in the tissues of patients treated with ganciclovir. The hypothesis should therefore be extended to include CMV late proteins as candidate antigens for stimulation of the pathologic host cell response in the lung.¹⁸

CMV-specific cytotoxic T-lymphocyte (CTL) response has been implicated in resistance to viral infection. Reusser reported that CTL were detected in only 10 of 20 patients after BMT. None of these 10 patients developed CMV pneumonia, whereas 6 of the 10 patients with an undetectable CMV-specific CTL response after bone marrow transplantation died with CMV pneumonia. Approaches to reconstitute CMV immunity in BMT patients by adoptive transfer of CMV-specific CD8+CTL clones derived from the bone marrow donor are now being pursued.¹⁹

The delineation of the role CMV plays in the pathogenesis of pneumonia is further complicated by the identification of coexisting pathogens such as Pneumocystis carinii. Indeed, these two organisms are isolated together so frequently that a synergistic relationship between them may exist. CMV may itself contribute to further diminishing cellular immunity which may result in superinfection or reactivation of other opportunistic pathogens. Animal studies have shown increased susceptibility to bacterial and fungal pathogens after murine CMV infection and impaired function of lymphocytes and phagocytic cells.^20,21 These data suggest an association between CMV infection and the subsequent development of severe bacterial infection.

Pathology

The characteristic pathologic features of CMV infection are cytolysis, focal necrosis, mononuclear cell infiltration with obliteration of alveolar spaces and thickening of alveolar septa, and the formation of cytomegalic cell. The typical cytomegalic cell is very much enlarged with eosinophilic intranuclear inclusion body that pushes the chromatin to the periphery, creating the "owl's eyes" appearance. These cytomegalic cells are seen in the alveolar and bronchial epithelium and interstitium and are associated with mononuclear cell inflammation. Pulmonary alveolar macrophage may express viral Ag and contain CMV-DNA.

In situ hybridization and in situ PCR for determining CMV in lung tissue and the histological examination showed the pathological lesions of HCMV infection had three different patterns: 1, necrotic granuloma type, 2, diffuse type, and 3, scattered type. HCMV-DNA was identified in all the inclusion bodies and dense, homogenous CMV-DNA(+) material was found in the nuclei and cytoplasm of the cytomegalic cells. In addition, some morphologically intact cells were found positive for CMV DNA in both nuclei and cytoplasm.^22,23

Clinical Manifestations

The clinical manifestations and X-rays findings are nonspecific. CMV pneumonia in non-immune children is a diffuse pneumonitis characterized by a prolonged clinical course, frequent co-infection with other opportunistic pathogens, most often Pneumocystis carinii (PC) and fungus. It may occur as the only disease manifestation or be part of a disseminated CMV infection. In immunocompromised patients, CMV pneumonitis usually occurs several months after the initiation of the immunosuppressive therapy or 1-4 months after the transplantation. It begins with symptoms of fever and dry, nonproductive cough. ft can then progress over 1 to 2 weeks to tachypnea, dyspnea, nasal flaring, retractions, diffuse rales, wheezing, hypoxia and acute respiratory failure.²⁴ These findings while necessary to establish the diagnosis, are very nonspecific and do not allow differentiation of CMV from P. carinii or other causes of diffuse pneumonia.

In patients with immune deficiency, particularly AIDS patients, in addition to pneumonitis, CMV can cause hepatitis, severe retinitis, gastroenteritis, colitis, meningoencephalitis, encephalitis and periodontal disease.

The radiographic appearance of CMV pneumonia is most often diffuse with interstitial infiltrates, but peribronchial infiltrates with hyperinflation and nodular pulmonary infiltrates, focal subsegmental opacity, lobar consolidation and pleural effusion were also observed.²⁵

CT findings include ground-glass attenuation, dense consolidation, bronchial wall thickening or bronchiectasis, and interstitial reticulation without air-space disease. Histopathologic findings correlate well with CT appearances.²⁶

Diagnosis

A number of methods are currently available for the detection of CMV in clinical specimens. These include isolation of virus, antigen detection, nucleic acid hybridization, DNA amplification, cytology and histology.^27-33

The clinical specimens come from sputum, deep tracheal and broncheal aspirates, BAL, transbronchial biopsy and open-lung biopsy. BAL may be superior to specimens obtained by lung biopsy because the primary sites of CMV infection in the lung are alveolar lining cells, which are easily recovered by lavage.^34,35 BAL was the most sensitive technique for detecting CMV whereas transbronchial biopsy is relatively insensitive.³⁶

Culture of the virus remains the most sensitive and specific test for the presence of CMV in lung tissues or BAL fluid, but the standard tube culture usually takes more than a week to show evidence of the virus, so the method is too slow to help in making therapeutic decision. The new shell viral technique greatly increases the sensitivity and speed of culture. The sensitivity is increased 80 fold and the speed of identification of CMV is greatly increased by using fluorescein-labeled monoclonal antibodies to detect early viral antigens. This permits identification of the virus within 16-24 hours. The combination of BAL and centrifugation culture is a highly sensitive and specific technique to detect CMV infection among bone marrow transplant recipients with pneumonitis, approaching that of viral culture of lung tissue. Recovery of virus correlates fairly closely with histopathological evidence of CMV pneumonia.³⁷ The recovery of CMV in culture from lung tissue of patients without pneumonitis has been uncommon.

Monoclonal antibodies directed against specific CMV antigen, such as immediate-early antigen, greatly enhanced the diagnosis of CMV pneumonia, when labeled with fluorescein or enzymatic markers, these monoclonal antibodies are able to detect specific CMV antigen in sections of lung biopsies and in cells obtained by BAL.^38,39 This technique provides an answer within a few hours but the sensitivity of this technique is considerably less than that of shell viral culture technique.

In situ hybridization has revealed the presence of CMV even in cells that do not appear to be infected by standard histological criteria.⁴⁰ It is applicable to formalin-fixed, paraffin-embedded tissue. It could be used for the rapid diagnosis of biopsied tissue as well as for retrospective studies of autopsied tissue. Although in situ hybridization can be a rapid, useful method for detecting CMV in BAL specimens, cytology appear to be a more sensitive method. The current hybridization methods are not sufficiently sensitive to replace centrifugation culture and cytology.⁴¹

PCR was found to be the most sensitive method for detection of CMV in BAL fluid. Rapid diagnosis of CMV pneumonitis by PCR in BAL appears to be at least as sensitive as CMV culture. Only active CMV infection was detected with the PCR applied to BAL fluids as a rapid method to detect CMV lung infection.⁴²

A combination of PCR and detection of CMV antigen after short incubation and directly in alveolar cells is optimal for rapid identification of CMV.⁴³

PCR-based testing is easy and more sensitive than traditional tests, allowing detection of viral replication earlier than tissue culture in the posttransplant period. PCR could provide a powerful means of monitoring the immunocompromised patients in whom presumptive therapeutic intervention for CMV disease is desirable.⁴⁴

The value of serologic tests for CMV is limited., because most of children have been infected by CMV before the school age and have antibodies to the virus, and CMV disease in the immunosuppressed is most often due to reactivation of latent infection rather than primary infection. The detection of antibodies to CMV for diagnosis of interstitial pneumonia is problematical in the phase immediately after BMT because bone marrow recipients are severely immunosuppressed by radiation and cytotoxic drugs received before transplantation. Although the majority of primary infections in transplant recipients are associated with an IgM antibody response, some immunocompromised patients may have no detectable antibody response of any kind.⁴⁵ Thus, two more reasons for the clinical use of serologic tests for CMV are for determining susceptibility to primary infection and for screening blood and organ donors for previous exposure to CMV and therefore for the potential for transmitting latent CMV.⁴⁶

There are problems in interpreting various laboratory test and in understanding pathogenesis such as the clinical manifestation and X-ray findings were unspecific, 33% patients who had CMV cultured from lung washes did not show histologic evidence of CMV infection in AIDS patient and virus culture might be a too sensitive method to diagnose CMV pneumonia.³⁵ In studies on patients infected by HIV, the presence of CMV in BAL material was not found to be associated with an increased prevalence of subsequent pulmonary CMV disease.^47,48 There is no absolute definite diagnostic test for CMV pneumonia, and the strength of the diagnosis rests on weighing a variety of clinical and laboratory finding, such as:

1. High risk patients. Immunocompromised patient are at high risk for CMV pneumonia although there are few reports that apparently immunocompetent individuals had severe evolution of cytomegalovirus infection.^33,49,50

2. Presence of interstitial pneumonia. The typical clinical picture of diffuse pneumonia with hypoxemia is an important finding. The presence of abnormal liver function and anemia, leukopenia, and thrombocytopenia may be helpful in suggesting CMV infection.^51,52

3. Demonstration of CMV, its antigen, or its genome in good quantity. A combination of PCR, detection of CMV antigen and direct demonstration of CMV in alveolar cells should be used. When positive PCR is the only indicator of CMV infection, the findings must be carefully evaluated. Because of the frequent finding of CMV in asymptomatic immunosuppressed patients, the mere presence of the virus, its antigen or its genome is not sufficient evidence to establish the diagnosis of CMV pneumonia.^53,54

4. Equally important is the demonstration of many cytomegalic cells and typical pathologic abnormalities on examination of BAL fluid or lung tissue and the presence of CMV on BAL fluids and lung tissue obtained by biopsy.

5. The only presence of the virus in urine, blood, and other nonpulmonory sites is of little value in making the diagnosis of CMV pneumonia.⁵⁵

A lung culture that is positive for CMV supports a diagnosis of CMV pneumonitis, especially if characteristic viral inclusions are seen in the tissue. If CMV is recovered from the lung or indicated by antigen detection or nucleic acid hybridization and no other pathogen or pathologic process is identified, CMV is probably the cause of the pneumonitis.

It is reasonable to conclude that the presence of CMV in lung tissues or BAL washing, particularly when present in large amounts, is a strong supportive evidence for CMV as a possible etiologic agent.

Treatment

The antiviral agent 9-[(1, 3-dihydroxy-2-propoxy)-methyl]guanine (DHPG or ganciclovir) has shown good activity against CMV in vitro and has been shown to be therapeutically effective against CMV disease in man. Ganciclovir, the derivative of acyclovir is a synthetic acyclic nucleotide analog of guanine. It can inhibit viral DNA polymerase of the human herpes viruses. Ganciclovir is 10 times more active than acyclovir against CMV by in vitro testing.^56,57

DHPG has been successfully used to treat CMV retinitis and enteritis,^57,58 but its efficacy in treating CMV pneumonitis remain controversial. The use of DHPG for CMV pneumonia in recipients of solid organ transplants and AIDS patients may be associated with rapid clinical improvements and a favorable outcome in the majority of AIDS patients.^59,60 When used to treat CMV pneumonitis in bone marrow transplant recipients, ganciclovir alone is not as effective as when it is combined with immune globulin.⁶¹ Ganciclovir did not appear to benefit the immunocompromised patients with CMV pneumonia in pediatric intensive care units.⁶²

There was a study on the effect of treatment of CMV-induced interstitial pneumonia (IP) established in RCMV-infected immunosuppressed BN rat. Although DHPG treatment induced a significant decrease in RCMV titers in the lungs, the effect of DHPG had only a minor and non-significant effect on both mononuclear cell infiltration and thickness of the alveolar septa and without effect on vascular dilatation, alveolar edema and hemorrhages.⁶³ The findings are consistent with the reports about the limited effect of DHPG treatment on HCMV-induced IP in allogeneic bone marrow transplant recipient patients.^59,64 The failure of response to antiviral treatment emphasizes the role of immunopathologic responses in the pathogenesis of CMV-induced IP.^15,66

Intravenous treatment with hyper immune serum (HIS) or hyper immune globulin(HIG) containing antibodies directed against CMV significantly reduced both mortality and virus titters in lungs. In an open study, mortality, which historically is 85%, was reduced to 50%. This is probably because of the strong beneficial effect of antibodies on the immunopathological response in the lungs. Patients who did not respond to this therapy were already being ventilated before treatment suggesting that prompt treatment was necessary. DHPG and HIS treatment minimized virus titer in internal organs and CMV-induced IP. In man, various reports indicate the improved therapeutic effect on CMV-induced IP after combined treatment with HIS and DHPG in allogeneic bone marrow transplant recipients.^63-67

It is well known from transplant patients that once CMV pneumonia is established with PO₂< 9 KPa and with diffuse bilateral infiltrates on chest X-rays, the clinical effect of treatment is poor. For treatment to be effective, it must be initiated early in the respiratory infection. Fibreoptic bronchoscopy should therefore be performed as soon as the patient presents with dry cough and if possible, before the start or during the very first days of treatment. The early treatment results in a more favorable outcome. The use of a dosage of ganciclovir that did not produce profound neutropenia and the use of CMV immune globulin in conjunction with ganciclovir may be two factors contributing to an enhanced survival.

Experiences in the use of DHPG in children is limited. The data available imply the efficacy and toxicity of ganciclovia treatment of CMV disease in children may be similar to that in adults.

Interferon and acyclovir used singly or in combination have proved unsuccessful.

Since almost all the studies of the efficacy of ganciclovir in treating CMV pneumonia have not been placebo-controlled, it is not easy to draw definite conclusion for its efficacy. There is a great need for controlled clinical trials of the efficacy of ganciclovir in patients with a well-established diagnosis of CMV pneumonia.

Prevention

Prophylactic measures can sometimes prevent or ameliorate CMV infection. Useful infection control measures include:

1. The use of blood product from CMV seronegative donors for immunocompromised children.

2. The use of CMV seronegative organ donors for seronegative recipients.

3. Passive immunoprophylaxis with a CMV specific immunoglobulin for the prevention of symptomatic cytomegalovirus disease in the transplant recipients.⁶⁸

4. Active immunization with CMV vaccine. Studies showed the Towne 125 vaccine is relatively safe and able to induce humoral and cellular immunity in both healthy and immunosuppressed subjects. A genetically engineered subunit vaccine based upon the surface glycoprotein of the virus shown to be targets for neutralizing antibody has been explored.⁶⁹

5. Prophylaxis use of antiviral agents. High-dose acyclovir failed to reduce the incidence of CMV disease, but significantly reduced the probability of dying at 1 year of follow up.⁷⁰ Recent study shows that CMV infection, as determined by rapid culture from prospective BAL specimens on day +35 has been identified as a formidable risk factor for the development of pneumonia after bone marrow transplantation. Preemptive therapy with ganciclovir alone prevents the evolution from infection to pneumonia in this subgroup while protecting most other patients from unnecessary drug exposure.⁷¹
   For patients who are CMV antibody positive and thus at the risk of activation of latent infection, using acyclovir intravenously or using GCV orally have good effect on prevention.⁵²

6. Approaches to reconstitute CMV immunity in BMT patients by adoptive transfer of CMV-specific CD8 +CTL clones derived from the bone marrow donor are now being pursued.¹⁹

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