Case 4 -

CMV Pneumonitis in Lung Transplant Patient Carol Farver Cleveland Clinic Foundation Cleveland, OH

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Clinical History The patient is a 57 year old white female, 4 months status post single lung transplantation (donor/recipient CMV+) for emphysema secondary to smoking. She presented with increasing shortness of breath which progressed from patchy infiltrates to complete opacification and diffuse infiltration of the transplanted lung by chest X-ray. The patient was brought to the OR for a surgical lung biopsy. The microscopic slides and images are from that procedure. Case 4 - Figure 1 - Low power of lung biopsy with hyaline membranes (Hematoxylin and eosin stain) Case 4 - Figure 2 - Hyaline membrane and type 2 pneumocyte hyperplasia (Hematoxylin and eosin stain) Case 4 - Figure 3 - High power view of type 2 pneumocyte atypia and inclusions (Hematoxylin and eosin stain) Case 4 - Figure 4 - Immunohistochemical study in area of Figure 3. (Hematoxylin counter stain). Diagnosis: Diffuse alveolar damage with cytomegalovirus infection Discussion Human cytomegalovirus is a member of the herpesvirus family and has many features similar to the other herpes viruses. The CMV virus can establish a latent or a persistent infection after a primary infection and may also be reactivated in an immunocompromised host. Infection with CMV is common in normal immunocompetent hosts, occurring in 50% to 80% of the population, and usually happens in one of three ways: In utero (congenital), at birth (perinatal) or later in life (postnatal). In recent years, the reactivation of CMV in the setting of either congenital or acquired defects in cellular immunity is the usual clinical setting in which a CMV infection is found. This is most frequent in patients who have undergone heart, lung, other solid organ and bone marrow transplantation. The role of CMV as a primary cause of serious pulmonary disease in immunocompromised patients became apparent as collections of these patients were studied. Its importance as a cause of fatal pneumonia was established originally in renal allograft recipients. In patients with allogeneic bone marrow transplants, it is now recognized as the most common form of pneumonia and is responsible for the highest fatality rate. Some studies report that up to 80% of bone marrow transplant patients will acquire CMV pneumonia within the first 30-90 days after their transplant. Risk factors for the disease include advanced age, the presence of acute graft-vs-host disease, intensive care regimens, and allografts. CMV infection and pneumonitis develop in the majority of isolated lung transplant recipients, who are serologically at risk, i.e. those seronegative recipients who receive seropositive donor lungs. Finally, the CMV infection is seen in the HIV (+) population though it is a much less common problem in these patients than in transplant patients. Clinically, patients with CMV pneumonia have sustained fever, nonproductive cough, and dyspnea. Rales may be detected and marked hypoxemia is usually present. Chest x-ray findings vary, but are usually bilateral, with diffuse infiltrates involving the mid and lower lung zones. Often the perihilar distribution of the infiltrate is suggestive of pulmonary edema. Progression to consolidation may occur. The clinical assessment of patients with suspected CMV pneumonia is complicated by their frequently having simultaneous pulmonary infections with other microbial agents. These include a variety of other bacterial, mycobacterial, viral, fungal and Pneumocystis carinii infections. In addition, noninfectious conditions are also common, including pulmonary malignancy or hemorrhage, the side effects of treatment therapies such as radiation therapy, chemotherapy or assisted ventilation and, in the transplant population, the effects of acute rejection. Pathology The diagnosis of CMV pneumonia depends on culture and pathologic examination of specimens obtained by bronchoalveolar lavage and biopsy. Finding the virus in respiratory secretions, urine or blood establishes that the patient has a CMV infection but does not show that the CMV is the causative agent of the pneumonia. Confirmation of the lung pathology requires tissue examination usually via transbronchial biopsy or open lung biopsy. The pathologic features include cellular enlargement (cytomegaly) with characteristic nuclear and cytoplasmic inclusions of CMV. (See Table 1). The intranuclear inclusion is a single dark basophilic body with a smooth border and a clear zone or halo around it. Typically, the nucleolus is retained within the inclusion body and the chromatin marginates and condenses on the inner aspect of the nuclear membrane outside of the halo. The intracyoplasmic inclusions are multiple and are small basophilic, irregular, granular structures in the perinuclear area. As they mature, they reach a maximum size of approximately 3 microns and coalesce near the cell membrane. They may be difficult to see when not fully developed, but will stain with a GMS stain which can be used to help identify them. CMV usually infects epithelial cells, endothelial cells, fibroblasts, T cells or macrophages in the lung. Most commonly, diagnostic viral inclusions can be found in the alveolar macrophages. The tissue reaction to the infection depends upon the immune status of the host. In general, CMV pneumonitis results in an interstitial pneumonia or in more severe infections diffuse alveolar damage. Techniques Used in the Diagnosis of CMV in Tissue Sections Examination of lung tissue by standard histologic methods has been a useful way of detecting cells containing CMV inclusions, however, the level of sensitivity of this method compares poorly with that of culture. More recently, nucleic acid in situ hybridization and immunohistochemistry using monoclonal antibodies against CMV (immediate) early antigens have proved to be valuable. Though previously limited to frozen tissue, both methods are now available for analysis of routinely obtained formaldehyde-fixed, paraffin-embedded tissue sections. More recently, new molecular diagnostic techniques are used for a more sensitive and rapid detection of CMV in various tissues and even quantitation of CMV viral load in patients. In general, most laboratories now use blood or bronchoalveolar lavage (BAL) samples to identify CMV RNA or DNA by Southern blot or PCR, if needed. If positive, detection of virus within tissue is sought, either by diagnostic viral inclusions on H & E stained tissue or by immunohistochemistry or in situ hybridization. In general, if BAL fluid only is obtained, there is a risk of over diagnosis, as approximately one third of the patients who have CMV detected in BAL fluid do not have CMV pneumonitis. The relationship of viral load in BAL fluid and blood to the presence of CMV inclusions in tissue has not been well-studied. Our laboratory measured the CMV viral load (number of copies/ml) by hybrid capture assay in BAL fluid and blood in lung transplant patients and compared this to the presence of CMV inclusions in lung transplant biopsies by H&E and immunohistochemitry (IHC). Our results revealed that a high number of virus copies of CMV in BAL fluid and blood from lung transplant patients was associated with the presence of typical (+IHC and cytomegalic) CMV inclusions in the lung tissue. An intermediate number of virus copies in BAL fluid correlated with the presence of atypical (+IHC but not cytomegalic) CMV inclusions in lung tissue from these patients. However, there was no correlation between an intermediate number of virus copies in the blood and the presence of inclusions in the lung tissue. (See Table 2.) Overall, these findings suggest that quantitation of CMV viral load may be useful in identifying patients with CMV viral inclusions in their lungs (high viral loads) and patients at risk to develop CMV viral inclusions in their lungs (intermediate viral loads), who may require close clinical follow-up. Table 1. Morphologic features of cytomegalovirus inclusions Laboratory Technique Nuclear Inclusions Cytoplasmic Inclusions Hematoxylin and eosin stain Large, amphophilic with halo Small, multiple, basophilic Histochemical stain   GMS (+); PAS (+) Immunohistochemistry Early antigen (Dako)   Ultrastructural analysis by electron microscopy Amorphous, incomplete virions Mature virion, 120-200 nm with electron dense core, capsid, surrounding envelope Table 2. Comparison of BAL fluid and blood CMV viral loads with CMV IHC staining results IHC staining for CMV BAL Fluid Blood   CMV viral load (no. of copies/ml) No. of samples CMV viral load (no. of copies/ml) No. of samples   Median Range   Median Range   Negative 0 0-193,442 26 0 0-40,587 20 Atypical 47,678 2,791-312,175 8 1,176 0-14,019 7 Typical 1,578,827 555,505-2,450,702 5 189,013 0-1,491,020 5 P value 0.001 0.019   0.028 0.15   From: Chemaly RF, Yen-Lieberman B, Castilla EA, Reilly A, Arrigain S, Farver C, Avery RC, Gordon SM, Procop GW. 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