Immunohistology of Infectious Disease

Hepatitis B

Hepatitis B virus infection constitutes an important cause of chronic hepatitis in a significant proportion of patients. In many instances, the morphologic changes induced by hepatitis B virus in hepatocytes are not typical enough to render a presumptive diagnosis of hepatitis B viral infection. In other instances, there may be so little hepatitis B surface antigen (HBsAg) that it cannot be demonstrated by techniques such as orcein staining. In these cases, immunohistochemical techniques to detect HBsAg are more sensitive than histochemical methods and are helpful in reaching diagnosis. Immunostaining for HBsAg has been used in the diagnosis of hepatitis B and in the study of carrier states. ^, Eighty percent or more of cases with positive serologic results for HBsAg demonstrate cytoplasmic HBsAg using immunohistochemistry. By immunoperoxidase localization, hepatitis B core antigen (HBcAg) can be demonstrated within the nuclei, the cytoplasm of hepatocytes, or both. Predominantly cytoplasmic expression of HBcAg is associated with a higher grade of hepatitis activity and diffuse immunostaining of nuclei for HBcAg generally suggests uncontrolled viral replication in the setting of immunosuppression. Immunostaining for HBsAg and HBcAg is useful in the diagnosis of recurrent hepatitis B infection in liver allografts, particularly when present with atypical histopathologic features.

Herpesviruses

Histologically, the diagnosis of herpes simplex virus (HSV) infection involves the detection of multinucleated giant cells containing characteristic molded, ground glass–appearing nuclei and Cowdry’s type A intranuclear inclusions. When there are abundant viral inclusions within infected cells, the diagnosis is usually straightforward. However, the diagnosis of HSV infection can be difficult when the characteristic intranuclear inclusions, multinucleated cells, or both are absent, or when the amount of tissue in a biopsy specimen is small. In these cases, immunohistochemistry using either polyclonal or monoclonal antibodies against HSV antigens has proven to be a sensitive and specific technique to diagnose HSV infections ( Fig. 3.1 ).

Although polyclonal antibodies against the major HSV glycoprotein antigens are sensitive, they do not allow distinction between HSV-1 and HSV-2 because these two viruses are antigenically similar. In addition; the histologic features of HSV infection are not specific and can occur in patients with varicella-zoster virus (VZV) infection. Monoclonal antibodies against the VZV envelope glycoprotein gp1 are sufficiently sensitive and specific to allow a clear-cut distinction between HSV and VZV infections. ^,

Immunohistochemistry has also been useful in demonstrating the association of human herpes virus 8 (HHV-8) with Kaposi sarcoma, primary effusion lymphoma, and multicentric Castleman disease. The diagnosis of Kaposi sarcoma may be problematic due to its broad morphologic spectrum and similar appearance to other benign and malignant neoplastic vascular lesions. Immunostaining for HHV-8 latent associated nuclear antigen-1 (LANA-1) is useful to confirm the diagnosis of Kaposi sarcoma, particularly in difficult early lesions that may mimick the appearance of interstitial granuloma annulare and when the neoplasm presents in an unusual location. In these instances, immunohistochemistry allows distinction of Kaposi sarcoma from several morphologically similar vasoproliferative lesions. Immunostaining is restricted to the nuclei of spindle cells and endothelial cells of the slit-like vascular spaces ( Fig. 3.2 ). Immunohistochemistry has also demonstrated expression of HHV-8 LANA-1 in mesothelial cells of human immunodeficiency virus (HIV)–associated recurrent pleural effusions.

Cytomegalovirus (CMV) continues to be an important opportunistic pathogen in immunocompromised patients; it is estimated that 30% of transplant recipients experience CMV disease. The range of organ involvement in post-transplant CMV disease is wide; hepatitis occurs in 40% of liver transplant recipients, and pneumonitis is more frequently seen in heart and heart-lung transplant patients.

Histologic diagnosis of CMV in fixed tissues usually rests on the identification of characteristic cytopathic effects, including intranuclear or cytoplasmic inclusions, or both. However, histologic examination lacks sensitivity, and in some cases atypical cytopathic features can be confused with reactive or degenerative changes. Additionally, up to 38% of patients with gastrointestinal CMV disease fail to demonstrate any inclusions. In these cases, immunohistochemistry using monoclonal antibodies against early and late CMV antigens allows the detection of CMV antigens in the nucleus and cytoplasm of infected cells ( Fig. 3.3 ). The sensitivity of immunohistochemistry for detecting CMV infection ranges from 78% to 93%. ^, In addition, immunohistochemistry may allow detection of CMV antigens early in the course of the disease when cytopathic changes have not yet developed ; for example, CMV early nuclear antigen is expressed 9 to 96 hours after cellular infection and indicates early active viral replication. Immunohistochemistry has played a key role in the detection of CMV infection in patients with steroid refractory ulcerative colitis, leading to recommendations for the routine use of immunohistochemistry for the detection of CMV in the evaluation of these patients. ^, CMV immunostaining has been used in detecting occult CMV infection of the central nervous system of liver transplant patients who develop neurologic complications. It has also been used to demonstrate a high frequency of CMV antigens in tissues from first trimester abortions. CMV is the most common opportunistic organism found in liver biopsies from transplant patients, but the incidence of CMV hepatitis appears to be decreasing due to better prophylactic treatments. Although CMV hepatitis presents with characteristic neutrophilic aggregates within the liver parenchyma, atypical features suggestive of acute rejection or changes indistinguishable from those of any other viral hepatitis are occasionally observed. In addition, parenchymal neutrophilic microabscesses have been described in cases with no evidence of CMV infection. It is in these cases where immunostaining for CMV antigens is most useful in diagnosing CMV.

The sensitivity of immunohistochemistry is better than light microscopic identification of viral inclusions and compares favorably with culture and in situ hybridization. ^{, , , ,} Immunohistochemical assays can be completed faster than the shell vial culture technique, allowing for rapid results that are important for early anti-CMV therapy.

Other herpesvirus infections that have been diagnosed using immunohistochemical methods include human herpesvirus 6 infection and Epstein-Barr viral infection. Immunohistochemistry has been used to identify Epstein-Barr virus (EBV) latent membrane protein-1 in cases of Hodgkin lymphoma and post-transplant lymphoproliferative disorder ( Fig. 3.4 ).

Adenoviruses

Adenovirus is increasingly recognized as a cause of morbidity and mortality among immunocompromised patients owing to transplant and congenital immunodeficiency. ^, Rarely has adenovirus infection been described in HIV-infected patients. Characteristic adenovirus inclusions are amphophilic, intranuclear, homogeneous, and glassy, however, in some cases the infection may contain only rare cells showing the characteristic cytopathic effect. In addition, other viral inclusions, including CMV, human papilloma virus, HSV, and VZV, can be mistaken for adenovirus inclusions and vice versa. In these circumstances, immunohistochemical assay may be necessary for a definitive diagnosis. A monoclonal antibody that is reactive with 41 serotypes of adenovirus has been used in an immunohistochemical technique to demonstrate intranuclear adenoviral antigen in immunocompromised patients ( Fig. 3.5 ). However, many other antibodies react with only a limited number of adenovirus serotypes. Histologic diagnosis of adenovirus colitis is difficult and is usually underdiagnosed. Moreover, in immunosuppressed patients the incidence of coinfection with other viruses is high, and the presence of adenovirus tends to be overlooked. Immunohistochemical staining has been of value in differentiating adenovirus colitis from CMV colitis. ^,

Parvovirus B19 Infection

Parvovirus B19 has been associated with asymptomatic infections, erythema infectiosum, acute arthropathy, aplastic crisis, hydrops fetalis, chronic anemia, and red cell aplasia. In addition, Parvovirus B19 infection has been recognized as an important cause of severe anemia in immunocompromised leukemic patients receiving chemotherapy. The diagnosis of parvovirus infection can be achieved by identifying typical findings in bone marrow specimens, including decreased or absent red cell precursors, giant pronormoblasts, and eosinophilic or amphophilic intranuclear inclusions in erythroid cells. ^, Because intravenous immunoglobulin therapy is highly effective, a rapid and accurate diagnostic method is important. Immunohistochemistry with a monoclonal antibody against VP1 and VP2 capsid proteins has been used as a rapid and sensitive method to establish the diagnosis of parvovirus B19 infection in formalin-fixed, paraffin-embedded tissues. Immunohistochemistry is of particular help in detecting parvovirus B19 antigen in cases with sparse inclusions, to study cases not initially identified by examination of routinely stained tissue sections, or in cases of hydrops fetalis where there is advanced cytolysis ( Fig. 3.6 ). ^{, ,} Several studies have found a good correlation between morphologic, immunohistochemical, in situ hybridization, and polymerase chain reaction (PCR) results. ^{, , ,}

Viral Hemorrhagic Fevers

Since the 1970s, numerous emerging and reemerging agents of viral hemorrhagic fevers have attracted the attention of pathologists. Pathologists have played an important role in the identification of these agents and in supporting epidemiologic, clinical, and pathogenetic studies of the emerging viral hemorrhagic fevers. ^{, ,} Viral hemorrhagic fevers are often fatal, and in the absence of bleeding or organ manifestations these diseases are clinically difficult to diagnose and frequently require handling and testing of potentially dangerous biological specimens. In addition, histopathologic features are not pathognomonic, and they can resemble other viral, rickettsial, and bacterial (e.g., leptospirosis) infections. Immunohistochemistry is essential and has been successfully and safely applied to the diagnosis and study of the pathogenesis of these diseases.

Several studies have established the utility of immunohistochemistry as a sensitive, safe, and rapid diagnostic method for viral hemorrhagic fevers such as yellow fever ( Fig. 3.7 ), dengue hemorrhagic fever, ^, Crimean-Congo hemorrhagic fever, Argentine hemorrhagic fever, Venezuelan hemorrhagic fever, and Marburg disease. Additionally, a sensitive, specific, and safe immunostaining method has been developed to diagnose Ebola hemorrhagic fever in formalin-fixed skin biopsies ( Fig. 3.8 ). Immunohistochemistry demonstrated that Lassa virus, the causative agent of Lassa fever, primarily targets endothelial cells, mononuclear inflammatory cells, and hepatocytes ( Fig. 3.9 ).

Polyomaviruses

BK virus infections are frequent during infancy. In immunocompetent individuals, the virus remains latent in the kidneys, central nervous system, and B lymphocytes. In immunocompromised patients, the infection reactivates and spreads to other organs. BK virus nephropathy is an important cause of graft failure in patients with renal transplant, with prevalence varying from 2% to 4.5% in different transplant centers. ^, Since specific clinical signs and symptoms are lacking in BK virus nephropathy, the diagnosis can only be made histologically in a graft biopsy. In the kidney, the infection is associated with mononuclear interstitial inflammatory infiltrates and tubular atrophy, findings that can be difficult to distinguish from acute rejection. Further, the cytopathic changes observed in BK virus infection are not pathognomonic and can be observed in other viral infections. Moreover, in early BK virus infection, there are minimal or no histologic changes, although immunohistochemistry can identify viral antigens. ^, In this setting, immunohistochemistry with an antibody against the large T antigen of SV40 virus has been effective in demonstrating BK virus infection ( Fig. 3.10 ). ^{, ,}

The human polyomavirus JC is a double-stranded DNA virus that causes progressive multifocal leukoencephalopathy (PML). This fatal demyelinating disease is characterized by cytopathic changes in oligodendrocytes and bizarre giant astrocytes. In addition to detection by antibodies to SV40-T antigen, immunohistochemistry using a polyclonal rabbit antiserum against the protein VP1 is a specific, sensitive, and rapid method for confirming the diagnosis of PML. JC virus antigen is usually seen within oligodendrocytes ( Fig. 3.11 ) and occasional astrocytes, and antigen-bearing cells are more commonly seen in early lesions.

Other Viral Infections

Immunohistochemistry has also been used to confirm the diagnosis of respiratory viral diseases such as influenza A, H1N1 (swine flu), H5N1 (avian flu), and respiratory syncytial virus infections ( Fig. 3.12 ) when cultures were not available.

The diagnosis of rabies relies heavily on histopathologic examination of tissues to demonstrate the characteristic cytoplasmic inclusions (Negri bodies). In a large number of cases, Negri bodies may be inconspicuous and so few that confirming the diagnosis of rabies may be extremely difficult. Furthermore, in non-endemic areas the diagnosis of rabies is usually not suspected clinically. Also, patients may present with ascending paralysis, rather than one of the more common presenting signs. In these settings, immunohistochemical staining is a very sensitive, safe, and specific diagnostic tool for rabies ( Fig. 3.13 ). Other viral agents that can be diagnosed using immunohistochemical methods include enteroviruses, eastern equine encephalitis virus, and rotavirus.

Immunohistochemical staining has been used in the histopathologic diagnosis of viral hepatitis C; however, immunohistochemistry for this virus is not superior to serologic assays and detection of hepatitis C virus (HCV) RNA in serum.

Helicobacter pylori Infection

Gastric infection by H. pylori results in chronic active gastritis and is strongly associated with lymphoid hyperplasia, gastric lymphomas, and gastric adenocarcinoma. Heavy infections with numerous organisms are easily detected on routine hematoxylin and eosin–stained tissues; however, the detection rate is only 66% with many false-positive and false-negative results. ^, Conventional histochemical methods such as silver stains are more sensitive than hematoxylin and eosin in detecting H. pylori . However, for the detection of scant numbers of organisms, immunohistochemistry has proved to be highly specific and sensitive, less expensive when all factors are considered, superior to conventional histochemical methods, and has low interobserver variation ( Fig. 3.14 ). ^, Treatment for chronic active gastritis and H. pylori infection can change the shape of the microorganism, making difficult its identification and differentiation from extracellular debris or mucin globules. In these cases, immunohistochemistry improves the rate of successful identification of the bacteria even when histologic examination and cultures are falsely negative. Experts currently recommend using IHC for identification of H. pylori when there is chronic active gastritis and H&E does not detect H. pylori organisms. In patients with chronic inactive gastritis and no H. pylori organisms visible on H&E, IHC use is recommended when there is gastroduodenal ulceration, underlying gastric MALT lymphoma or adenocarcinoma, duodenal lymphocytosis, or presence of well-formed lymphoid follicles with germinal centers in the stomach. Similarly, IHC is also recommended for patients previously treated for H. pylori infection in the presence of chronic inactive gastritis with no visible H. pylori organisms on H&E. Helicobacter heilmannii belongs to the Helicobacter family and is a less common causative agent of chronic gastritis found in a few gastric biopsies. This bacterium has been associated with mild chronic gastritis, peptic ulceration, and rarely gastric adenocarcinoma and MALT-lymphoma. Commercially available polyclonal antibodies against H. pylori cross-react with H. heilmannii antigens, allowing for detection of this microorganism in gastric biopsies with a paucity of bacteria ( Fig. 3.15 ). Recently, polyclonal antibodies against Treponema pallidum have been described to immunostain gastric biopsies with H. heilmannii gastritis.