Immunohistology of Infectious Diseases

INTRODUCTION

Since the 1980s, immunohistochemistry (IHC) has dramatically transformed the approach to histopathologic diagnosis, specifically in the diagnosis and classification of tumors, and more recently in the diagnosis of infectious diseases in tissue samples.¹

Pathologists play an important role in recognizing infectious agents in tissue samples from patients, providing a rapid morphologic diagnosis, and facilitating clinical decisions in patient treatment. When fresh tissue is not available for culture, pathologists can provide a rapid morphologic diagnosis and facilitate clinical decisions in patient treatment.² In addition, pathologists have played a central role in identifying emerging and reemerging infectious agents, describing the pathogenetic processes of emerging diseases (e.g., hantavirus pulmonary syndrome, viral hemorrhagic fevers, leptospirosis, and rickettsial and ehrlichial infections), and diagnosing anthrax during the bioterrorist attack of 2001.3., 4., 5., 6., 7.

Cultures and serologic assays are usually used for microbial identification in infectious diseases. However, fresh tissue is not always available, and culturing fastidious pathogens can be difficult and may take weeks or months to yield results. Moreover, culture alone cannot distinguish colonization from tissue invasion. In addition, serologic results can be difficult to interpret in the setting of immunosuppression or when only a single sample is available for evaluation. Some microorganisms have distinctive morphologic characteristics that allow their identification in formalin-fixed tissues using routine and special stains. Nevertheless, in many instances it is difficult or even impossible to identify an infectious agent specifically by conventional morphologic methods.

Immunohistochemistry is one of the most powerful techniques in surgical pathology. There has been an increasing interest in the use of specific antibodies to viral, bacterial, fungal, and parasitic antigens in the detection and identification of the causative agents in many infectious diseases. Coons and associates were the first to use a specific antibody to detect a microbial antigen to detect pneumococcal antigen in tissues.⁸ The advantages of IHC over conventional staining methods (Table 3.1 ) and the contributions of IHC in infectious diseases (Table 3.2 ) are substantial. In many instances, IHC has shown high specificity, allowing the differentiation of morphologically similar microorganisms.⁹ Immunohistochemistry is especially useful when microorganisms are difficult to identify by routine or special stains, are fastidious to grow, or exhibit atypical morphology (Table 3.3 ).10., 11., 12., 13., 14. It is important to understand that there may be widespread occurrence of common antigens among bacteria and pathogenic fungi, and both monoclonal and polyclonal antibodies must be tested for possible cross-reactivity with other organisms.¹⁵ Finally, it is important to emphasize that IHC has several steps, and that all of them can affect the final result; however, in general the only limitations are the availability of specific antibodies and the preservation of epitopes.¹⁶

TABLE 3.1.

Advantages of IHC for the Diagnosis of Infectious Diseases

1.Opportunity for rapid results

2.Reduced risk of exposure to serious infectious diseases by performance on formalin-fixed, paraffin-embedded tissue

3.High sensitivity allowing identification of infectious agents even before morphologic changes occur

4.Opportunities for retrospective diagnosis of individual patients and for in-depth study of the disease

5.Specific identification of infectious agents with many monoclonal antibodies and some polyclonal antibodies

TABLE 3.2.

Contributions of IHC to the Diagnosis of Infectious Diseases

1.Allows identification of new human pathogens

2.Allows microbiological-morphological correlation establishing the pathogenic significance of microbiological results

3.Provides a rapid morphologic diagnosis allowing early treatment of serious infectious diseases

4.Contributes to understanding of the pathogenesis of infectious diseases

5.Provides a diagnosis when fresh tissue is not available or when culture methods do not exist

TABLE 3.3.

Applications of IHC in the Diagnosis of Infectious Diseases

1.Identification of microorganisms that are difficult to detect by routine or special stains

2.Detection of microorganisms that are present in low numbers

3.Detection of microorganisms that stain poorly

4.Identification of microorganisms that are fastidious to grow or noncultivable

5.Identification of microorganisms that exhibit atypical morphology

Table 3.4 lists some commercially available antibodies for diagnostic use in surgical pathology.

TABLE 3.4.

Commercially Available Antibodies for Immunohistochemical Diagnosis of Infectious and Prion Diseases

Microorganism	Antibody/Clone	Dilution	Pretreatment	Source
Adenovirus	Mab/20/11 and 2/6	1:2000	Proteinase K	Chemicon
B. henselae	Mab	1:100	HIAR	Biocare Medical
BK virus	Mab/BK T.1	1:8000	Trypsin	Chemicon
C. albicans	Mab/1B12	1:400	HIAR	Chemicon
C. pneumoniae	Mab/RR402	1:200	HIAR	Accurate
Cryptosporidium	Mab/Mabc1	1:100	HIAR	Novocastra
CMV	Mab/DDG9/CCH2	1:50	HIAR	Novocastra
Clostridium spp.	Rabbit polyclonal	1:1000	None	Biodesign
G. intestinalis	Mab/9D5.3.1	1:50	HIAR	Novocastra
Hepatitis B core antigen	Rabbit polyclonal	1:2000	HIAR	Dako
Hepatitis B surface antigen	Mab/3E7	1:100	HIAR	Dako
Herpes simplex 1 and 2 viruses	Rabbit polyclonal	1:3200	HIAR	Dako
H. pylori	Rabbit polyclonal	1:40	Proteinase K	Dako
HHV 8	Mab/LNA-1	1:500	HIAR	Novocastra
K. pneumoniae	Rabbit polyclonal	1:200	Proteinase K	Biogenex
L. monocytogenes	Rabbit polyclonal	1:5000	Proteinase K	Difco
M. pneumoniae	Mab/1.B.432	1:25	HIAR	US Biological
Parvovirus B19	Mab/R92F6	1:500	HIAR	Novocastra
P. carinii	Mab/3F6	1:20	HIAR	Novocastra
P. falciparum	Mab/BDI400	1:1000	Proteinase K	Biodesign
Prion	Mab/3F4	1:200	Antigen retrieval	Dako
	Mab/12F10	1:1000	Proteinase K	Cayman Chemical
Mab/KG9	1:1000	Proteinase K	TSE Resource Center
Respiratory syncytial virus	Mab/5H5N	1:200	HIAR	Novocastra
S. aureus	Rabbit polyclonal	1:500	Proteinase K	Biodesign
T. pallidum	Rabbit polyclonal		HIAR	Biodesign
T. gondii	Rabbit polyclonal	1:320	HIAR	Biogenex
West Nile virus	Mab/5H10	1:400	Proteinase K	Bioreliance

VIRAL INFECTIONS

Immunohistochemistry has played an important role not only in the diagnosis of a large number of viral infections but also in the study of their pathogenesis and epidemiology. Conventionally, the diagnosis of viral infections has relied on cytopathic changes observed by routine histopathologic examination. Several viral pathogens produce characteristic intracellular inclusions, which allow pathologists to make a presumptive diagnosis of viral infection. However, for some viral infections the characteristic cytopathic changes are subtle and sparse, requiring a meticulous search.¹⁷ Moreover, only 50% of known viral diseases are associated with characteristic intracellular inclusions.¹⁸ In addition, formalin, which is the most commonly used fixative in histopathology, is a poor fixative for demonstrating the morphologic and tinctorial features of viral inclusions.¹⁹ When viral inclusions are not detected in hematoxylin and eosin–stained sections or when the viral inclusions present cannot be differentiated from those of other viral diseases, immunohistochemical techniques offer a more reliable approach to reach a specific diagnosis.

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 a diagnosis.²⁰ Immunostaining for HBsAg has been used in the diagnosis of hepatitis B and in the study of carrier states.21., 22. Eighty percent or more of cases with positive serologic results for HBsAg demonstrate cytoplasmic HBsAg using IHC.²³ By immunoperoxidase localization, hepatitis B core antigen (HBcAg) can be demonstrated within the nuclei or the cytoplasm of hepatocytes, or both. Cytoplasmic expression of HBcAg usually is associated with a higher grade of hepatitis activity,²³ and diffuse immunostaining of nuclei for HBcAg generally suggests uncontrolled viral replication in the setting of immunosuppression.²⁴ 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.²⁵

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 abundant viral inclusions exist within infected cells, the diagnosis is usually straightforward. However, diagnosis can be difficult when the characteristic intranuclear inclusions or multinucleated cells, or both, are absent or when the amount of tissue in a biopsy specimen is small.²⁶ In these cases, IHC using either polyclonal or monoclonal antibodies against HSV antigens has proven to be a sensitive and specific technique used to diagnose HSV infections (Fig. 3.1 ).27., 28., 29., 30.

FIGURE 3.1.

Photomicrograph of cervical biopsy from a patient with herpes simplex virus infection showing abundant nuclear and cytoplasmic antigen. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Although polyclonal antibodies against major HSV glycoprotein antigens are sensitive, they do not allow distinction between HSV-1 and HSV-2; this is because the two viruses are antigenically similar.³¹ In addition, the histologic features of HSV infection are not specific and can also occur in patients with varicella-zoster (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.27., 32., 33.

Immunohistochemistry has also been useful in demonstrating the association of human herpes virus 8 (HHV-8) with Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease.34., 35., 36., 37., 38. Diagnosis of Kaposi’s sarcoma may be problematic because of its broad morphologic spectrum and similar appearance to other benign and malignant neoplastic vascular lesions. Immunostaining of latent associated nuclear antigen-1 (LANA-1) is useful to confirm the diagnosis of Kaposi’s sarcoma, particularly when difficult early lesions closely resemble the appearance of interstitial granuloma annulare and when the neoplasm presents in an unusual location. Immunostaining also allows distinction of Kaposi’s sarcoma from several morphologically similar vasoproliferative lesions.39., 40., 41. Immunostaining is restricted to the nuclei of spindle cells and endothelial cells of the slitlike vascular spaces (Fig. 3.2 ). Immunohistochemistry has also demonstrated expression of HHV-8 LANA-1 in mesothelial cells of HIV-associated recurrent pleural effusions.⁴²

FIGURE 3.2.

Lymph node biopsy from a patient with Kaposi’s sarcoma. The spindle cells show strong nuclear staining for HHV-8 LANA-1 antigen. Endothelial cells of well formed vascular spaces are negative. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Cytomegalovirus (CMV) continues to be an important opportunistic pathogen in immunocompromised patients; it is estimated that 30% of transplant recipients experience CMV disease.⁴³ The range of organ involvement in post-transplant CMV disease is wide; hepatitis occurs in 40% of liver transplant recipients,⁴⁴ and pneumonitis is more frequently seen in heart and heart-lung transplant patients.⁴⁵ Other organs that are commonly affected are the gastrointestinal tract and the peripheral and central nervous systems. Histologic diagnosis of CMV in fixed tissues usually rests on identifying characteristic cytopathic effects including intranuclear inclusions, cytoplasmic inclusions, or both. However, histologic examination lacks sensitivity, and in some cases atypical cytopathic features can be confused with reactive or degenerative changes.⁴⁶ Additionally, up to 38% of patients with gastrointestinal CMV disease fail to demonstrate any inclusions.⁴⁷ In these cases, IHC 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 IHC for detecting CMV infection ranges from 78% to 93%.47., 48. In addition, IHC may allow detection of CMV antigens early in the course of the disease when cytopathic changes have not yet developed.49., 50., 51., 52., 53., 54. For example, CMV early nuclear antigen is expressed 9 to 96 hours after cellular infection and indicates early active viral replication. Immunohistochemistry has been used to detect CMV infection in patients with steroid refractory ulcerative colitis, and the routine use of IHC for the detection of CMV in the evaluation of these patients is now recommended.55., 56. CMV immunostaining has been used to detect occult CMV infection of the central nervous system in liver transplant patients who develop neurologic complications.⁵⁷ It has also been used to demonstrate a high frequency of CMV antigens in tissues from first-trimester abortions.⁵⁸ CMV is the most common opportunistic organism found in liver biopsies from transplant patients; nonetheless, the incidence of CMV hepatitis appears to be decreasing owing to better prophylactic treatments.⁵⁹ 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.⁶⁰ In addition, parenchymal neutrophilic microabscesses have been described in cases with no evidence of CMV infection.⁶¹ In these cases, immunostaining for CMV antigens is most useful in determining the diagnosis of CMV infection.⁶²

FIGURE 3.3.

Colon biopsy of a patient with steroid-refractory ulcerative colitis. Rare epithelial cells show intranuclear CMV antigen. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

The sensitivity of IHC is better than light microscopic identification of viral inclusions and compares favorably with culture and in situ hybridization.49., 51., 52., 54., 63. Additionally, immunohistochemical assays can be completed faster than the shell vial culture technique, allowing for rapid results that are important for early anti-CMV therapy.⁵⁴

Other herpesvirus infections that have been diagnosed using immunohistochemical methods include human herpesvirus 6 infection⁶⁴ and Epstein-Barr viral infection.⁶⁵ Immunohistochemistry has been used to identify EBV latent membrane protein-1 in cases of Hodgkin’s lymphoma and post-transplant lymphoproliferative disorder (Fig. 3.4 ).⁶⁶

FIGURE 3.4.

Epstein-Barr virus LMP-1 within cytoplasm of characteristic Reed-Sternberg cells in a case of Hodgkin’s lymphoma. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Adenoviruses

Adenovirus has been increasingly recognized as a cause of morbidity and mortality among immunocompromised patients owing to transplant and congenital immunodeficiency.67., 68. Rarely adenovirus infection has been described in HIV-infected patients.69., 70., 71. 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.⁷⁰ In addition, other viral inclusions, including CMV, human papillomavirus (HPV), 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 all 41 serotypes of adenovirus has been used in an immunohistochemical technique to demonstrate intranuclear adenoviral antigen in immunocompromised patients (Fig. 3.5 ).70., 71., 72., 73., 74. Histologic diagnosis of adenovirus colitis is difficult, and it 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.70., 75.

FIGURE 3.5.

Adenovirus pneumonia in a heart transplant patient who developed ARDS and respiratory failure. Infected cells within necrotizing exudate show intranuclear reactivity with antibody to adenovirus antigen. Some cells show inclusions with a clear halo around them, making a differential diagnosis from CMV difficult on H&E stain. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Parvovirus B19 Infection

Parvovirus B19 has been associated with asymptomatic infections, erythema infectiosum, acute arthropathy, aplastic crisis, hydrops fetalis, and 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.⁷⁶

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.77., 78. Because intravenous immunoglobulin therapy is 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.79., 80., 81., 82. 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 with advanced cytolysis (Fig. 3.6 ).79., 83., 84. Several studies have found a strong correlation among results obtained from morphologic, immunohistochemical, in situ hybridization (ISH), and polymerase chain reaction (PCR) methods.78., 79., 82., 84.

FIGURE 3.6.

Hydrops fetalis caused by parvovirus B19 infection. Normoblasts within the villous capillaries show intranuclear viral antigen. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Viral Hemorrhagic Fevers

Since the 1980s, numerous emerging and reemerging agents of viral hemorrhagic fevers have attracted the attention of pathologists.3., 4., 5. Investigators have played an important role in identifying these agents and in supporting epidemiologic, clinical, and pathogenetic studies of emerging viral hemorrhagic fevers.4., 5., 7. Viral hemorrhagic fevers are often fatal. They are clinically difficult to diagnose (in the absence of bleeding or organ manifestations) 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 IHC as a sensitive, safe, and rapid diagnostic method for the diagnosis of viral hemorrhagic fevers such as yellow fever (Fig. 3.7 ),85., 86., 87. dengue hemorrhagic fever,87., 88. Crimean-Congo hemorrhagic fever,⁸⁹ Argentine hemorrhagic fever,⁹⁰ Venezuelan hemorrhagic fever,⁹¹ and Marburg disease.⁹² Additionally, a sensitive, specific, and safe immunostaining method has been developed to diagnose Ebola hemorrhagic fever in formalin-fixed skin biopsies (Fig. 3.8 ).⁹³ Immunohistochemistry demonstrated that Lassa virus targets primarily endothelial cells, mononuclear inflammatory cells, and hepatocytes (Fig. 3.9 ).93., 94., 95.

FIGURE 3.7.

Yellow fever. Abundant yellow fever viral antigens are seen within hepatocytes and Kupffer cells. (Immunoperoxidase staining with AEC and hematoxylin counterstain; ×400.)

FIGURE 3.8.

Extensive Ebola viral antigens are seen primarily within fibroblasts in the dermis of a skin specimen from a fatal case of Ebola hemorrhagic fever. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×20.)

FIGURE 3.9.

Lassa fever. Liver from a patient with Lassa fever. Scattered hepatocytes and reticuloendothelial cells show reactivity with monoclonal antibody to Lassa virus. (Naphthol fast red substrate and hematoxylin counterstain; original magnification ×100.)

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 a renal transplant,⁹⁶ with a prevalence varying from 2% to 4.5% in different transplant centers.96., 97. Since specific clinical signs and symptoms are lacking in BK virus nephropathy, the diagnosis can only be made histologically in a graft biopsy.⁹⁸ 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.⁹⁸ 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 IHC can identify viral antigen.99., 100. In this setting, IHC with an antibody against the large T antigen of SV40 virus has been effective in demonstrating BK virus infection (Fig. 3.10 ).96., 99., 101., 102., 103.

FIGURE 3.10.

Immunohistochemical detection of SV40-T antigen in the nuclei of tubular cells in a renal transplant patient with BK virus–associated nephropathy. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

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, IHC using a polyclonal rabbit antiserum against the protein VP1 is a specific, sensitive, and rapid method used to confirm the diagnosis of PML.104., 105., 106., 107. 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.

FIGURE 3.11.

Progressive multifocal leukoencephalopathy: SV40-T antigen in the nuclei of enlarged oligodendrocytes in a patient with JC virus infection. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Other Viral Infections

Immunohistochemistry has also been used to confirm the diagnosis of respiratory viral diseases such as influenza A virus and respiratory syncytial virus infections (Fig. 3.12 ) when cultures were not available.108., 109., 110., 111.

FIGURE 3.12.

Immunostaining of RSV antigens in desquamated bronchial and alveolar lining cells using a monoclonal antibody. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

The diagnosis of rabies relies heavily on histopathologic examination of tissues to demonstrate its characteristic cytoplasmic inclusions (Negri bodies). In a significant percentage of cases, Negri bodies are inconspicuous and so few that confirming the diagnosis of rabies is extremely difficult.¹¹² Furthermore, in non-endemic areas the diagnosis of rabies usually is not suspected clinically, or the patient may present with ascending paralysis. In these settings, immunohistochemical staining is a very sensitive, specific, and safe diagnostic tool for rabies (Fig. 3.13 ).112., 113., 114., 115., 116. Other viral agents that can be diagnosed using immunohistochemical methods include enteroviruses,117., 118., 119., 120. eastern equine encephalitis virus,121., 122., 123. and rotavirus.124., 125., 126.

FIGURE 3.13.

Immunostaining of rabies viral antigens in neurons of the CNS using a rabbit polyclonal antibody. Red precipitate corresponds to Negri inclusions by H&E staining. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×40.)

Immunohistochemical staining has been used in the histopathologic diagnosis of viral hepatitis C; however, IHC for this virus is not as effective as serologic assays and detection of HCV RNA in serum.

BACTERIAL INFECTIONS

Among bacterial infections, the greatest number of immunohistochemical studies has been performed in the investigation of Helicobacter pylori. A few studies have evaluated the use of IHC for other bacterial, mycobacterial, rickettsial, and spirochetal infections.

Antigen retrieval is generally not required for the immunohistochemical demonstration of bacteria in fixed tissue. However, interpreting the results can be complicated because many of these antibodies cross-react with other bacteria. Moreover, antibodies may react with only portions of the bacteria, and they may label remnants of bacteria or spirochetes when viable organisms are no longer present.

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.127., 128. Conventional histochemical methods such as silver stains are more sensitive than hematoxylin and eosin in detecting H. pylori. Nonetheless, for detecting scant numbers of organisms, it has been proven that IHC has high specificity and sensitivity, is less expensive when all factors are considered, is superior to conventional histochemical methods, and has a low interobserver variation (Fig. 3.14 ).¹²⁷ Treatment for chronic active gastritis and H. pylori infection can change the shape of the microorganism. This can make it difficult to identify and differentiate the organism from extracellular debris or mucin globules. In these cases IHC improves the rate of successful identification of the bacteria, even when histologic examination and cultures are falsely negative.129., 130., 131., 132.

FIGURE 3.14.

Numerous curved H. pylori in the superficial gastric mucus are clearly demonstrated by immunoperoxidase staining in this patient with chronic active gastritis. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Whipple’s Disease

Whipple’s disease affects primarily the small bowel and mesenteric lymph nodes and less commonly other organs such as the heart and central nervous system. Numerous foamy macrophages characterize the disease, and the diagnosis usually relies on the demonstration of PAS-positive intracytoplasmic bacteria. Nevertheless, the presence of PAS-positive macrophages is not pathognomonic; they can be observed in other diseases such as Mycobacterium avium infections, histoplasmosis, Rhodococcus equi infections, and macroglobulinemia. Tropheryma whipplei is a rare cause of endocarditis that shares many histologic features with other culture-negative endocarditides such as those caused by Coxiella burnetii and Bartonella sp.¹³³ The development of specific antibodies against these microorganisms has significantly enhanced the ability to detect them in the heart valves of patients with culture-negative endocarditis.¹³⁴ Immunohistochemical staining with rabbit polyclonal antibody provides a sensitive and specific method for the rapid diagnosis of intestinal and extraintestinal Whipple’s disease and for follow-up of treatment response.135., 136., 137.

Rocky Mountain Spotted Fever

Confirmation of Rocky Mountain spotted fever (RMSF) usually requires the use of serologic methods to detect antibodies to spotted fever group (SFG) Rickettsiae; however, most patients with RMSF lack diagnostic titers during the first week of disease. Immunohistochemistry has been successfully used to detect SFG Rickettsiae in formalin-fixed tissue sections, and it is superior to histochemical methods (Fig. 3.15 ).138., 139. Several studies illustrate the value of IHC in diagnosing suspected cases of RMSF using skin biopsies with high specificity and sensitivity, and in confirming fatal cases of seronegative RMSF.10., 140., 141., 142., 143., 144. R. rickettsii cannot be distinguished from other spotted fever group Rickettsiae such as R. parkeri or R. conorii because they cross-react.

FIGURE 3.15.

Immunohistologic demonstration of R. rickettsii within vascular endothelium in the pons of a patient with fatal Rocky Mountain spotted fever. (Immunoperoxidase staining with AEC and hematoxylin counterstain; ×600.)

Bartonella Infections

Bartonella are slow-growing, fastidious gram-negative, Warthin-Starry–stained bacteria associated with bacillary angiomatosis, peliosis hepatis, cat-scratch disease, trench fever, relapsing bacteremia, and disseminated granulomatous lesions of liver and spleen.¹⁴⁵ Bartonella are important agents of blood culture–negative endocarditis. Traditional techniques such as histology, electron microscopy, and serology have been employed to identify the agents of culture-negative endocarditis. However, Bartonella sp., C. burnetii, and T. whipplei endocarditis share many morphologic features that do not allow for a specific histologic diagnosis.¹⁴⁶ Besides, serologic tests for Bartonella sp. may show cross-reactivity with C. burnetii and Chlamydia sp.¹⁴⁷ Immunostaining has been successfully used to identify Bartonella henselae and B. quintana in the heart valves of patients with blood culture–negative endocarditis and has significantly enhanced the ability to establish a specific diagnosis in these cases.148., 149. A polyclonal rabbit antibody that does not differentiate between B. henselae and B. quintana has also been used to detect these microorganisms in cat-scratch disease (Fig. 3.16 ), bacillary angiomatosis, and peliosis hepatis.150., 151. A commercially available monoclonal antibody specific for B. henselae is also available and has been used to demonstrate the organism in a case of spontaneous splenic rupture caused by this bacterium.¹⁵²

FIGURE 3.16.

Photomicrograph of a lymph node biopsy from a patient with cat-scratch disease showing abundant extracellular, clumped coccobacilli of B. henselae in necrotic foci. (Immunoalkaline phosphatase with monoclonal antibody against B. henselae, naphthol fast red substrate and hematoxylin counterstain; ×200.)

Courtesy of Dr. Suimin Qiu, University of Texas Medical Branch.

Syphilis

Syphilis continues to be a public health problem caused by T. pallidum, a fastidious organism that has not been cultivated.¹⁵³ The diagnosis of syphilis relies on serology and the identification of T. pallidum by dark-field microscopy. However, these methods have low sensitivity and specificity,¹⁵⁴ and serologic methods can be negative in early stages of the disease and in immunosuppressed patients such as those coinfected with human immunodeficiency virus (HIV).¹⁵⁵ In tissue sections, the usual method for detecting spirochetes is through silver impregnation stains (Warthin-Starry or Steiner). These stains, however, can be technically difficult to perform and interpret, are nonspecific, and frequently show marked background artifacts because silver stains also highlight melanin granules and reticulin fibers. Detection rates of spirochetes using silver stains vary from 33% to 71%.¹⁵⁶ It has been shown that immunostaining of biopsy specimens with anti–T. pallidum polyclonal antibody (Fig. 3.17 ) is more sensitive and specific than silver staining methods, with sensitivities ranging from 71% to 94%.153., 156., 157.

FIGURE 3.17.

Syphilis. Skin biopsy from a patient with secondary syphilis. Scattered intact T. pallidum are easily visible with a rabbit polyclonal antibody against T. pallidum. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Mycobacterium Tuberculosis Infection

Identification of M. tuberculosis is routinely achieved by acid-fast bacilli (AFB) staining, culture of biopsy specimens, or both. Nevertheless, AFB staining has a low sensitivity, and it is not specific because it does not differentiate mycobacterial species.¹⁵⁸ Furthermore, cultures may take several weeks, and sensitivity is low in paucibacillary lesions.¹⁵⁹ In the histologic diagnosis of mycobacterial infections, IHC with anti-BCG polyclonal antibody has shown better sensitivity than AFB staining. However, in paucibacillary lesions it is inferior to AFB staining and cannot differentiate between M. tuberculosis and other mycobacteria.¹⁶⁰ Recently a polyclonal antibody against the M. tuberculosis–secreted antigen MPT64 was used in cases of mycobacterial lymphadenitis. This method showed 90% sensitivity and 83% specificity and performed better than AFB staining in cases of paucibacillary disease and comparably to nested PCR.¹⁶¹

Other Bacterial Infections

Other bacterial diseases that can be identified by IHC in formalin-fixed tissue include leptospirosis, a zoonosis that usually presents as an acute febrile syndrome but occasionally can have unusual manifestations such as pulmonary hemorrhage with respiratory failure or abdominal pain.162., 163., 164. Rabbit polyclonal antibodies have been used in IHC to detect leptospiral antigens in the gallbladder and lungs from patients with unusual presentations (Fig. 3.18 ).162., 163., 164., 165.

FIGURE 3.18.

Leptospira. Immunostaining of intact leptospires and granular forms of leptospiral antigens in kidney of patient who died of pulmonary hemorrhage. (Immunoalkaline phosphatase with rabbit polyclonal antisera with naphthol fast red substrate and hematoxylin counterstain; original magnification ×63.)

Lyme disease has protean clinical manifestations, and Borrelia burgdoferi is difficult to culture from tissues and fluids. In addition, cultures are rarely positive before 2 to 4 weeks of incubation. Borrelia burgdorferi can be identified in tissues by immunostaining with polyclonal or monoclonal antibodies. Although IHC is more specific than silver impregnation staining, the sensitivity of immunostaining is poor, and the microorganisms are difficult to detect owing to the low numbers present in tissue sections.166., 167.

Q fever is a zoonosis caused by Coxiella burnetii and is characterized by protean and non-specific manifestations. Acute Q fever can manifest as atypical pneumonia or granulomatous hepatitis, frequently with characteristic fibrin ring granulomas. This microorganism is recognized as one agent that causes blood culture–negative chronic endocarditis.¹⁶⁸ A monoclonal antibody has been used to specifically identify C. burnetii in cardiac valves of patients with chronic Q fever endocarditis.12., 169.

Recently IHC has been successfully used to identify Streptococcus pneumoniae in formalin-fixed organs with an overall sensitivity of 100% and a specificity of 71% when compared with cultures.¹⁷⁰ Immunohistochemical assays are used to identify Clostridium sp., S. aureus, and S. pyogenes; 171., 172. Haemophilus influenzae; 173., 174., 175. Chlamydia species;176., 177., 178. Legionella pneumophila and L. dumoffii; 179., 180., 181. Listeria monocytogenes; 182., 183., 184. Salmonella; 185., 186. and rickettsial infections other than Rocky Mountain spotted fever such as boutonneuse fever, epidemic typhus, murine typhus,¹⁸⁷ rickettsialpox,188., 189. African tick bite fever,¹³⁸ and scrub typhus.¹⁹⁰

FUNGAL INFECTIONS

The great majority of fungi are readily identified by hematoxylin and eosin staining alone or in combination with histochemical stains (e.g., periodic acid-Schiff [PAS], Gomori’s methenamine silver [GMS]). However, these stains cannot distinguish morphologically similar fungi with potential differences in susceptibility to antimycotic drugs. In addition, several factors may influence the appearance of fungal elements, which may appear atypical in tissue sections because of steric orientation, age of the fungal lesion, effects of antifungal chemotherapy, type of infected tissue, and host immune response.¹⁹¹ Currently the final identification of fungi relies on culture techniques; however, culture may take several days or longer to yield a definitive result, and surgical pathologists rarely have access to fresh tissue.

In past years, IHC has been used to identify various fungal elements in paraffin-embedded, formalin-fixed tissue.192., 193., 194. Immunohistochemical methods have the advantage of providing rapid and specific identification of several fungi and allowing pathologists to identify unusual filamentous hyphal and yeast infections and to accurately distinguish them from confounding artifacts.193., 195. In addition, IHC allows pathologists to correlate microbiological and histologic findings of fungal infections and to distinguish them from harmless colonization. Immunohistochemistry can also be helpful when more than one fungus is present; in these cases dual immunostaining techniques can highlight the different fungal species present in the tissue.¹⁹⁶ An important limitation of IHC in the identification of fungi is the well-known, widespread occurrence of common antigens among pathogenic fungi that frequently results in cross-reactivity with polyclonal antibodies and even with some monoclonal antibodies.193., 195., 196., 197. Therefore, assessing cross-reactivity using a panel of fungi is a very important step in the evaluation of immunohistochemical methods.193., 194.

Candida species are often stained weakly with hematoxylin and eosin, and sometimes the yeast form may be difficult to differentiate from Histoplasma capsulatum, Cryptococcus neoformans, and even Pneumocystis carinii. Polyclonal and monoclonal antibodies against Candida genus antigens are sensitive and strongly reactive and do not show cross-reactivity with other fungi tested.193., 194., 198., 199. In particular, two monoclonal antibodies against Candida albicans mannoproteins show high sensitivity and specificity. Monoclonal antibody 3H8 recognizes primarily filamentous forms of C. albicans, whereas monoclonal antibody 1B12 highlights yeast forms.199., 200., 201., 202., 203.

Identification of Cryptococcus neoformans usually is not a problem when the fungus produces a mucicarmine-positive capsule. However, infections by capsule-negative strains are more difficult to diagnose, and the disease can be confused with histoplasmosis, blastomycosis, or torulopsis. Also, in long-standing infections the yeast often appear atypical and fragmented. Polyclonal antibodies raised against C. neoformans yeast cells are sensitive and specific.193., 194. More recently, monoclonal antibodies have been produced that allow identification and differentiation of varieties of C. neoformans in formalin-fixed tissue. The antibodies are highly sensitive (97%) and specific (100%) and can differentiate C. neoformans var. neoformans from C. neoformans var. gattii.204., 205.

Sporothrix schenckii may be confused in tissue sections with Blastomyces dermatitidis and fungal agents of phaeohyphomycosis. In addition, yeast cells of Sporothrix schenckii may be sparsely present in tissues. Antibodies against yeast cells of S. schenckii are sensitive but demonstrate cross-reactivity with Candida species; however, after specific adsorption of the antibody with Candida yeast cells, the cross-reactivity of the antibodies is eliminated.193., 194.

Invasive aspergillosis is a frequent cause of fungal infection with high morbidity and mortality rates in immunocompromised patients.²⁰⁶ The diagnosis is often difficult and relies heavily on histologic identification of invasive septate hyphae and culture confirmation. Nevertheless, several filamentous fungi such as Fusarium species, Pseudallescheria boydii, and Scedosporium species share similar morphology with Aspergillus species in hematoxylin and eosin–stained tissues.²⁰⁷ A precise and rapid diagnosis of invasive aspergillosis is important because early diagnosis is associated with improved clinical response, and it allows planning of the correct duration and choice of antimycotic therapy. Researchers have shown that the yield of cultures in histologically proven cases is low, ranging from 25% to 50%.206., 208., 209., 210., 211. Several polyclonal and monoclonal antibodies against Aspergillus antigens have been tested in formalin-fixed tissues with variable sensitivities, and most of them cross-react with other fungi.197., 212., 213. More recently, monoclonal antibodies (WF-AF-1, 164G, and 611F) against Aspergillus galactomannan have shown high sensitivity and specificity in identifying A. fumigatus, A. flavus, and A. niger in formalin-fixed tissues without cross-reactivity with other filamentous fungi.211., 214., 215.

Cysts and trophozoites of Pneumocystis jirovecii can be detected in bronchoalveolar lavage specimens using monoclonal antibodies that yield results that are slightly more sensitive than GMS, Giemsa, or Papanicolaou staining (Fig. 3.19 ).194., 216., 217. Antibodies are most helpful in cases of extrapulmonary pneumocystosis or in the diagnosis of P. jirovecii pneumonia when atypical pathologic features are present (e.g., hyaline membranes or granulomatous pneumocystosis where microorganisms are usually very sparse).

FIGURE 3.19.

Immunodeficient patient with P. jirovecii pneumonia. Cohesive aggregates of cyst forms and trophozoites within alveolar spaces are demonstrated by a monoclonal antibody against pneumocystis with an immunoperoxidase technique. (Immunoperoxidase staining with DAB and hematoxylin counterstain; ×400.)

Penicillium marneffei can cause a disseminated infection in immunocompromised patients.218., 219. Morphologically the organisms must be differentiated from H. capsulatum, C. neoformans, and C. albicans. The monoclonal antibody EBA-1 against the galactomannan of Aspergillus species cross-reacts with and detects P. marneffei in tissue sections.209., 220. Immunohistochemistry has also been used to detect Blastomyces, Coccidioides, and Histoplasma.193., 194., 221. However, the antibodies have significant cross-reactivity with several other fungi.

PROTOZOAL INFECTIONS

Protozoa usually can be identified in tissue sections stained with hematoxylin and eosin or Giemsa stain; however, because of the small size of the organisms and the subtle distinguishing features, an unequivocal diagnosis cannot always be made. The role of IHC in the detection of protozoal infections has been particularly valuable in cases in which the morphology of the parasite is distorted by tissue necrosis or autolysis. In addition, in immunocompromised patients, toxoplasmosis can have an unusual disseminated presentation with numerous tachyzoites without bradyzoites (Fig. 3.20 ).222., 223. Immunohistochemistry has also been useful in cases with unusual presentation of the disease.²²⁴

FIGURE 3.20.

HIV-infected patient with toxoplasmic encephalitis. Immunoperoxidase staining highlights cyst forms and scattered tachyzoites. (DAB substrate with hematoxylin counterstain; ×400.)

The diagnosis of leishmaniasis in routine practice usually is not difficult; however, in certain circumstances pathologic diagnosis may be problematic, as is the case in chronic granulomatous leishmaniasis with small numbers of parasites, when the microorganism presents in unusual locations, or when necrosis distorts the morphologic appearance of the disease.²²⁵ In these cases, immunohistochemical staining has been a valuable diagnostic tool.225., 226., 227., 228. The highly sensitive and specific monoclonal antibody p19-11 recognizes different species of Leishmania and allows differentiation from morphologically similar microorganisms (Toxoplasma, Trypanosoma cruzi, and P. marneffei).²²⁵

Immunohistochemical assays using polyclonal antibodies specific for Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba sp. are used to demonstrate amebic trophozoites and cysts in areas of necrosis and can allow their differentiation from macrophages in cases of amebic meningoencephalitis.²²⁹

Immunohistochemistry has also been used to identify Cryptosporidium, ²³⁰ Entamoeba histolytica, ²³¹ Trypanosoma cruzi, 232., 233., 234. babesia,²³⁵ Giardia lamblia, ²³⁶ Plasmodium falciparum, and P. vivax in fatal cases of malaria²³⁷ in formalin-fixed paraffin-embedded tissue samples.

EMERGING INFECTIOUS DISEASES

In 1992 the Institute of Medicine defined emerging infectious diseases (EIDs) as caused by new, previously unidentified microorganisms or those whose incidence in humans has increased within the previous two decades or threatens to increase in the near future.²³⁸ The list of pathogens newly recognized since 1973 is long and continues to increase. Recognizing emerging infections is a challenge, and many new infectious agents remain undetected for years before emerging as an identified public health problem.²³⁹ EIDs are a global phenomenon that requires a global response. The Centers for Disease Control and Prevention (CDC) has defined the strategy to prevent and detect EIDs.²³⁹ The anatomic pathology laboratory plays a critical role in the initial and rapid detection of EIDs.240., 241. Besides assisting in the identification of new infectious agents, IHC has contributed to the understanding of the pathogenesis and epidemiology of EIDs.

Hantavirus Pulmonary Syndrome

In 1993 in the southwestern United States, several previously healthy individuals died of rapidly progressive pulmonary edema, respiratory insufficiency, and shock.242., 243. Immunohistochemistry played a central role in identifying the viral antigens of an unknown hantavirus,244., 245. detecting the occurrence of unrecognized cases of hantavirus pulmonary syndrome prior to 1993, and showing the distribution of viral antigen in endothelial cells of the microcirculation, particularly in the lung (Fig. 3.21 ).244., 246.

FIGURE 3.21.

Hantavirus antigen–containing endothelial cells of the pulmonary microvasculature in the lung of an HPS patient as detected by immunohistochemistry using a mouse monoclonal antibody. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×100.)

West Nile Virus Encephalitis

West Nile virus (WNV) was originally identified in Africa in 1937, and the first cases of WNV encephalitis in the United States were described in 1999. The clinical picture is variable and non-specific. It can range from subclinical infection to flaccid paralysis and encephalitis characterized morphologically by perivascular mononuclear cell inflammatory infiltrates, neuronal necrosis, edema, and microglial nodules, particularly prominent in the brainstem, cerebellum, and spinal cord.247., 248., 249., 250., 251. The diagnosis of WNV encephalitis is usually established by identifying virus-specific IgM in CSF and/or serum and by demonstrating viral RNA in serum, CSF, or other tissue.²⁵² Immunostaining with monoclonal or polyclonal antibodies has been successfully used to diagnose WNV infection in immunocompromised patients with an inadequate antibody response (Fig. 3.22 ).²⁴⁸

FIGURE 3.22.

West Nile virus. Immunostaining of flaviviral antigens in neurons and neuronal processes in the central nervous system from an immunosuppressed patient who died of West Nile virus encephalitis. (Flavivirus-hyperimmune mouse ascitic fluid naphthol fast red substrate with hematoxylin counterstain; original magnification ×40.)

Enterovirus 71 Encephalomyelitis

Enterovirus 71 (EV71) has been associated with hand, foot, and mouth disease; herpangina; aseptic meningitis; and poliomyelitis-like flaccid paralysis. More recently EV71 has been associated with unusual cases of fulminant encephalitis, pulmonary edema and hemorrhage, and heart failure.253., 254. Severe and extensive encephalomyelitis of the cerebral cortex, brainstem, and spinal cord has been described. Immunohistochemical staining with monoclonal antibody against EV71 has played a pivotal role in the linking of EV71 infection to fulminant encephalitis (Fig. 3.23 ). Viral antigen is observed within neurons, neuronal processes, and mononuclear inflammatory cells.255., 256., 257.

FIGURE 3.23.

Enterovirus 71. Positive staining of EV71 viral antigens in neurons and neuronal processes of a fatal case of enterovirus encephalitis. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×40.)

Nipah Virus Infection

Nipah virus is a recently described paramyxovirus that causes an acute febrile encephalitic syndrome with a high mortality rate.258., 259., 260. Pathology played a key role in identifying the causative agent. Histopathologic findings include vasculitis with thrombosis, microinfarctions, syncytial giant cells, and viral inclusions.258., 260. Although characteristic of this disease, syncytial giant endothelial cells are seen only in 25% of cases,²⁵⁸ and viral inclusions of similar morphology can been seen in other paramyxoviral infections. Immunostaining provides a useful tool for unequivocal diagnosis of the disease, demonstrating viral antigen within neurons and endothelial cells of most organs (Fig. 3.24 ).5., 258.

FIGURE 3.24.

Nipah virus. Immunostaining of Nipah virus antigens in neurons and neuronal processes in CNS of a fatal case of Nipah virus encephalitis. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×63.)

Ehrlichioses

Tick-transmitted intracellular gram-negative bacteria belonging to the genera Ehrlichia and Anaplasma are the agents of human monocytotropic ehrlichiosis and human granulocytotropic anaplasmosis, respectively. The acute febrile illnesses usually present with cytopenias, myalgias, and mild to moderate hepatitis.261., 262., 263., 264.

Diagnosis of ehrlichiosis depends upon finding the characteristic monocytic and/or granulocytic cytoplasmic inclusions (morulae), PCR analysis of blood, and detection of specific antibodies in blood. However, morulae are rare and often missed on initial evaluation; hematoxylin and eosin–stained sections often fail to show organisms even when IHC reveals abundant ehrlichial antigen; and antibody titers may take several weeks to rise to diagnostic levels.261., 265. Additionally, immunocompromised patients may not develop anti-ehrlichial antibodies prior to death.261., 263. In these cases, immunostaining for Ehrlichia or Anaplasma is a sensitive and specific diagnostic method.261., 263., 264., 266.

Immunohistochemistry has been a very valuable tool used to identify and study several other EIDs such as Ebola hemorrhagic fever;93., 94., 95. Hendra virus encephalitis;5., 267., 268. leptospirosis;163., 164., 165. emerging tick-borne rickettsioses such as R. parkeri ²⁶⁹ and R. africae; ²⁷⁰ and, recently, a new coronavirus associated with severe acute respiratory syndrome (SARS).271., 272. SARS was first recognized during a global outbreak of severe pneumonia that occurred in late 2002 in Guangdong Province, China, and then erupted in February 2003 with cases in more than two dozen countries in Asia, Europe, North America, and South America. Early in the investigation, clinical, pathologic, and laboratory studies focused on previously known agents of respiratory illness. Subsequently, a virus was isolated from the oropharynx of a SARS patient and identified by ultrastructural characteristics as belonging to the family Coronaviridae.271., 271. Various reports have described diffuse alveolar damage as the main histopathologic finding in SARS patients, and SARS-associated coronavirus (SARS-CoV) has been demonstrated in human and experimental animal tissues by immunohistochemical (Fig. 3.25 ) or ISH assays.273., 274., 275., 276., 277., 278., 279., 280., 281., 282.

FIGURE 3.25.

SARS. Coronavirus antigen–positive pneumocytes and macrophages in the lung of a SARS case. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×63.)

PATHOLOGISTS, IMMUNOHISTOCHEMISTRY, AND BIOTERRORISM

There is increasing concern about the use of infectious agents as potential biological weapons. Biological warfare agents vary from rare exotic viruses to common bacterial agents, and the intentional use of biologic agents to cause disease can simulate naturally occurring outbreaks or may have unusual characteristics.²⁸³ The CDC has issued recommendations for a complete public health response to a biological attack.284., 285., 286. Two important components of this response plan include the rapid diagnosis and characterization of biological agents. Pathologists using newer diagnostic techniques such as IHC, ISH, and PCR will have a direct impact on the rapid detection and control of emerging infectious diseases from natural or intentional causes.²⁸⁷ Immunohistochemistry provides a simple, safe, sensitive, and specific method for the rapid detection of biological threats (at the time of investigation or retrospectively), thereby facilitating the rapid implementation of effective public health responses.

Anthrax

Immunohistochemical staining of Bacillus anthracis with monoclonal antibodies against cell wall and capsule antigens has been successfully used in the recognition of bioterrorism-related anthrax cases and is an important step in early diagnosis and treatment (Fig. 3.26 A-C).5., 288., 289., 290., 291., 292. Gram staining and culture isolation of B. anthracis are usually used to diagnose anthrax; however, previous antibiotic treatment will affect culture yield and Gram stain identification of the bacteria.²⁹⁰ Immunohistochemistry has demonstrated high sensitivity and specificity for the detection of B. anthracis in skin biopsies, pleural biopsies, transbronchial biopsies, and pleural fluids (see Fig. 3.26).289., 290., 291., 293.

FIGURE 3.26.

Anthrax. (A) Photomicrograph of a pleural effusion cell block from a patient with bioterrorism-associated inhalation anthrax showing bacillary fragments and granular antigen-staining using the anti–B. anthracis capsule antibody. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×63.) (B) Skin biopsy from a patient with cutaneous anthrax showing abundant granular antigen and bacillary fragments using B. anthracis cell-wall antibody. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×40.) (C) Photomicrograph of mediastinal lymph node from a patient with inhalational anthrax showing abundant granular antigen and bacillary fragments using anti–B. anthracis cell-wall antibody. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×63.)

In addition, immunostaining has been very useful for determining the route of entry of the bacteria and identifying the mode of spread of the disease.290., 294.

Tularemia

Immunohistochemical staining is also valuable for rapid identification of Francisella tularensis in formalin-fixed tissue sections. Tularemia can have a variable clinical and pathologic presentation that can simulate other infectious diseases such as anthrax, plague, cat-scratch disease, or lymphogranuloma venereum. Moreover, the microorganisms are difficult to demonstrate in tissue sections, even with Gram stain or silver staining methods. A mouse monoclonal antibody against the lipopolysaccharide of F. tularensis has been used with high sensitivity and specificity to demonstrate intact bacteria and granular bacterial antigen in the lungs, spleen, lymph nodes, and liver (Fig. 3.27 ).295., 296.

FIGURE 3.27.

Tularemia. Immunohistochemistry of lymph node showing a stellate abscess with F. tularensis antigen-bearing macrophages in the central necrotic area using a mouse monoclonal antibody against the lipopolysaccharide of F. tularensis. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×40.)

Plague

A mouse monoclonal antibody directed against the fraction 1 antigen of Yersinia pestis has been used to detect intracellular and extracellular bacteria in dermal blood vessels, lungs, lymph nodes, spleen, and liver (Fig. 3.28 ).297., 298., 299., 300., 301., 302. This technique is potentially useful for the rapid diagnosis of plague in formalin-fixed skin biopsies. In addition, IHC can differentiate primary and secondary pneumonic plague by identifying Y. pestis in different lung locations (e.g., alveoli vs. interstitium).²⁹⁷

FIGURE 3.28.

Immunohistochemical stain of lung containing abundant bacterial and granular Yersinia pestis antigen in the alveolar spaces using a mouse monoclonal antibody against F1 capsular antigen. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×20.)

Immunohistochemical methods using polyclonal or monoclonal antibodies have been used to identify several other potential biological terrorism agents, including the causative agents of brucellosis,⁵ Q fever,5., 138., 168., 169. viral encephalitides (eastern equine encephalitis) (Fig. 3.29 ),5., 121., 122., 123. rickettsioses (typhus and Rocky Mountain spotted fever),138., 139., 140., 141., 187. and viral hemorrhagic fevers (Ebola and Marburg hemorrhagic fever).5., 89., 90., 91., 92., 93., 94., 95.

FIGURE 3.29.

Immunostaining of viral antigens in neurons and neuronal processes in CNS using a mouse anti-eastern equine encephalitis virus antibody. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×10.)

BEYOND IMMUNOHISTOLOGY: MOLECULAR DIAGNOSTIC APPLICATIONS

Since the 1980s, an enormous advancement in molecular technology has dramatically influenced the diagnosis and study of infectious diseases. The application of molecular probes to the study and diagnosis of infectious diseases is a great adjunct to IHC as a diagnostic method and often allows for even more rapid and specific identification of organisms.303., 304., 305., 306., 307., 308., 309. Along with rapid advances in molecular diagnostic techniques, there has been an increased interest in the use of paraffin-embedded specimens for nucleic acid hybridization assays. The two main hybridization formats used in the diagnostic pathology laboratory for the diagnosis of infectious diseases are ISH and PCR. In situ hybridization is analogous to IHC in that it allows the cellular identification and localization of microbial pathogens. Instead of microbial antigens, the targets of ISH are specific RNA or DNA sequences. Many viruses (FIGURE 3.30, FIGURE 3.31, FIGURE 3.32, FIGURE 3.33 ), bacteria (Fig. 3.34 ), and other microorganisms can be localized in tissues by ISH; these include Epstein-Barr virus, HPV (Fig. 3.35 ), polyomaviruses (Fig. 3.36 ), Mycobacterium leprae, Legionella, Haemophilus influenzae, zygomycetes, and Aspergillus. 108., 114., 207., 261., 282., 310., 311., 312., 313., 314., 315., 316., 317., 318., 319., 320., 321., 322., 323., 324., 325., 326., 327., 328., 329., 330., 331., 332. PCR has the advantage of increased sensitivity, minimal tissue requirements, and potential sequencing of the amplified product for specific identification of the microbial genotype or strain of the agent involved. There are PCR assays for most microorganisms that have been or can be adapted for use on formalin-fixed tissues.11., 124., 172., 293., 333., 334., 335., 336., 337., 338., 339., 340., 341., 342., 343., 344., 345.

FIGURE 3.30.

Crimean-Congo hemorrhagic fever (CCHF). Localization of CCHF viral RNA as seen in a single CCHF-infected hepatocyte. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×250.)

FIGURE 3.31.

Influenza A. In situ hybridization showing localization of viral nucleic acids in bronchial epithelium using an influenza A hemagglutinin digoxigenin-labeled probe. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×158.)

FIGURE 3.32.

Parvovirus infection. Confirmation of B19-infected cells in bone marrow of an HIV-infected patient by using a digoxigenin-labeled B19 riboprobe and in situ hybridization. Staining is mainly nuclear and seen in multiple cells containing classic parvovirus inclusions. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×250.)

FIGURE 3.33.

SARS. Lung showing diffuse alveolar damage and SARS-CoV nucleic acids primarily in pneumocytes as seen by colorimetric ISH. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×158.)

FIGURE 3.34.

Ehrlichia chaffeensis. Organisms appear as red inclusions within monocytes by in situ hybridization. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×250.)

FIGURE 3.35.

Human papillomavirus (HPV). In situ hybridization for HPV in a patient with a benign cervical lesion. HPV RNA is localized within nucleus and cytoplasm of koilocytotic cells. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×250.)

FIGURE 3.36.

Polyomavirus (BK virus) infection. BK virus–infected cells in urothelium as seen by using a colorimetric ISH and a DNA probe. Staining is both nuclear and cytoplasmic and seen in multiple cells containing classic viral inclusions. (Immunoalkaline phosphatase with naphthol fast red substrate and hematoxylin counterstain; original magnification ×100.)

In summary, IHC, ISH, and PCR should be regarded as complementary diagnostic methods for use in the diagnostic pathology laboratory. The laboratory must consider the advantages and limitations of each method and how they apply to each case and the common needs of the laboratory. This ever-expanding field behooves all pathologists interested in the field of infectious diseases to keep abreast of the changing technology and its ever-increasing application in the arena of diagnosis.