3.2 Immunostaining for B. cereus pneumonia using B. cereus antiserum

B. cereus pneumonia is characterized by a lethal necrotic and hemorrhagic lesion infrequently seen in an immunocompromised or immunocompetent patient [27]. The autopsied lung was obtained from a female aged 60's long suffering from chronic lymphoplasmacytic leukemia. Gram-positive bacilli with spore formation

#### Figure 1.

BCG immunostaining I (upper panels, cerebral tuberculoma; lower panels, old calcified nodule in the lung; left, H&E; right, BCG immunostaining). Mycobacterial antigens were clearly demonstrable not only in caseous necrosis but also in a fibrous nodule of old tuberculosis. Mycobacterial antigenic substances on destroyed bacterial fragments are detectable by the antiserum.


were multifocally clustered in the necrohemorrhagic lesion. Immunostaining (the amino acid polymer method, Simple Stain-Max, Nichirei, Tokyo, Japan) using B. cereus rabbit antiserum (Abcam) diluted at 1:500 after heat-induced antigen retrieval in 10 mM citrate buffer, pH 6, demonstrated positive signals on the spore of the bacilli (Figure 4) [8, 19, 28]. Similar findings were obtained in case of

BCG immunostaining III (left, HE; right, BCG immunostaining). BCG antigens are very stable against fixation. Dense-positive signals are seen in a pulmonary exudative tuberculosis lesion fixed in formalin for 70 years. Of note is that the nuclei are poorly stained with hematoxylin due to prolonged fixation.

BCG immunostaining II (upper panels, non-tuberculous mycobacterial lymphadenitis in AIDS; lower panels, lepromatous leprosy; left, H&E; right, BCG immunostaining; inset, acid-fast staining). Striated histiocytes in Mycobacterium avium-intracellulare (MAI) infection in AIDS and globi in lepromatous (multibacillary) leprosy in a biopsied skin lesion are strongly labeled. Fite modification of acid-fast staining is requested for

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens…

DOI: http://dx.doi.org/10.5772/intechopen.85055

Warthin-Starry's silver method is technically difficult, frequently with a false-

3.3 Immunostaining for syphilitic lesions using T. pallidum antiserum

negative result in case of treponemal infection. In contrast, immunostaining

opportunistic soft tissue gangrene caused by B. cereus [9].

Figure 2.

Figure 3.

75

demonstrating M. leprae.

\* Fite modification employing oil-xylene for deparaffinization required.

Acid-fast staining and BCG immunostaining were compared using three types of granulomatous lesions embedded in paraffin.

#### Table 2.

Comparative detectability of mycobacteria with acid-fast staining and immunostaining for BCG antigens.

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.85055

#### Figure 2.

3.2 Immunostaining for B. cereus pneumonia using B. cereus antiserum

Immunohistochemistry - The Ageless Biotechnology

Figure 1.

Tuberculosis

Leprosy

\*

74

paraffin.

Table 2.

Sarcoid-type granuloma

bacterial fragments are detectable by the antiserum.

B. cereus pneumonia is characterized by a lethal necrotic and hemorrhagic lesion infrequently seen in an immunocompromised or immunocompetent patient [27]. The autopsied lung was obtained from a female aged 60's long suffering from chronic lymphoplasmacytic leukemia. Gram-positive bacilli with spore formation

BCG immunostaining I (upper panels, cerebral tuberculoma; lower panels, old calcified nodule in the lung; left, H&E; right, BCG immunostaining). Mycobacterial antigens were clearly demonstrable not only in caseous necrosis but also in a fibrous nodule of old tuberculosis. Mycobacterial antigenic substances on destroyed

Disease Acid-fast staining BCG immunostaining

Exudative lesion 5/6 (83%) 6/6 (100%) Caseous granuloma 3/11 (27%) 5/11 (45%) Non-caseous granuloma 0/6 (0%) 1/6 (17%) Encapsulated caseous focus, non-calcified 0/9 (0%) 5/9 (56%) Encapsulated caseous focus, calcified 0/11 (0%) 8/11 (73%) Fibrous focus, calcified 0/1 (0%) 1/1 (100%) Total 8/44 (18%) 26/44 (59%)

Lepromatous leprosy (multibacillary form) 2/2 (100%)\* 2/2 (100%) Tuberculoid leprosy (paucibacillary form) 0/3 (0%) 0/3 (0%)

Sarcoidosis 0/7 (0%) 0/7 (0%) Sarcoid-like reaction in lymph node 0/3 (0%) 0/3 (0%)

Acid-fast staining and BCG immunostaining were compared using three types of granulomatous lesions embedded in

Comparative detectability of mycobacteria with acid-fast staining and immunostaining for BCG antigens.

Fite modification employing oil-xylene for deparaffinization required.

BCG immunostaining II (upper panels, non-tuberculous mycobacterial lymphadenitis in AIDS; lower panels, lepromatous leprosy; left, H&E; right, BCG immunostaining; inset, acid-fast staining). Striated histiocytes in Mycobacterium avium-intracellulare (MAI) infection in AIDS and globi in lepromatous (multibacillary) leprosy in a biopsied skin lesion are strongly labeled. Fite modification of acid-fast staining is requested for demonstrating M. leprae.

#### Figure 3.

BCG immunostaining III (left, HE; right, BCG immunostaining). BCG antigens are very stable against fixation. Dense-positive signals are seen in a pulmonary exudative tuberculosis lesion fixed in formalin for 70 years. Of note is that the nuclei are poorly stained with hematoxylin due to prolonged fixation.

were multifocally clustered in the necrohemorrhagic lesion. Immunostaining (the amino acid polymer method, Simple Stain-Max, Nichirei, Tokyo, Japan) using B. cereus rabbit antiserum (Abcam) diluted at 1:500 after heat-induced antigen retrieval in 10 mM citrate buffer, pH 6, demonstrated positive signals on the spore of the bacilli (Figure 4) [8, 19, 28]. Similar findings were obtained in case of opportunistic soft tissue gangrene caused by B. cereus [9].

#### 3.3 Immunostaining for syphilitic lesions using T. pallidum antiserum

Warthin-Starry's silver method is technically difficult, frequently with a falsenegative result in case of treponemal infection. In contrast, immunostaining

(Simple Stain-Max, Nichirei) using T. pallidum rabbit antiserum (Biocare) diluted at 1:1000, after heat-induced antigen retrieval in 1 mM EDTA solution, pH 8, is highly sensitive and reproducible in demonstrating long coiled microbes among the lesion [4, 7–10, 19]. Figure 5 illustrates a neck skin papule richly infiltrated by plasma cells, biopsied from a middle-aged Japanese male patient in a remission state of malignant lymphoma 2 years after chemotherapy. Clinicians suspected of skin recurrence of malignancy, but immunostaining clearly demonstrated dense infection of coiled bacteria in the epidermis. Plasma cell-rich appearance microscopically suggested the possibility of syphilis, and thus immunostaining for T. pallidum was

performed. The diagnosis of stage II syphilis was subsequently confirmed by sero-

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens…

In Figure 6, a biopsied penile lesion with painless ulceration (chancre) in stage I and excised syphilitic granulomatous lymphadenitis in stage III are presented. In the penis, numerous spiral microbes were clustered mainly in the basal part of the squamous mucosa and around the dermal capillary vessels. In the stage III lesion with granulomatous reaction, coiled spirochetes were infrequently observed.

T. pallidum immunostaining II (left and center, stage I penile chancre; right, stage III syphilitic lymphadenitis; left and right, H&E; center and inset, immunostaining using T. pallidum antiserum). In the penis, numerous spiral microbes are clustered mainly in the basal part of the squamous mucosa and around the dermal capillary vessels. In the stage III lesion accompanying granulomatous reaction with multinucleated giant cells, coiled

T. pallidum immunostaining III (left, tonsil; center, gastric mucosa; right, aortic valve). Immunohistochemical visualization of spirochetes in the biopsy specimens using T. pallidum antiserum significantly contributes to confirming the clinical and serological diagnosis of syphilis haphazardly affecting the tonsil, gastric mucosa, and

logical test for syphilis.

DOI: http://dx.doi.org/10.5772/intechopen.85055

Figure 6.

Figure 7.

aortic valve.

77

spirochetes are infrequently identified (inset).

#### Figure 4.

Lethal B. cereus pneumonia (left, H&E; center, Gram stain; right, immunostaining using B. cereus rabbit antiserum). Gram-positive rods are clustered in the necrohemorrhagic lung tissue, and B. cereus antigens are localized in the spore of the bacteria.

#### Figure 5.

T. pallidum immunostaining I (left, H&E; right, immunostaining using T. pallidum antiserum; inset, gross appearance of skin eruption on the neck). The papular skin lesion in a middle-aged male is richly infiltrated by plasma cells, and thus the possibility of syphilis was suspected histopathologically. Immunostaining discloses infection of spiral-shaped long bacteria in the epidermis, confirming the diagnosis of clinically unsuspected syphilis.

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.85055

performed. The diagnosis of stage II syphilis was subsequently confirmed by serological test for syphilis.

In Figure 6, a biopsied penile lesion with painless ulceration (chancre) in stage I and excised syphilitic granulomatous lymphadenitis in stage III are presented. In the penis, numerous spiral microbes were clustered mainly in the basal part of the squamous mucosa and around the dermal capillary vessels. In the stage III lesion with granulomatous reaction, coiled spirochetes were infrequently observed.

#### Figure 6.

(Simple Stain-Max, Nichirei) using T. pallidum rabbit antiserum (Biocare) diluted at 1:1000, after heat-induced antigen retrieval in 1 mM EDTA solution, pH 8, is highly sensitive and reproducible in demonstrating long coiled microbes among the lesion [4, 7–10, 19]. Figure 5 illustrates a neck skin papule richly infiltrated by plasma cells, biopsied from a middle-aged Japanese male patient in a remission state of malignant lymphoma 2 years after chemotherapy. Clinicians suspected of skin recurrence of malignancy, but immunostaining clearly demonstrated dense infection of coiled bacteria in the epidermis. Plasma cell-rich appearance microscopically suggested the possibility of syphilis, and thus immunostaining for T. pallidum was

Immunohistochemistry - The Ageless Biotechnology

Lethal B. cereus pneumonia (left, H&E; center, Gram stain; right, immunostaining using B. cereus rabbit antiserum). Gram-positive rods are clustered in the necrohemorrhagic lung tissue, and B. cereus antigens are

T. pallidum immunostaining I (left, H&E; right, immunostaining using T. pallidum antiserum; inset, gross appearance of skin eruption on the neck). The papular skin lesion in a middle-aged male is richly infiltrated by plasma cells, and thus the possibility of syphilis was suspected histopathologically. Immunostaining discloses infection of spiral-shaped long bacteria in the epidermis, confirming the diagnosis of clinically unsuspected

Figure 4.

Figure 5.

syphilis.

76

localized in the spore of the bacteria.

T. pallidum immunostaining II (left and center, stage I penile chancre; right, stage III syphilitic lymphadenitis; left and right, H&E; center and inset, immunostaining using T. pallidum antiserum). In the penis, numerous spiral microbes are clustered mainly in the basal part of the squamous mucosa and around the dermal capillary vessels. In the stage III lesion accompanying granulomatous reaction with multinucleated giant cells, coiled spirochetes are infrequently identified (inset).

#### Figure 7.

T. pallidum immunostaining III (left, tonsil; center, gastric mucosa; right, aortic valve). Immunohistochemical visualization of spirochetes in the biopsy specimens using T. pallidum antiserum significantly contributes to confirming the clinical and serological diagnosis of syphilis haphazardly affecting the tonsil, gastric mucosa, and aortic valve.

Immunostaining was considerably supportive and useful in the clinical practice, when treponemal microbes were visualized in the tonsillar, gastric, and aortic

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens…

3.4 Immunostaining for infection of E. coli and related bacteria using E. coli

Immunostaining (Simple Stain-Max, Nichirei) using E. coli rabbit antiserum (Dako) diluted at 1:20,000 after proteinase K pretreatment is applicable to demonstrating infection of E. coli or related enterobacteria in paraffin sections. E. colirelated antigens were immunodetected in colonic erosion and malacoplakia in the rectal mucosa (Figure 8) [4, 7, 8, 10, 28]. Malacoplakia is a variant of xanthogranulomatous inflammation caused by E. coli, and Michaelis-Gutmann bodies, a microscopic hallmark of malacoplakia, are immunoreactive for E. coli antigens [28]. E. coli-like organisms were immunohistochemically detected in xanthogranulomatous proctitis, cholecystitis, and cholangitis, as well as abscess-forming epididymitis [4, 29, 30]. Positive granular signals in E. coli-induced bronchopneumonia

[31] and emphysematous pyelonephritis are illustrated in Figure 9.

causative pathogen. In such situations, immunostaining employing the

4. Immunohistochemical demonstration of bacterial antigens using four kinds of antibacterial antisera with a wide cross-reactivity

We diagnostic pathologists encounter lesions strongly suggestive of infection but with poor clinical information or with difficulty in microscopically supposing a

abovementioned four kinds of rabbit antisera is worthy of application [8, 19]. We can prove the existence of pathogens in a certain part of the lesion. Background staining is negligible, whereas B. cereus antiserum may be cross-reactive to the nuclei of human cells in some cases (see Figure 16). In this type of application, we must abandon the specificity of immunostaining, and instead we welcome to accept

Regrettably enough, the availability of antisera against microbes from commercial sources has become limited. In fact, antisera against BCG and E. coli are no longer available from Dako (Agilent) company [16]. The author is afraid that this may hamper the standardization of immunohistochemical diagnosis of infectious

Table 3 summarizes reactivities of various microbes to the four kinds of rabbit

Hamster liver experimentally infected with Leptospira interrogans is shown in Figure 10. Not only Leptospira antiserum (the gift from Prof. Shinichi Yoshida, Department of Bacteriology, Faculty of Medical Sciences, Kyushu University, Fukuoka) but also E. coli antiserum were reactive to spiral-shaped bacteria in the hepatic sinusoid [19]. Pathogens phagocytized by activated Kupffer cells were visualized only by Leptospira antiserum [32]. E. coli antiserum was not crossreactive with T. pallidum in the syphilitic lesion, while T. pallidum antiserum was

valvular biopsy specimens (Figure 7).

DOI: http://dx.doi.org/10.5772/intechopen.85055

to a variety of bacteria

the sensitivity of detection.

antibacterial antisera.

4.1 E. coli antigens in leptospirosis

unreactive with Leptospira in the hamster liver.

diseases.

79

antiserum

#### Figure 8.

E. coli immunostaining I (upper panels, rectal erosion; lower panels, rectal malacoplakia; left, H&E; right, immunostaining using E. coli antiserum). E. coli-related antigens are detectable in eroded surface of rectal mucosa and in malacoplakia of the rectum. Michaelis-Gutmann bodies (arrows) in the cytoplasm of macrophages, the microscopic hallmark of malacoplakia, are round, basophilic, and immunoreactive to E. coli antigens.

#### Figure 9.

E. coli immunostaining II (upper panels, opportunistic E. coli-induced pneumonia; lower panels, E. coliinduced emphysematous pyelonephritis in a diabetic patient; left, H&E; right, immunostaining using E. coli antiserum). Positive rod-shaped signals are clearly seen in the cytoplasm of phagocytes in nosocomial bronchopneumonia and in severely affected kidney with numerous colonies of gas-forming bacteria.

Low-Specificity and High-Sensitivity Immunostaining for Demonstrating Pathogens… DOI: http://dx.doi.org/10.5772/intechopen.85055

Immunostaining was considerably supportive and useful in the clinical practice, when treponemal microbes were visualized in the tonsillar, gastric, and aortic valvular biopsy specimens (Figure 7).

## 3.4 Immunostaining for infection of E. coli and related bacteria using E. coli antiserum

Immunostaining (Simple Stain-Max, Nichirei) using E. coli rabbit antiserum (Dako) diluted at 1:20,000 after proteinase K pretreatment is applicable to demonstrating infection of E. coli or related enterobacteria in paraffin sections. E. colirelated antigens were immunodetected in colonic erosion and malacoplakia in the rectal mucosa (Figure 8) [4, 7, 8, 10, 28]. Malacoplakia is a variant of xanthogranulomatous inflammation caused by E. coli, and Michaelis-Gutmann bodies, a microscopic hallmark of malacoplakia, are immunoreactive for E. coli antigens [28]. E. coli-like organisms were immunohistochemically detected in xanthogranulomatous proctitis, cholecystitis, and cholangitis, as well as abscess-forming epididymitis [4, 29, 30]. Positive granular signals in E. coli-induced bronchopneumonia [31] and emphysematous pyelonephritis are illustrated in Figure 9.
