4.9 Application to nocardiosis

Nocardiosis is caused by filamentous Gram-positive saprophytic bacteria, Nocardia spp., mainly in the lung. This weakly acid-fast aerobe infects mainly in the immunocompromised patients. N. asteroides is the most frequent isolate, but other species may also be encountered [40]. A subpleural nodule in the left upper lobe

was pointed out in a diabetic Japanese male aged 70's who also suffered from nephrotic syndrome with steroid administration. Video-assisted thoracic surgery disclosed necrotic nodule grossly resembling a tuberculous lesion. Microscopically, abscess formation with foamy cells clustering and occasional multinucleated giant cell reaction was confirmed, and N. beijingensis was isolated and molecularly confirmed by analyzing 16S ribosomal RNA. Grocott, periodic acid-Schiff, Gram, and Ziehl-Neelsen stains were all negative. Immunostaining using antisera against B. cereus and BCG clearly demonstrated filamentous or aggregated bacteria phagocytized by foamy cells and multinucleated giant cells (Figure 18). Some bacteria

Pulmonary nocardiosis caused by Nocardia beijingensis (left upper, gross picture; right upper, low-powered H&E; inset, high-powered H&E; left lower, B. cereus antigens; right lower, BCG antigens). Subpleural abscess seen in a diabetic aged with steroid therapy was surgically excised. Necrotic lesions reveal focal clusters of foamy macrophages and a few multinucleated giant cells. Grocott stain failed to identify the pathogen. Antisera against B. cereus and BCG clearly demonstrate filamentous and aggregated microbes in the cytoplasm of foamy

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

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

reacted to T. pallidum antiserum, while E. coli antiserum was unreactive.

antisera against BCG and T. pallidum (Figure 19) [19].

4.11 Application to granulomatous mastitis

Cervical lymphadenopathy in cat-scratch disease, infection of Bartonella henselae, is microscopically characterized by suppurative granuloma formation. Not only cat scratch but also bite by cat flea can provoke bartonellosis [41]. Splenic abscess is occasionally seen as systemic manifestation of cat-scratch disease [42]. Splenectomy was performed from a male patient aged 40's. Monoclonal antibody H2A10 to B. henselae (not cross-reactive to non-henselae Bartonella) [41] demonstrated a few microbes in the cytoplasm of macrophages accumulated in the abscess cavity. The positive findings were more easily obtained by immunostaining with

Granulomatous mastitis is seen in childbearing women 2–4 years after breastfeeding [43]. The causative agent is lipophilic Gram-positive bacillus,

4.10 Application to bartonellosis

Figure 18.

85

macrophages and giant cells.

#### Figure 16.

Haemophilus influenzae-induced tonsillitis (left, H&E; center left, B. cereus antigens; center right, BCG antigens; right, T. pallidum antigens). Antisera against B. cereus, BCG, and T. pallidum demonstrate H. influenzae colonizing the tonsillar ulcer. B. cereus antigens are expressed with the strongest reactivity, while cross-reactivity to the nucleus of human cells is seen.

#### Figure 17.

Actinomyces israelii colonizing the sequestration of the jaw bone (left upper, H&E; center upper, Grocott stain; right upper, reactivity with A. israelii monoclonal antibody 396AN1; left lower, B. cereus antigens; center lower, BCG antigens; right lower, T. pallidum antigens). The marrow space among dead bone trabeculae is occupied by Grocott-positive filamentous bacteria but with little inflammatory reaction. The pathogen is immunolocalized not only by the monoclonal antibody but also by antisera against B. cereus and BCG. T. pallidum antigens are scarcely expressed.

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

#### Figure 18.

4.8 Application to actinomycosis

Immunohistochemistry - The Ageless Biotechnology

4.9 Application to nocardiosis

Figure 17.

84

Figure 16.

T. pallidum antigens are scarcely expressed.

antisera against T. pallidum and E. coli was faint.

while cross-reactivity to the nucleus of human cells is seen.

Secondary dense infection of Actinomyces israelii is occasionally observed in biopsied sequestrum of the alveolar bone of the jaw [39]. Grocott-positive filamentous bacteria occupy the marrow space but with little inflammatory reaction. The microbes were visualized not only with A. israelii monoclonal antibody (396AN1) but also with antisera against BCG and B. cereus (Figure 17) [19]. Reactivity with

Nocardiosis is caused by filamentous Gram-positive saprophytic bacteria, Nocardia spp., mainly in the lung. This weakly acid-fast aerobe infects mainly in the immunocompromised patients. N. asteroides is the most frequent isolate, but other species may also be encountered [40]. A subpleural nodule in the left upper lobe

Haemophilus influenzae-induced tonsillitis (left, H&E; center left, B. cereus antigens; center right, BCG antigens; right, T. pallidum antigens). Antisera against B. cereus, BCG, and T. pallidum demonstrate H. influenzae colonizing the tonsillar ulcer. B. cereus antigens are expressed with the strongest reactivity,

Actinomyces israelii colonizing the sequestration of the jaw bone (left upper, H&E; center upper, Grocott stain; right upper, reactivity with A. israelii monoclonal antibody 396AN1; left lower, B. cereus antigens; center lower, BCG antigens; right lower, T. pallidum antigens). The marrow space among dead bone trabeculae is occupied by Grocott-positive filamentous bacteria but with little inflammatory reaction. The pathogen is immunolocalized not only by the monoclonal antibody but also by antisera against B. cereus and BCG.

Pulmonary nocardiosis caused by Nocardia beijingensis (left upper, gross picture; right upper, low-powered H&E; inset, high-powered H&E; left lower, B. cereus antigens; right lower, BCG antigens). Subpleural abscess seen in a diabetic aged with steroid therapy was surgically excised. Necrotic lesions reveal focal clusters of foamy macrophages and a few multinucleated giant cells. Grocott stain failed to identify the pathogen. Antisera against B. cereus and BCG clearly demonstrate filamentous and aggregated microbes in the cytoplasm of foamy macrophages and giant cells.

was pointed out in a diabetic Japanese male aged 70's who also suffered from nephrotic syndrome with steroid administration. Video-assisted thoracic surgery disclosed necrotic nodule grossly resembling a tuberculous lesion. Microscopically, abscess formation with foamy cells clustering and occasional multinucleated giant cell reaction was confirmed, and N. beijingensis was isolated and molecularly confirmed by analyzing 16S ribosomal RNA. Grocott, periodic acid-Schiff, Gram, and Ziehl-Neelsen stains were all negative. Immunostaining using antisera against B. cereus and BCG clearly demonstrated filamentous or aggregated bacteria phagocytized by foamy cells and multinucleated giant cells (Figure 18). Some bacteria reacted to T. pallidum antiserum, while E. coli antiserum was unreactive.

## 4.10 Application to bartonellosis

Cervical lymphadenopathy in cat-scratch disease, infection of Bartonella henselae, is microscopically characterized by suppurative granuloma formation. Not only cat scratch but also bite by cat flea can provoke bartonellosis [41]. Splenic abscess is occasionally seen as systemic manifestation of cat-scratch disease [42]. Splenectomy was performed from a male patient aged 40's. Monoclonal antibody H2A10 to B. henselae (not cross-reactive to non-henselae Bartonella) [41] demonstrated a few microbes in the cytoplasm of macrophages accumulated in the abscess cavity. The positive findings were more easily obtained by immunostaining with antisera against BCG and T. pallidum (Figure 19) [19].

#### 4.11 Application to granulomatous mastitis

Granulomatous mastitis is seen in childbearing women 2–4 years after breastfeeding [43]. The causative agent is lipophilic Gram-positive bacillus, Corynebacterium kroppenstedtii, and the lesion is microscopically featured by lipid droplet-centered abscess or epithelioid granuloma. Antisera against BCG, B. cereus, and T. pallidum demonstrated bacterial cross-reactive antigens mainly in the lipid droplet surrounded by abscess and/or granuloma (Figure 20) [8, 16, 19]. The antigens were occasionally seen in the cytoplasm of macrophages clustered in the inflammatory lesion. E. coli antigens were infrequently seen in the lesion.

It should be of note that the positive signals were identified at the site of the disease-specific lesion. Polymerase chain reaction (PCR) analysis using DNA extracted from paraffin sections confirmed the nucleotide sequence of

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

Rhinoscleroma is localized and indolent infective nodule in the nasal cavity, endemic in Egypt, South America and eastern Europe. The main causative microbe has been reported to be Klebsiella rhinoscleromatis. Microscopically, foamy macrophages phagocytizing gram-negative rods, so-called Mickulicz cells, are observed in chronic inflammatory infiltrates [44]. A Japanese male aged 70's complained of a single elevated nodule in the nasal vestibule. Biopsy revealed localized infiltration

vacuoles with short rod-like material were occasionally seen in the macrophages or Mickulicz cells [45]. Immunostaining disclosed positive findings with monoclonal antibody 70-2 against Klebsiella spp., and the antiserum against B. cereus gave similar positivity. Fewer cells were also reactive to BCG antiserum. Antisera against T. pallidum and E. coli failed to detect the intracellular microbes (Figure 21). Negativity to E. coli antiserum was an unexpected finding, because of the close

In certain lesions, B. cereus antiserum was reactive to the pathogen, while the other three antisera were poorly reactive. It is plausible that antiserum against B. cereus, a Gram-positive spore-forming rod, shows the widest cross-reactivity including Gram-positive bacteria. Bacterial microbes positive for B. cereus-related antigens were demonstrated in brain abscess, placental chorioamnionitis, and Propionibacterium acnes-induced folliculitis of the skin (Figure 22) (refer also to Figure 45 for P. acnes folliculitis). Positive signals of B. cereus antigens in a lethal adult case of Pseudomonas aeruginosa-induced pneumonia/septicemia are

Rhinoscleroma caused by Klebsiella rhinoscleromatis (left upper: H&E, right upper: reactivity with monoclonal antibody 70-2 to Klebsiella spp., left lower: B. cereus antigens, right lower: E. coli antigens, inset: BCG antigens). Foamy macrophages with intracytoplasmic vacuoles containing short rod-like material (Mickulicz cells) are immunoreactive with monoclonal antibody 70-2 to Klebsiella spp. and B. cereus antiserum. Fewer

cells are labeled with BCG antiserum, while E. coli antiserum is unreactive.

of foamy macrophages in the background of chronic inflammation. Round

4.13 Immunostaining with B. cereus antiserum showing the widest

C. kroppenstedtii [16].

4.12 Application to rhinoscleroma

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

relationship between the two enterobacteria.

cross-reactivity among four

demonstrated in Figure 23 [19].

Figure 21.

87

#### Figure 19.

Splenic bartonellosis (left upper, H&E; right upper, reactivity with Bartonella henselae monoclonal antibody H2A10; left lower, BCG antigens; right lower, T. pallidum antigens). Suppurative granulomas are formed in the spleen. Macrophages in the abscess cavity focally reveal dot-like positive signals not only by the monoclonal antibody but also by antisera against BCG and T. pallidum. Of note is that more signals are seen with the two antisera than with the monoclonal antibody.

#### Figure 20.

Granulomatous mastitis caused by Corynebacterium kroppenstedtii (left upper, H&E; right upper, Gram stain; left lower, BCG antigens; right lower, T. pallidum antigens). This lactation-related infection is microscopically featured by lipid droplet-centered abscess or epithelioid granuloma. Gram stain and immunostaining for BCG, B. cereus, and T. pallidum demonstrate bacterial colonies in the lipid droplet surrounded by inflammatory reactions.

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

It should be of note that the positive signals were identified at the site of the disease-specific lesion. Polymerase chain reaction (PCR) analysis using DNA extracted from paraffin sections confirmed the nucleotide sequence of C. kroppenstedtii [16].
