1. Introduction

Infectious diseases kill a significant number of people in the world. Of the top 10 leading causes of death in low-income countries in 2016, pneumonia, diarrheal diseases, acquired immune deficiency syndrome (AIDS), malaria, and tuberculosis are listed up. More than half of deaths in low-income countries were due to communicable diseases, maternal causes, conditions arising during pregnancy and childbirth, and nutritional deficiencies, while such causes shared less than 7% of deaths in high-income countries [1]. It is no double to say that the detection of infectious agents in the lesion is essential for the histopathological diagnosis of infectious diseases [2, 3]. For the correct diagnosis and appropriate treatment of the patient, immunohistochemical demonstration of pathogens within the lesion must be suitable and desirable [4–10].

Needless to say, the most important factor, the "life," in the immunohistochemical analysis should be a high-specificity antibody for exactly demonstrating the corresponding antigen. A variety of immunohistochemical techniques for

increasing the sensitivity of detection have been developed, in order to localize the antigens under highly specific and highly sensitive conditions in routinely processed (formalin-fixed, paraffin-embedded) sections [11–13]. Immunohistochemical approach is quite fitted to the histopathological diagnosis of infectious diseases, since the antigens of the pathogen are absent from the human tissue specimens [4, 7–10].

2. Commercially available antibodies against pathogens: the author's

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

In Table 1, commercially available antibodies against pathogens are listed up, simply for the convenience of the readers. The catalog is solely based on the author's experience, so that the antibodies may not be most suitable for detecting pathogens in routinely prepared sections [8, 19]. Some antibodies may be no longer available, simply because of limited market. The specificity of the antibodies is categorized

Pathogen Type (clone) Company Dilution Pretreatment Specificity

Actinomyces Mo (396AN1) DSHB 1:5 CB6 High Bacillus cereus Rabbit Abcam 1:500 CB6 Low Bartonella henselae Mo (H2A10) Biocare 1:100 EDTA High BCG Rabbit Dako 1:5000 None Low Campylobacter jejuni Mo (4080) Novocastra 1:50 CB6 Moderate

Escherichia coli Rabbit Dako 1:20,000 PK Low E. coli Mo (MAB706) Millipore 1:50 CB6 High E. coli (LPS) Mo (2D7/1) Abcam 1:5000 CB6 Moderate Enterococcus Rabbit Abcam 1:2000 None Moderate Helicobacter pylori Rabbit Dako 1:50 PK Moderate Helicobacter pylori Mo (UCL3R) Novocastra 1:100 EDTA High Klebsiella pneumoniae Mo (70–2) Monosan 1:300 None High

Pneumococcus Mo (128/390) Chemicon 1:1000 None Moderate Pneumolysin Mo (9.1/2/3/6) Novocastra 1:50 EDTA High Protein A Mo (SPA-27) Sigma 1:100 PK Moderate Protein G Rabbit Abcam 1:500 PK Moderate

Streptococcus Goat BioReagents 1:500 PK Moderate Treponema pallidum Rabbit Biocare 1:1000 EDTA Low Treponema pallidum Mo (J010J) Thermo 1:50 CB6 High

Aspergillus Rabbit Biocare 1:200 EDTA Moderate

Mo (B104.1) Biomeda 1:5 CB6 High

Rabbit Denka Seiken 1:500 EDTA High

Rabbit Abcam 1:800 CB6 High

Mo (B11) Biogenesis 1:800 EDTA High

Chemicon 1:500 PK Moderate

Mo (MAB738) Chemicon 1:3000 EDTA High

experience

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

Antibacterial antibody

Chlamydia trachomatis

Legionella pneumophila

Mycobacterium tuberculosis

M. tuberculosis (MPT64)

Pseudomonas aeruginosa

Antifungal antibody

71

Staphylococcus Mo (STAPH11–

248.2)

However, pathogens express multiple antigens, and the antigens are often crossreactive among different pathogens [14]. The pathogen in a single category further reveals a variety of serum types [15]. It is more difficult for us pathologists than expected to detect a certain pathogen using a single antibody [4, 7]. It is next to impossible to prepare and keep specific antibodies in hand for immunohistochemical diagnosis in a single institution simply because there are too many species of microbes pathogenic to humans. We have a limited number of commercially available antibodies against pathogens. Useful commercial antibodies may soon disappear from the market because of a simple reason: the dead stock [16].

In the routine practice of histopathological services using hematoxylin and eosin (H&E) staining, it is often the situation that the pathologists are requested to judge if the lesion is infective or not. Namely, the presence of some sort of pathogenic organisms within the lesion should be shown with the maximal priority in making the diagnosis, and the detailed identification of the species name should be analyzed using different technologies afterward. For example, if a lesion showing massive necrosis is experienced, we must comment that the necrosis is infective in origin or tumor-related. In such a situation, an antibody widely cross-reactive to bacteria but unreactive to human tissue is valuable. A monoclonal antibody H9 (a gift of Prof. Shigeru Kamiya, Department of Microbiology, Kyorin University School of Medicine, Mitaka, Tokyo) against bacteria-specific heat shock protein (HSP)-60, not cross-reactive animal mitochondrial HSP-60, is quite valuable [7, 17], but it is not commercially available. Lipopolysaccharide (LPS) or endotoxin, located in the outer layer of the bacterial wall, should be the good marker of Gram-negative bacteria (refer to Figure 11) [18], but it is practically difficult for us to get a monoclonal or polyclonal antibody detecting widely cross-reactive LPS.

In order to visualize pathogens in formalin-fixed, paraffin-embedded sections, we do not necessarily need to prepare antibodies with high specificity [4, 7, 19]. The author routinely uses four kinds of commercially available rabbit antisera raised against Bacillus Calmette-Guérin (BCG; Mycobacterium bovis), Bacillus cereus (B. cereus),Treponema pallidum (T. pallidum), and Escherichia coli (E. coli) [16, 19]. For immunoperoxidase staining, the amino acid polymer technique or indirect immunoperoxidase method is employed. Immunostaining using these lowspecificity antimicrobial antisera commonly yields clear high-sensitivity signals with low background, because of poor cross-reactivity of bacterial antigens to human cells and tissues.

The second approach for detecting unknown pathogens in the histopathological sections is the use of patient's serum [4, 7, 8, 20–23]. Patients'sera diluted at 1:500 or 1:1000 become convenient probes for indirect immunoperoxidase localization of pathogens in formalin-fixed, paraffinembedded sections, particularly when cellular tissue reactions have been confirmed under the microscope. Serum antibody titer should be high in the recovery or chronic stage of illness. The existence of inflammatory tissue reaction, such as an abscess or granuloma, indicates that immune cells in the patient have been activated against the causative pathogen. The second approach is of high value for protozoan and helminthic disorders.
