5.3.4 Free-living amebic meningoencephalitis

Free-living amoeba widely seen in soil and water may cause lethal meningoencephalitis as an opportunistic or non-opportunistic infection [78]. A Japanese male

#### Figure 48.

Visceral leishmaniasis (left, H&E; right, reactivity with patient's own serum; inset, high-powered view). During the stay in India, a Japanese businessman manifested headache, high fever, thrombocytopenia, and liver dysfunction. Liver biopsy shows small epithelioid granulomas. A panel of immunohistochemical analysis has failed to identify the causal agent. The patient's own serum demonstrates red cell-sized positive signals in the cytoplasm of epithelioid cells, strongly suggesting visceral leishmaniasis (kala azar) endemic in India. The final diagnosis was made by the serological study and successful treatment.

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

aged 60's suffering from alcoholic liver cirrhosis manifested left hemiparesis [79]. Computed tomography disclosed multifocal low densities in his right hemisphere. Herpetic encephalitis was clinically suspected. HIV antibody was negative. Progressive intracranial edema necessitated decompressive craniotomy with brain biopsy. The brain tissue microscopically showed perivascular chronic active inflammation, with ameba-like cells somewhat resembling macrophages being scattered. The 1:500 diluted patient's serum clearly reacted with the protozoan bodies, and mouse antiserum to Acanthamoeba culbertsoni (a gift from Prof. Yuji Tachibana, Department of Infectious Diseases, Tokai University School of Medicine, Isehara) also gave positivity (Figure 49) [7, 8, 21, 23, 77]. No reactivities for A. polyphaga and A. castellanii were noted. High immunofluorescence titer against A. culbertsoni was serologically confirmed in the patient's serum. Detailed microscopic observation of H&E-stained preparations disclosed the presence of acanthamebic trophozoites and cysts in the brain tissue. The final diagnosis was opportunistic acanthamebic meningoencephalitis associated with liver cirrhosis.

Balamuthia mandrillaris may cause amebic meningoencephalitis in both immunocompromised and immunocompetent individuals [80]. An amoeba-induced skin nodule may be formed before the onset of meningoencephalitis [81]. A healthy Japanese farmer housewife aged 50's suddenly complained of progressive consciousness disturbance and seizure. She daily grew vegetables. Two weeks after onset, the patient expired. At autopsy, the basal side of the brain grossly revealed hemorrhagic meningoencephalitis. Microscopically, large-sized, basophilic amebic trophozoites were clustered mainly in Virchow-Robin's spaces. Smaller-sized cysts were focally observed. PCR analysis disclosed infection of B. mandrillaris. Immunostaining using both the patient's own serum and the patient's serum of the abovementioned acanthamebic meningoencephalitis gave distinct positivity (Figure 50) [23]. Balamuthia-infected skin nodule seen in another patient gave clear positivity of the microbe using these two patients'sera. Cross-reactivity of the acanthamebic antigens to Balamuthia species was thus confirmed, but the serum

#### Figure 49.

of patient with hepatic ascariasis reacted with the dead worm body, confirming the diagnosis of ectopic splenic ascariasis. The serum functioned as an immunohisto-

Visceral leishmaniasis, sandfly-mediated systemic infection of Leishmania donovani, kills more than 20,000 persons in 2015 but has been classified by the World Health Organization as a neglected tropical disease [76]. A Japanese businessman aged 30's stayed in a sequential order in Australia, Thailand, Singapore, and finally India [77]. During his stay in India, he manifested headache, high fever, thrombocytopenia, and liver dysfunction. The patient was hospitalized in Japan, but his general condition was poor. In order to confirm the diagnosis, liver biopsy was performed. Small epithelioid granulomas were identified, and the possibility of Q fever in Australia, melioidosis in Thailand, brucellosis, miliary tuberculosis, and non-tuberculous mycobacteriosis was considered histopathologically. No positive findings were obtained in immunohistochemical analysis using a panel of antibodies. The patient's own serum diluted at 1:500 demonstrated red cell-sized positive signals in the cytoplasm of epithelioid cells (Figure 48) [7, 8, 21, 23, 77]. The size and shape of the pathogen strongly suggested visceral leishmaniasis (kala azar) endemic in India. High serum immunofluorescence titer against L. donovani was serologically confirmed thereafter. Administration of pentavalent antimony compound saved his life. This is the real case, in which immunostaining using the patient's own serum was practical maximally.

Free-living amoeba widely seen in soil and water may cause lethal meningoencephalitis as an opportunistic or non-opportunistic infection [78]. A Japanese male

Visceral leishmaniasis (left, H&E; right, reactivity with patient's own serum; inset, high-powered view). During the stay in India, a Japanese businessman manifested headache, high fever, thrombocytopenia, and liver dysfunction. Liver biopsy shows small epithelioid granulomas. A panel of immunohistochemical analysis has failed to identify the causal agent. The patient's own serum demonstrates red cell-sized positive signals in the cytoplasm of epithelioid cells, strongly suggesting visceral leishmaniasis (kala azar) endemic in India. The final

diagnosis was made by the serological study and successful treatment.

chemical probe specific to A. lumbricoides [21].

Immunohistochemistry - The Ageless Biotechnology

5.3.4 Free-living amebic meningoencephalitis

Figure 48.

106

5.3.3 Visceral leishmaniasis

Acanthamebic meningoencephalitis (left upper, low-powered H&E; right upper, high-powered H&E; inset, an amebic cyst; left lower, reactivity with patient's own serum; right lower, reactivity with mouse antiserum to Acanthamoeba culbertsoni). An HIV-negative Japanese male suffering from alcoholic liver cirrhosis manifested left hemiparesis. Progressive intracranial edema necessitated decompressive craniotomy with brain biopsy. The brain tissue microscopically shows perivascular chronic active inflammation, and amebic trophozoites and cysts (inset) are scattered. The diluted patient's serum clearly reacts with the amebic bodies. A. culbertsoni infection has been confirmed by using a panel of mouse antisera against different Acanthamoeba subspecies.

helminthic infection, the specificity was much narrower with limited crossreactivities, and once the specificity is known, the patients'sera turn to become specific primary antibodies for identifying pathogens in the following new cases. In the latter approach, what one should do is, instead of ordering an expensive antibody of unknown quality, to make a brief phone call to clinicians or laboratory technicians to ask to save a small aliquot of patients'sera, soon after microscopic confirmation of the host response in histopathology specimens. This is particularly true when specific antibodies are not listed in the commercial catalog. Of note is that informed consent is unnecessary when the patient's serum is utilized primarily

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

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

for making a diagnosis of the patient's own. When the serum is applied to immunostaining for another case as the primary antibody, the author strongly

histopathological diagnosis of infectious diseases in the readers' laboratories.

The author sincerely hopes that the approaches shown here will be applied to the

The author deeply thanks many colleagues of technicians who supported the authors' idea and kindly immunostained specimens. The author has no granting for

recommends linking the preserved serum non-anonymously.

The author has no conflict of interest in the present study.

Acknowledgements

the present study.

Author details

Yutaka Tsutsumi

109

Pathos Tsutsumi, Toyoake, Aichi, Japan

provided the original work is properly cited.

\*Address all correspondence to: pathos223@kind.ocn.ne.jp

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Conflict of interest

#### Figure 50.

Balamuthia meningoencephalitis (left, H&E; center, reactivity with patient's own serum; right, reactivity with the serum of patient of acanthamebic meningoencephalitis). A healthy Japanese farmer housewife complained of progressive consciousness disturbance and seizure. At autopsy, hemorrhagic meningoencephalitis was observed. Large-sized, basophilic amebic trophozoites are microscopically clustered in Virchow-Robin's spaces. PCR analysis has disclosed infection of B. mandrillaris. Immunostaining using both the patient's own serum and the patient's serum of the abovementioned acanthamebic meningoencephalitis gives distinct positivity. Of note is that heat-induced antigen retrieval is needed for visualizing B. mandrillaris antigens.

was not cross-reactive to Naegleria fowleri (brain-eating amoeba) seen in the autopsied brain of another patient. Regarding acanthamebic keratitis, see Figure 33.

Of note is an exception that Balamuthia antigens were detectable by the diluted patient's serum only after heating pretreatment of deparaffinized sections in 10 mM citrate buffer, pH 6. Without heat-induced antigen retrieval, signals were not observed at all. Therefore, the author strongly recommends employing the heating pretreatment for immunostaining using patients'sera in order to avoid unexpected false negativity. Silane-coated glass slides should thus be used for preventing section detachment.
