／【Ｋ：】Ｓｅｒｖｅｒ／１７０１０３６２ 新潟厚生連農業協同組合／本文／１６五十嵐

０７３‐０７５2017.02.10 19.42.4

Genetic confirmation of Entamoeba histolytica by the 厚生連医誌
real-time polymerase
第２６巻 chain
１号reaction（rPCR）
７３〜７５ ２０１７
with formalin-fixed paraffin-embedded specimen（FFPE）of colorectal biopsy

Brief report

Genetic confirmation of Entamoeba histolytica
by the real-time polymerase chain reaction（rPCR）
with formalin-fixed paraffin-embedded specimen（FFPE）
of colorectal biopsy
Nagaoka General Central Hospital, Department of pathology ; Pathologist

Toshihiko Ikarashi
Background : Entamoeba histolytica should be rapidly
confirmed by genetic analysis. In this report wecould establish the rapid and convenient rPCRFFPE method and reported our case.
Case report : ５
１-year-old male patient revealed rectal
macrophages phagocytizing amebas, which wereconfirmed as Entamoeba histolytica by both theusual PCR-FFPE method followed by gel electrophoresis and fluorescence reaction（usual PCRFFPE method）and the rPCR-FFPE method.
Conclusion : The rPCR-FFPE method provided a definitive diagnosis of Entamoeba histolytica more readily than the usual PCR method.
Key words : Entamoeba histolytica, genetic analysis, thereal-time polymerase chain reaction with formalinfixed paraffin-embedded specimen（rPCR-FFPEmethod）, usual PCR method followed by gel electrophoresis and fluorescence reaction（usual PCRFFPE method）

bris on the surface of rectal epithelium. The intracytoplasmic inclusions suggested amebas microscopically. TheFFPE specimen was routinely processed with two types ofprimers ; pathogenetic pairs and non-pathogenetic ones
（１,３）
. Both the usual PCR-FFPE method and the rPCRFFPE method were done, the latter of which was donewith real-time PCR : Mx３０００P Real-Time QPCR system（Agilent）and SYBRTM Green I method, SYBRTM Premix
EX TaqTM（Tli RNaseH Plus）RP４２０A（Takara）（Table１）. The usual PCR-FFPE process required １１ hours toget the expected bands at １００ base-pair. The rPCR-FFPE
method against pathogenetic ameba under the annealing
temperature at ６０℃ showed the specific peak at ７７℃
with a small non-specific peak on the chart of dissociation
curve within the total curse of４hours. The latter non-specific peak could be reduced to avoid the false-positive
peak under the annealing temperature at ６５℃. No peak ofpathogenetic ameba could be found under the annealing
temperature over ６５℃. Non-pathogenetic amebas showeda single peak at ７２℃ under the annealing temperature at
６０℃ and no peak under the annealing temperature over６０℃（Figure１,２）.

Background
Infectious enterocolitis by Entamoeba histolytica ispathologically diagnosed by the confirmation of macrophages phagocytizing amebas, which is often difficult tofind microscopically because of the interruption-driven environment with marked inflammatory debris and degeneration. More accurate diagnostic tool has been required
pathologically. The PCR-FFPE method of colorectal lesions was tried to detect Entamoeba histolitica, and we,furthermore, compared its superiority between the usualPCR-FFPE method and the rPCR-FFPE method in this report.

Case report

Discussion
The usual PCR-FFPE method required quite acomplicated procedures and lot of works. In contrast, therPCR-FFPE method could shave time off and was moreconvenient method to get the final diagnosis than theusual PCR-FFPE method.
In the rPCR-FFPE method, the preferable annealing temperatures are ６５℃ for pathogenetic ameba and６０℃ for non-pathogenetic ameba. The single screening
test for both pathogenetic amebas and non-pathogenetic
ones could be used under the annealing temperature at
６０℃ and differentiated by the dissociation curve with
their different peak temperatures.

５１-year-old male patient complained of diarrhea
and rectal erosions were identified during endoscopy. Several macrophages were histologically found in necrotic de-― 73 ―／【Ｋ：】Ｓｅｒｖｅｒ／１７０１０３６２ 新潟厚生連農業協同組合／本文／１６

厚生連医誌

第２６巻

五十嵐

０７３‐０７５2017.02.10 19.42.4

１号 ７
３〜７５ ２０１７Referrence

和 文 抄

１．Ikarashi, T et al. Detection of monoclonality in Band T-cell lymphoma by the use of polymerase chain
reaction of formalin-fixed paraffin-embedded tissues.
Niigata-ken Koseiren Med J２０００;１０:１０−５．
２．Ikarashi, T. Trial of rapid diagnosis of ifection andindividual confirmation against tissue contamination byreal-time PCR（polymerase chain reaction）of SYBRGreen method to paraffin-embedding specimens. Niigata-ken Koseiren Med J２５;１２７−３０;２０１６．
３．Tachibana, H et al. Distinguishing pathogenic isolates
of Entamoeba histolytica by polymerase chain reaction.
J Inf Dis１９９１;１６４:８２５−６．

録

短報
結腸直腸生検のホルマリン固定パラフィン包埋材料（FFPE）を使ったリアルタイム・ポリメラーゼ連鎖反応（rPCR）による赤痢アメーバの遺伝子学的同定
長岡中央綜合病院、病理部；病理医
いか らしとしひこ

五十嵐俊彦
背景：赤痢アメーバを rPCR-FFPE によって迅速に同定することができたので、１症例を報告した。
症例報告：５１歳の男性患者は、直腸生検材料においてアメーバを貪食した大食細胞を示し、ゲル電気泳動・蛍光反応による従来の PCR-FFPE 検査とrPCR-FFPE 検査において赤痢アメーバを同定できた。
結論：rPCR-FFPE 検査は、従来の PCR 検査に比較して、容易に赤痢アメーバを同定できる。
キーワード：赤痢アメーバ、遺伝子分析、ホルマリン固定パラフィン包埋標本を使ったリアルタイ
ム・ポリメラーゼ連鎖反応検査（rPCR-FFPE）、ゲル電気泳動・蛍光反応による従来の PCR 検
査

Figure １. Chart of dissociation curve in the rPCR-FFPE method under the annealing temperature
at６０℃..
Two peaks at ７７℃ of specific peak and ８２℃ of small non-specific one in pathogenetic ameba.
Single peak at７２℃ in non-pathogenetic ameba.
circle : PCR using primers for pathogenetic ameba, square : PCR using primers for non-pathogenetic ameba, x-axis : temperature（℃）, y-axis : fluorescent degree.

― 74 ―

／【Ｋ：】Ｓｅｒｖｅｒ／１７０１０３６２ 新潟厚生連農業協同組合／本文／１６五十嵐

０７３‐０７５2017.02.10 19.42.4

Genetic confirmation of Entamoeba histolytica by the real-time polymerase chain reaction（rPCR）
with formalin-fixed paraffin-embedded specimen（FFPE）of colorectal biopsy

Figure ２. Chart of dissociation curve in the rPCR-FFPE method under the annealing temperatureat６５℃..
Single peak at７７℃of specific peak and a small negligible non-specific hump in pathogenetic
ameba.
circle : PCR using primers for pathogenetic ameba, x-axis : temperature（℃）, y-axis : fluorescentdegree.

Table１. Procedure of PCR（２０１６／０６／２３受付）― 75 ―