380
CUSTER ET AL.
methods, volume I. Sixteenth Edition. Philadelphia, WB Saunders, 1979, pp 594-595
4. Li PK, Lee JT, MacGillivray MH, Schaefer PA, Siegel JH: Direct,
fixed-time kinetic assays for 0-hydroxybutyrate and acetoacetate with a centrifugal analyzer or a computer-backed spectrophotometer. Clin Chem 1980; 26:1713-1717
5. Moore JJ, Marcus M, Sax SM: Kinetic assay of /8-hydroxybutyrate
in plasma with a COBAS-BIO centrifugal analyzer. Clin Chem
1982;28:702-703
6. Schoen I, Fisher C, Winter S, Barnett R: Patient preparation and
specimen collection and handling including storage stability.
A.J.C.P. • September 1983
Medical Usefulness Subcommittee of CAP Standards Committee, March 1974
7. Stephens JM, Sulway MJ, Watkins JP: Relationship of blood acetoacetate and 3-hydroxybutyrate in diabetes. Diabetes 1971;
20:485-489
8. WildenhofTKE: Diurnal variations in the concentrations of blood
acetoacetate, 3-hydroxybutyrate and glucose in normal persons. Acta Med Scand 1972; 191:303-306
9. Williamson DH, Mellanby J, Krebs HA: Enzymatic determination of D(-)j3-hydroxybutyric acid and acetoacetic acid in
blood. Biochem J 1962; 82:90-96
Frequency of Entamoeba Gingivalis in
Human Gingival Scrapings
ANH H. DAO, M.D., D. PAUL ROBINSON, B.A., AND SONG W. WONG, M.D.
A survey was made of gingival scrapings stained by the Papanicolaou method to assess the occurrence of Entamoeba gingivalis, a nonpathogenic-oral amoeba. Positive findings were
recorded in 59% of 113 dental patients, and 32% of 96 healthy
controls. These figures showed no significant changes during
the last 20 years when compared with data published in 1960
and 1963.
The existence of E. gingivalis and its rare appearance in the
sputum should be known to cytologists because of the morphologic resemblance to Entamoeba histolytica, a pathogenic
amoeba. Morphologic features are described to differentiate
E. gingivalis from similar structures found in sputum. (Key
words: E. gingivalis; Amoeba; Oral protozoa) Am J Clin Pathol
1983; 80: 380-383
ENTAMOEBA GINGIVALIS is a human oral protozoon occurring frequently in persons with dental and
gingival disease. According to Kofoid,8 Gros first recovered this organism in 1849 from dental tartar, making
it the first parasitic amoeba. It was described in detail
by von Prowazek in 1904, according to Belding.3 Various investigators, 12 subsequently attempted to link its
presence causally with periodontal diseases, leading to
the treatment of some of these conditions with emetine
hydrochloride. Others 5,6 have been unable to verify this
causal relation, though they agreed that this amoeba
tended to be more prevalent in patients with poor oral
hygiene.
E. gingivalis, because of its widespread distribution
and morphologic resemblance to the pathogenic Entamoeba histolytica, can cause a diagnostic problem if
Received June 1, 1982; received revised manuscript and accepted
for publication February 7, 1983.
Address reprint requests to Dr. Dao: Department of Pathology,
Vanderbilt University School of Medicine, Nashville, Tennessee
37232.
Departments of Pathology, Metropolitan Nashville General
Hospital and Vanderbilt University School of Medicine,
Nashville, Tennessee
found in the sputum of patients studied for pulmonary
masses. Amebic abscess is then a distinct possibility and
needs consideration.
E. gingivalis is not encountered frequently in sputum.
During the year of 1981, with 646 sputa examined, our
Cytopathology Service registered only one case, the one
described in this report. Recent statistics concerning the
occurrence of this parasite in sputa were not found in
the medical literature. There were, however, studies
"dealing with the high frequency in the general population of different countries, including the United States.4'6,8
These studies were done almost 20 years ago. Therefore,
we decided to conduct a survey to assess the present
frequency of this particular amoeba in our area.
Survey Material and Methods
During the two months from mid-July through midSeptember 1981, 113 patients at the Oral Surgery Clinic
were examined for the presence ofE. gingivalis. A sterile
swab dipped in sterile saline was rubbed around the gingival line and between the teeth of each patient before
the dental procedure began. The swab then was rolled
on a glass slide, which immediately was placed in 95%
ethanol. Patients with tooth extractions had additional
slides made from the removed teeth. All slides were
stained by the Papanicolaou progressive method 7 with
EA-50 counterstain and examined for the presence of
amoeba. Each patient had at least one, and as many as
four, slides. The amoeba were confirmed by at least two
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BRIEF SCIENTIFIC REPORTS
Vol. 80 • No. 3
Table I. Occurrence of E. gingivalis
in Different Studies
Authors
Number of Cases
Per Cent Positive
Wantland and
Wantland(1960)
Jaskoski(1963)
700
39%
Patients—240
Controls—265
367
Patients—113
Controls—96
61%
42%
37%
59%
32%
Carneri (1964)
Daoelal. (1981)
observers. All questionable specimens were recorded as
negative. As controls, a group of 96 healthy persons associated with the hospital were swabbed and the slides
examined similarly.
Results
Of the 113 patients examined for amoeba, 67 (59%)
were positive. Of the 96 controls with good oral con-
381
dition, 31(32%) were positive. Table 1 compares our
results with those of other investigators.
Discussion
Our interest in this problem arose because of the following case. A 54-year-old man was admitted for evaluation of a pulmonary lesion discovered on chest roentgenogram. His teeth were in poor condition. Cytologic
examination of the sputum revealed atypical cells and
several amoebic trophozoites (Fig. 1) later identified as
E. gingivalis. Biopsy from the lung lesion showed a
poorly differentiated carcinoma.
Only the trophozoite of E. gingivalis is known (Fig.
2). It ranges from 12-30 nm in diameter. The granular
endoplasm has numerous food vacuoles containing degenerated, dark-staining nuclei and nuclear fragments
from epithelial cells, lymphocytes, and polymorphonuclears. Bacteria occasionally are seen. The nucleus is
FIG. 1 (left). E. gingivalis trophozoite in sputum smear, exhibiting the characteristic Entamoeba nucleus (arrow).
Papanicolaou method with EA-50 counterstain (XI,000).
FIG. 2 (center). A group of three trophozoites in buccal smear, with debris in the cytoplasm and a barely visible nucleus
in the amoeba marked by arrow. (Papanicolaou method with EA-50 counterstain (X 1,000).
FIG. 3 (right). The trophozoite cytoplasm (arrow) is loaded with nuclear fragments,
completely masking the amoebic nucleus. (Papanicolaou method with EA-50 counterstain (X 1,000).
DAO, ROBINSON, AND WONG
382
Table 2. Clinical Conditions Associated
with E. gingivalis
Clinical Conditions
Number of
Cases
Dental caries
Periodontal disease
Mandibular deformities
Oral abscess
Extraction of wisdom tooth
34
11
9
8
5
51%
17%
13%
12%
7%
Total
67
100%
Percentage
spheric and is smaller than that of E. histolytica. The
nuclear membrane is thick, and the central or sometimes
eccentric karyosome is surrounded by a granular halo.
The nucleus stains faintly with chromatin stains.
Morphologically, E. gingivalis is similar to E. histolytica. The major differences we noted is the presence
of ingested nuclear material in the cytoplasm of E. gingivalis. In many instances, these inclusions, which
stained intensely with basic dyes, were so numerous that
they completely masked the amoebic nucleus (Fig. 3).
E. histolytica, however, is more selective; only red blood
cells are found within the cytoplasmic mass.
A cystic form of E. gingivalis has not been described.
This stage of development is obviated because the organism can be transmitted among individuals by direct
contact.
The frequency of E. gingivalis varies with the studies
but remains consistently high in populations of dental
patients. Wantland and Wantland10 found 39% in 700
subjects. Jaskoski6 found 61% in a group of 240 patients
with poor oral hygiene and 42% in 265 students with
good oral condition. Levine9 quoted an occurrence as
high as 62% in Budapest. Carneri4 found E. gingivalis
in only 37% of 367 women in Italy.
Our own results ranged from 59% in dental patients
to 32% in healthy controls. These results are similar to
those reported earlier. They have remained high, despite
recent efforts to improve oral hygiene and reduction of
dental caries by fluoridation during the last two decades.
The culture method was used for all other studies
cited, but ours employed only the swab technic with
direct smear on a glass slide. Cultures are both expensive
and time consuming.
Earlier studies implicated E. gingivalis as the agent
of gingival pyorrhea.12 It appears now that this organism
is acting as a scavenger, living in crevices between the
teeth and gum to feed on food particles and cellular
materials collected in these areas. This concept correlates well with the frequent occurrence of the organism
in people with poor dental condition as well as the absence of disease when the amoeba is found in persons
with healthy teeth.
A.J.C.P. • September 1983
Table 2 lists the conditions associated with E. gingivalis in our patients. The highest rate of infestation is
found in people with carious teeth in need of extraction
(51%). The next highest rate is with periodontal
disease (17%).
Despite its relatively high frequency in this country,
E. gingivalis rarely is found in cytologic examinations
of sputum. This absence may be due to the fact that this
organism lives in recesses between the teeth and gingival
tissue and will not appear in the sputum unless the sputum is contaminated massively by saliva and dental debris. Another reason is that E. gingivalis is relatively
unknown to cytologists and may be confused with other
cells when found in a sputum smear.
From the cytologic standpoint, E. gingivalis could be
mistaken easily for a macrophage, because of the ingested debris in the cytoplasm of the macrophage. Careful observation of the nucleus will help make the distinction: the nucleus of the macrophage is larger, often
bean shaped, and does not possess the central karyosome
surrounded by a halo. Multinucleated histiocytes or epithelial cells may cause some problems, but the nuclei
in these cells are approximately the same size and possess
a definite chromatin pattern, as opposed to the intensely
stained, often irregular, nuclear fragments found in E.
gingivalis.
Cannibal cells found in cases of cancer of the respiratory tract represent phagocytosis of cancer cells by
their fellows. In this instance, whole cells are engulfed
and not only nuclear fragments. Atypical features often
are detected in nuclei of both phagocytizing and phagocytized ceils.
Plant cells possess numerous cytoplasmic granules
that may be mistaken for ingested nuclear debris. However, often they are found in clusters with large, dark,
irregular, homogeneous nuclei, mimicking tumor cells
more than amoebae.
Bronchial cells in cases of viral pneumonia may exhibit nuclear enlargement and nucleolar prominence,
bearing some resemblance to the amoebic nuclei. Their
cytoplasmic masses remain homogeneous, however, devoid of ingested foreign debris.
Acknowledgments. The generous help of Charles R. Means, DDS,
and Martin G. Netsky, M.D. who reviewed the manuscript gratefully
is acknowledged.
References
1. Barrett MT: The protozoa of the mouth in relation to pyorrhea
alveolaris. Dent Cosmos 1914; 56:948-953
2. Bass CC, Johns FM: Pyorrhea dentalis and alveolaris. Specific
cause and treatment. JAMA 1915; 64:553-558
3. Belding DL: Textbook of parasitology. Third ed. New York, Appleton-Century-Crofts, 1965, pp 98-99
Vol. 80 • No. 3
BRIEF SCIENTIFIC REPORTS
383
Chicago, American Society of Clinical Pathologists, 1977; pp
4. de Carneri I, Giannone R: Frequency of Trichomonas vaginalis.
301-302
Trichomonas tenax and Entamoeba gingivalis in Italian women.
8. Kofoid CA: The protozoa of the human mouth. J Parasitol. 1929;
Am J Trop Med Hyg 1964; 13:261-264
15:151-174
5. Goodey T, Wellings AW: Observations on E. gingivalis from the
9. Levine ND: Protozoan parasites of domestic animals and of man.
human mouth. Parasitology 1917; 9:537-559
Second ed. Minneapolis, Burgess Publishing Co, 1975
6. Jaskoski BJ: Incidence of oral protozoa. Trans Am Microbiol Soc
10. Wantland WW, Wantland EM: Incidence, ecology and reproduc1963;82:418-420
tion of oral protozoa. J Dent Res 1960; 39:863
7. Keebler CM, Reagan JW: A manual of cytotechnology. Fifth ed.
Inability of Counterimmunoelectrophoresis to
Detect Echovirus in Cerebrospinal Fluid
KENNETH BROMBERG, M.D., PETER R. SHANK, PH.D., STEPHEN H. ZINNER, M.D.,
AND GEORGES PETER, M.D.
Methods for rapid detection of viral antigens in cerebrospinal
fluid (CSF) are needed to aid in the differentiation of viral from
bacterial meningitis. The formation of precipitin bands in patients with suspect viral meningitis utilizing viral antisera in
a counterimmunoelectrophoresis (CIE) system has been described. To investigate further the possible value of CIE in the
diagnosis of viral meningitis, the specificity of the CSF precipitin bands was studied. Precipitin bands were formed between commercially available type-specific antisera and cell
culture supernatant fluids. Precipitin bands were also formed
when control CSF was used as an antigen. Using type-specific
antisera produced against purified virus, enteroviral antigens
were not detected in CSF from patients from whom CSF viruses
had been isolated. CIE lacks sufficient sensitivity for the detection of echovirus 11 antigens in CSF. (Key words: Counterimmunoelectrophoresis; Echovirus; Cerebrospinal fluid; Viral
meningitis) Am J Clin Pathol 1983; 80: 383-385
DEFINITIVE DIAGNOSIS of enteroviral meningitis
requires cell culture or mouse innoculation for the isolation of virus from the cerebrospinal fluid (CSF). Because results of viral isolation may not be available until
one to two weeks after the onset of the patient's illness,
methods for rapid detection of viral antigens in CSF are
needed to aid in the differentiation of viral from bacterial
meningitis. The feasibility of developing such techniques
is suggested by the ultimate recovery of coxsackie and
echoviruses in as many as 60% of patients whose spinal
fluid is cultured in the first few days of their illness.4,6
Brown et al.' recently described the formation of precipitin bands in 79% of samples from patients with sus-
Department of Medicine and Pediatrics, Rhode Island
Hospital; Department of Medicine, Roger Williams General
Hospital, and Sections of Pediatrics, Medicine, and
Microbiology, Division of Biology and Medicine, Brown
University, Providence, Rhode Island
pected viral meningitis utilizing a battery of viral antisera in a counterimmunoelectrophoresis (CIE) system.
This work suggested that CIE may be useful in the rapid
detection of viral antigens for the early diagnosis of viral
meningitis. However, in that study, precipitin bands
were also detected in 19% of patients with bacterial
meningitis and in 4% of patients whose CSF was not
consistent with infection. In those cases where enteroviruses were isolated from CSF, the antigens detected
were not necessarily consistent with the virus type isolated by cell culture.
To investigate further the possible value of CIE in the
diagnosis of viral meningitis, the specificity of the CSF
precipitin bands was studied. Four different antisera
were tested against the supernatant fluid from echovirus
11 infected tissue culture as well as CSF from control
patients and patients with documented echovirus 11,
echovirus 24, or bacterial meningitis.
Materials and Methods
Cerebrospinal Fluids
Received December 8, 1982; received revised manuscript and accepted for publication January 31, 1983.
Supported by a grant from the Rhode Island Foundation.
Address reprint requests to Dr. Bromberg: Department of Pediatrics,
Division of Infectious Diseases; B-6 King County Hospital, 451 Clarkson Avenue, Brooklyn, New York 11203.
Aliquots of CSF from 12 patients with culture-proven
echovirus 11 meningitis, 3 patients with echovirus 24
meningitis, 10 patients with bacterial meningitis (5.
pneumonia 4, N. meningitidis 3, H. influenzae type b
3), and 20 patients without known infection (in whom
0002-9173/83/0900/0383 $00.95 © American Society of Clinical Pathologists