566
ADENOVIRUS
AND
INTUSSUSCEPTION
may be dangerous.’ Bacterial agglutination ti-ters generally become positive from 14 to 31
days after infections; an agglutination titer of
1 :80 or greater is considered diagnostic.8
Sero-logic crossreactions occur with brucella, pro-teus OX-19 and heterophil antigens.6 Direct immunofluorescence of biopsied or aspirated material is highly specific, and since it depends neither on seroconversion nor viable organisms, allows the early initiation of antimicrobial
ther-2 Dermal hypersensitivity as measured by
the tularemia skin test occurs earlier than
sero-conversion.3 However, the intradermal skin test
antigen is not available for general use.
Streptomycin remains the drug of choice for
the treatment of tularemia.8 Prompt clinical re-sponse is expected only if therapy is initiated within the first two weeks of illness.
MARSHALL E. BLOOM,
M.D.
WILLIAM
T.
SHEARER, M.D., PH.D. LESLIEL.
BARTON,M.D.
Edward
Mallinckrodt
Department of Pediatrics
Washington University School of Medicine Division of Infectious Diseases
St. Louis Children’s Hospital
St. Louis, Missouri
Dr. Shearer is the recipient of United States
Pub-lic Health Special Research Fellowship
1-FO3-A153856-01 from the National Institutes of Allergy
and Infectious Diseases.
We are deeply indebted to Ms. B. Pittman at the Center for Disease Control, Atlanta, Ceorgia, who
performed the cultural and immunofluorescent
studies of the l)i01)Sied lymph node. Dr. J. Kissane
reviewed the bio1)sy specimens. Dr. P. R. Dodge
critically reviewed the manuscript.
ADDRESS FOR REPRINTS:
(
L.L.B.)
500 South Kingshighway, St. Louis, Missouri 63110.REFERENCES
1. Brooks, C. F., and Buchanan, T. M.: Tularemia
in the United States: Epidemiologic aspects
in the 1960s and follow-up of the outbreak of
tiilaremia in Vermont. J. Infect. Dis., 121:
357, 1970.
2. White, J. D., and McCavran, M. H. :
Identifica-tion of Pasteurella tularensi.c by
immunofluo-rescence. JAMA, 194:180, 1965.
3. Buchanan, T. M., Brooks, C. F., and Brachman, P. S. : The tularemia skin test. 325 skin tests
in 210 persons: Serologic correlation and
re-view of the literature. Ann. Intern. Med., 74:
336, 1971.
4. Hughes, W. T., and Etteldorf, J. N. :
Orophar-vngeal titlaremia. J. Pediat., 51:363, 1957.
5. Hughes, W. T. : Tularemia in children. J. Pe-diat., 62:495, 1963.
6. Levy, H. B., Webb, C. H., and Wilkinson,
J. D. : Tularemia as a pediatric problem. PEDI-ATRICS, 6:113, 1950.
7. Meyer, K. F. : Pasteurella infections. Pediat.
Clin. N. Amer., 2:3, 1955.
8. Hughes, W. T. : Oculoglandular tularemia:
transmission from rabbit, through dog and
tick to man. PEDIATRICS, 36:270, 1965.
9. Overholt, E. L., Tigertt, W. D., Kadull, P. J., Ward, M. K., Charkes, N. D., Rene, R. M.,
Salzman, T. E., and Stephens, NI. : An
analy-sis of forty-two cases of laboratory-acquired
tularemia. Amer. J. Med., 30:785, 1961.
Electron
Microscopic
Demonstration
of
Adenovirus
in Appendix
Vermiformis
in a Case
of
Ileocecal
Intussusception
Since the isolation of adenovirus from
hyper-trophied adenoidsl and from mesenteric lymph nodes of nonspecific lymphadenitis, adenovirus
has been isolated frequently from children with
intussusception38 and has been thought to play
a role in some cases of intussusception in child-hood by increasing bowel motility and produc-ing hyperplasia of the lymphoid tissue of the intestine. Adenovirus has also been recovered from the appendices of children with acute
ap-pendicitis.1#{176} However, as far as we know, the
virus has not been visualized in any of these sites. In a case of intussusception we found in-tranuclear inclusion bodies in appendiceal epi-thelial cells. Using the routine histologic section
of the appendix containing the inclusion bodies
we were able to demonstrate under the electron microscope viral particles morphologically
iden-tical to those of adenovirus.
CASE REPORT
A 3A2-year-old white boy began to have
inter-mittent colic in the evening on January 2, 1971.
The pain became regular, occurring every 20 to 30 minutes. There was no vomiting or fever. He was
brought to the emergency room 20 hours after the
onset of the symptoms. He had a normal stool on
the day of admission. Physical examination
re-vealed a well-developed and well-nourished boy in
no acute distress. The temperature was 37.2C.
There were no signs of an upper-respiratory tract
infection. The bowel sounds were normal. The
spleen and liver were palpable 1.0 cm below the
left and right costal margins and there was some
EXPERIENCE AND REASON-BRIEFLY RECORDED 567
FIG. 1. The appendiceal mucosa shows focal re-generation of the surface epithelium and groups of
desquamated epithelial cells. Hyperplasia of the
lymphoid tissue is evident
(
hematoxylin-eosinstain, reduced to x83 from X 128).
hemoglobin was 12.2 gm/100 ml; hematocrit 36%; white blood cell count 11,400/cu mm with 70
neu-trophils, 27 lymphocytes, and 3 monocytes. The
abdominal roentgenograms suggested the presence
of a mass on the right side. Roentgenograms with
barium enema revealed a typical ileocecal intussus-ception extending to the hepatic flexure. Reduction of intussusception was accomplished without
diffi-culty by barium enema, using three feet of
hydro-static pressure. Following this procedure his
abdo-men became soft. During the afternoon of January 3, the abdominal pains recurred, at which time he cried out in pain; hyperactive bowel sounds were
s1;iwrif
‘#{248},
S
FIG. 2. A higher magnification of desquamated
epithelial cells shows intranuclear inclusion bodies
with
(
large open arrow)
or without(
large solidarrow
)
halos, and “smudge cells”(
small arrow)(
hematoxylin-eosin stain, reduced to X400 fromx
640).heard. The white cell count was virtually
sin-changed. On the evening of January 3, a
laparot-omy revealed edema in the ileocecal area but
nei-ther intussusception, nor a Meckel’s diverticulum
was found. The mesenteric lymph nodes were
en-larged. An appendectomy was performed, and two
mesenteric lymph nodes were excised. The
postop-erative course was characterized b intermittent
fe-ver up to 39.9C until the fifth postoperative day.
He was given intravenous fluid and aspirin. The
fever was thought to be due to a viral infection on
the basis of the demonstration of intranuclear
in-clusion bodies in the appendix. He was discharged on the seventh postoperative day.
METHODS
The appendix was fixed in formaldehyde solu-tion ( Formalin ),embedded in paraffin and
sec-tions were stained with hematoxylin and eosin
for light microscopy. For electron microscopy,
the hematoxylin and eosin stained slide in which intranuclear inclusion bodies were seen was
uti-lized according to the method described by
Pinkerton
and
Carroll.hl In this technique, the cover slip is removed and the slide rinsed inmixtures of xylol and propylene oxide. The
tis-sue is covered with Epon-araldite and placed in the oven. Upon hardening, the tissue becomes attached to the sheet of Epon-araldite and is re-moved from the slide by prying with a razor blade. The desired area is cut out under the
microscope, attached to the tip of a Beem cap-sule block of Epon-araldite and sectioned for
electron microscopy. \Ve resorted to this
tech-nique after futile attempts to demonstrate
in-clusions in preparations from the wet
formalde-hyde-fixed specimen or from paraffin sections of
the appendix adjacent to those in which the
inclusions were seen.
RESULTS
The appendix was not remarkable grossly.
Microscol)ically, the mucosa showed a marked
hvperplasia of lvmphoid tissue with active
ger-minal centers. Focal regeneration of the surface
epithelium was seen in the mucosa adjacent to
a few groups of desquamated epithelial cells
(Fig. 1). In these areas two types of cytologic
changes were observed ( Fig. 2 ). A few nuclei
contained eosinophilic intranuclear inclusion
l)odies surrounded by halos and distinct nuclear
membranes. In some, eosinophilic inclusion
bod-ies displaced the chromatin to the periphery
and filled the entire nuclei. Some cells (“smudge
homogene-568
ADENOVIRUS
AND
INTUSSUSCEPTION
ously basophilic nuclei without visible nuclear
membrane and with scant eosinophiic
cyto-plasma (Fig. 2) . Two mesenteric lymph nodes
showed hyperplasia of lymph follicles with
ac-live germinal centers but no inclusions were
found. By electron microscopy, crystalline
ar-rays of viral particles identical to those of
adeno-virus’2 were demonstrated in both types of
in-clusion bodies (Fig. 3) . Many nuclei were
al-most entirely filled
by crystalline
arrays ofuni-form particles, but smaller groups of virions were also found in the cytoplasm of some cells.
The virions appeared round to polygonal in shape, and averaged 67 mu in greatest dimen-sion, and also by center-to-center measurements of contiguous virions. The dense central core, the nucleoid, was surrounded by an outer coat, the capsid. In several places fibrillar structures appeared to be attached to the angles of the capsid (Fig. 4) possibly representing the fibers at the apex of the penton in the adenovirus icosahedron.
DIscussIoN
The etiology of intussusception in younger
children is obscure in the majority of cases. Demonstrable causes, such as a Meckel’s
diver-ticulum, a neoplasm or an ectopic pancreatic
nodule have been described in only 2% to 8% of the cases at laparotomy.13 Im more than 90% of
cases, intussusception begins at the ileocecal
valve or in the distal end of the ileum and
char-acteristically affects children under two years of age.’3 Many etiologic factors have been de-scribed. These include adenovirus infection,3’’8 hyperplasia of the lymphoid tissue in the ileum, the greater disproportion between the caliber of
the terminal ileum and cecum in infants’s and dietary habits of infants) It is most likely that more than one etiologic factor is involved in the pathogenesis of intussusception . Adenovirus
in-fection may contribute to the occurrence of
in-tussusception l)y either altering the intestinal
motility or producing hyperplasia of lymphoid
tissue of the ileum amid mesentery, or by both.
Much interest has centered on the isolation of adenovirus from children with intussuscep-tion,35 mesenteric adenitis and acute
appendi-citis.mm Adenoviruses have been isolated from
stools, throat swabs,’ and mesenteric
lymph nodes3 of children with intussusception. In well-controlled studies, adenoviruses were isolated in 65%, 68%, amid 26% of cases of intus-susception, whereas adenovirus isolation rate in
FIG. 3. Electron micrograph of an appendiceal
epithelial cell showing a portion of a nucleus
con-taming crystalline arrays of viral particles. The
chromatin is displaced to the nuclear membrane
(
arrow)
. A few virions are present in the cytoplasm (C), (reduced to x41,000 from x74,000).control groups ranged from 2.7% to
Adeno-virus was isolated from mesenteric lymph nodes in 28% of cases of non-specific mesenteric
adeni-tis.’ Serotypes of adenoviruses recovered from
cases of intussusception and controls included
types 1, 2, 3, 5, 6, and 7; the types 1, 2 and 5
comprising more than 85% of adenoviruses re-covered.
Even though adenoviruses have been
iso-lated from cases of intussusception, the etiologic
significance of the adenovirus in such situations
has been questioned because of the prevalence
of inapparent infections by this virus.
Adeno-virus infections are common in young
chil-dren16 and adenoviruses types 1, 2, 5, and 6
FIG. 4. A higher magnification of the virions shows
a dense core, the nucleoid, surrounded by an outer
coat, the capsid. Fibrillar structures appear to be
attached to the angle of the capsid
(
arrow heads),EXPERIENCE
AND
REASON-BRIEFLY
RECORDED
569
are particularly endemic in communities, asso-ciated with inapparent infections or mild upper
respiratory illnesses. Adenoviruses types 1, 2,
and 5 have also been isolated from 26% of
ton-sils and
46%
of adenoids removed forhyper-trophy.19 Furthermore, adenovirus infections
are frequently followed by a prolonged excre-tion of the virus, as long as over 900 days.18 In a significant number of cases3’5 17 serologic studies have indicated that at
the
time ofintus-susception there was evidence of concurrent or
recent infection by adenovirus. In addition, chil-dren with intussusception showed less serologic
evidence of previous infections with
adeno-viruses type 1, 2, 5, and 6 in the acute phase serum, indicating the susceptibility of children
with intussusception to these types of viruses.17
Results of adenovirus isolation from children
with acute appendicitis have been
inconsis-tent.5’9’10’2#{176}In one series of 20 children with acute appendicitis who also had concurrent
pharyngitis and mesenteric adenitis, adenovirus
type 1 or 6 was isolated in 6 of 20 appendices.#{176} However, in a well-controlled study, no
adeno-virus was isolated from 51 appendices removed
from children with acute appendicitis and only 1 of 44 mesenteric lymph nodes yielded
adeno-virus.20
The ubiquity of adenovirus has made it
diffi-cult to assess the role of the virus in the
situa-lions in which it is found. Although no viral
cul-hire was done in our patient,
the demonstration
of intranuclear inclusions containing viral
parti-des identical to those of adenovirusil indicated
that an adenovirus infection was present in the appendix and probably also in the terminal ileum when the intussusception occurred.
In spite
of extensive
virologic,
serologic
and
epidemiological studies of adenovirus infection, morphologic studies of adenovirus infection in humans are largely limited to adenovirus pneu-monia.11’21 Necrotizing bronchiolitis and pneu-monia with two types of intranuclear inclusion
bodies have been considered diagnostic of adenovirus infection. The two types of intranu-clear inclusion bodies, namely eosinophilic
in-clusions with or without halos and the so-called
“smudge cells” showing homogeneous basophilic
inclusions were present in our case. The
demon-stration of intranuclear inclusion bodies in
viro-logically documented adenovirus infections has
been reported in the liver,22,23 pancreatic acini,23
peribronchial lymph node,23 esophageal mucous
gland,24 mucosa of the tongue25 and brain,26
but to
our
knowledge, no inclusion bodies have been demonstrated in intussusception, mesen-teric adenitis or adenovirus enteritis. Our studysuggests that intranuclear inclusion bodies may
be found if a diligent search is made in the latter cases As shown here, when suitable tis-sues are not available for electron microscopy
or isolation of virus, a routine histologic section
can be used for the demonstration of viral
parti-des.
SUMMARY
Viral particles identical to those of adeno-virus
were
demonstrated
in the intranuclear
in-clusion bodies of the epithelial cells of an ap-pendix vermiformis removed from a boy with a recent history of intussusception. Viral particles were demonstrated by electron microscopy
fol-lowing
reprocessing
of
a formaldehyde-fixed,
paraffin embedded and hematoxylin-eosin stained section. This simple method permits
pre-cise selection of
the
tissue
for electron
micro-scopy and in instances such as this givessuffi-cient tissue preservation to demonstrate viral
particles. Our findings support the idea
that
some cases of intussusception may be the result
of the adenovirus infection.
EDUARDO
J.
YUNIS,M.D.
YOSHIE HASHIDA, M.D.
Department of Pathology
Children’s Hospital of Pittsburgh
School of
Medicine
of the
University
of
Pittsburgh
Pittsburgh, Penneylvanki
Mr. Rocco Agostini and Mrs. Janet Walpusk gave technical assistance. This work was supported by Crant 5-S01-FR-05507 and by the Junior
Com-mittee, Children’s Hospital of Pittsburgh.
REFERENCES
1. Rowe, W. P., Huebner, R. J., Gilmore, L. K.,
Parrott, R. H.,
and
Ward, T. C.: Isolation ofa cytopathogenic agent from human
ade-noids undergoing spontaneous degeneration in tissue culture. Proc. Soc. Exp. Biol. Med., 84:570, 1953.
2. Kjell#{233}n,L., Sterner, C., and Svedmyr, A. : On
the occurrence of adenoviruses in Sweden. Acta Paediat. Scand., 46: 164, 1957.
3. Cardner, P. S., Knox, E. G., Court, S. D., and Green, C. A. : Virus infection and
intussus-ception in childhood. Brit. Med.
J.,
2:697,1962.
4. Ross,
J.
C., Potter, C. W., and Zachary, R. B.:in-tussusception in infancy. Lancet, 2:221,
1962.
5. Bell, T. M., and Steyn, J. H. : Viruses in lymph nodes of children with mesenteric adenitis and intussusception. Brit. Med. J., 2:700, 1962.
6. Potter, C. W. : Adenovirus infection as an aeti-ological factor in intussusception of infants and young children. J. Pathol. Bact., 88:263,
1964.
7. Clarke, E. J., Jr., Phillips, I. A., and Alexander, E. R. : Adenovirus infection in intussuscep-tion in children in Taiwan. J.A.M.A., 208:
1671,
1969.
8. White, D. 0., and Solomon, J. R.: Adenovirus and intussusception. Med. J. Aust., 1:447, 1966.
9. Kulcs#{225}r,C., Vutskits, Z., N#{225}sz,I., Dan, P., and L#{233}b,J.: Viruses isolated from appendicitis cases in childhood. Zbl. Bakt. (Orig. ), 215:
506, 1970.
10. Bonard, E. C., and Paccaud, M. F. : Abdominal
adenovirosis and appendicitis. Helv. Med.
Acta. 33:164, 1966.
11. Pinkerton, H., and Carroll, S.: Fatal
adenovi-rus pneumonia in infants. Correlation of
his-tologic and electron microscopic observa-tions. Amer. J. Path., 65:543, 1971.
12. Davis, B. D., Dulbecco, R., Eisen, H.,
Cins-berg, H. S., and Wood, W. B., Jr. :
Micro-biology. New York: Harper & Row, p. 1222, 1967.
13. Ravitch, M. M. : Intussusception. In Mustard,
W. T., Ravitch, NI. M., Snyder, W. H., Jr.,
Welch, K.
J.,
and Benson, C. D., eds. :Pedi-atric Surgery, ed. 2. Chicago: Year Book
Medical Publishers Inc., p. 917, 1969.
14. COmes,
J.
S., and Dawson, I. M. : Papillarylymphoid hvperplasia at the ileocaecal valve
as a cause of acute intussusception in
in-fancy. Arch. Dis. Child., 38:89, 1963. 15. Knox, E. C., Court, S. D., and Cardner, P. S.:
Aetiology of intussusception in children.
Bnt. Med. J., 2:692, 1962.
18. Jordan, W. S., Jr., Badger, C. F., and Dingle,
J.
H. : A study of illness in a group of Cleve-land families: XV. Acquisition of type-spe-cific adenovirus antibodies in the first 5 years of life-Implications for the use ofadeno-virus vaccine. N. Eng. J. Med., 258:1041,
1958.
17. Potter, C. W., Shedden, W. I., and Zachary,
R. B.: A comparative study of the incidence
of adenovirus antibodies in children with
intussusception with that in a control group.
J. Pediat., 63:420, 1963.
18. Spigland, I., Fox, J. P., Elveback, L. R.,
Was-sermann, F. E., Ketler, A., Brandt, C. D.
and Kogon, A.: The virus watch program : A
continuing surveillance of viral infections in
metropolitan New York families. II. Labora-tory methods and preliminary report on
in-fections revealed by virus isolation. Amer. J.
Epidem., 83:413, 1966.
19. Israel, M. S. : The viral flora of enlarged tonsils and adenoids. J. Path. Bact., 84: 169, 1962. 20. Jackson, R. H., Cardner, P. 5., Kennedy,
J.,
and McQuillin, J.: Viruses in the aetiology of acute appendicitis. Lancet, 2:711, 1966. 21. Becroft, D. M. : Histopathology of fatal
adeno-virus infection of the respiratory tract in
young children. J. Clin. Path., 20:561, 1967.
22. Benyesh-Melnick, M., and Rosenberg, H. S.:
The isolation of adenovirus type 7 from a
fatal case of pneumonia and disseminated
disease. J. Pediat., 64:83, 1964.
23. Wigger, H. J., and Blanc, W. A. : Fatal hepatic
and bronchial necrosis in adenovirus infec-tion with thymic alymphoplasia. N. Eng.
J.
Med., 275:870, 1966.24. Chany, C., L#{233}pine, P., Lelong, M., Le-Tan-Vinh, Satg#{233},P., and Virat, J.: Severe and fa-tal pneumonia in infants and young children associated with adenovirus infections. Amer.
J. Hygiene, 67:367, 1958.
25. Kawai, K. : Pathology and pathologic anatomy
of adenovirus infection. Based on three
au-topsy cases of infantile pneumonia. Jap. J.
Exp. Med., 29:359, 1959.
26. Chou, S. M., Burrell, R., Harley,
J.,
Cutmann,L., and Roos, R. : Subacute focal advenovi-n’s encephalitis. (Abstract.
) J.
Neuropath. Exp. Neurol. 31:173, 1972.The
Response
of Leukocytes
in the
Peripheral
Blood
During
and
Following
Exchange
Transfusion
in
the
Newborn
The red cell picture of the blood1 and
eryth-ropoiesis,2 following exchange transfusion, have
been studied. Very little is known, however, about the white blood cell response to this pro-cedure. There appears to be only one study by Phibbs3 who reported on the changes, mainly in the total white blood cell count, in the pe-ripheral blood of 16 jaundiced babies during exchange transfusion and the five hours
follow-ing it.
This paper reports on the response of the white blood cells during exchange transfusion
in the newborn and for seven to nine days
thereafter.
MATERIAL AND METHODS
Twenty-seven exchange transfusions were
