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The isolation of adenovirus type 7 from a fatal case of pneumonia and disseminated disease
7"he Journal o[ P E D I A T R I C S
83
The isolation of adenovirus type 7from a fatal case ofpneumonia and disseminated disease The isolation o[ adenovirus type 7 in high titers from postmortem organs and serum from a 5-month-old girl with pneumonia and disseminated disease is reported. The clinical history and bacteriologic, pathologic, and virologic findings are presented. The morphologie differential diagnosis is discussed.
Matilda Benyesh-Melnick, M.D.,* and Harvey S. Rosenberg, M.D. HOUSTON,
TEXAS
T H E association of adenoviruses a n d atypical p n e u m o n i a of infancy a n d c h i l d h o o d has been d e m o n s t r a t e d in recent years. 1-1~ T h e clinical p a t t e r n in infants is suggestive of disseminated disease with localization in the lung. T h e r e m a y / be paroxysmal respiratory distress, central nervous system findings, gastrointestinal symptoms, acute nephritis, and a m o r b i l l i f o r m rash. T h e basic pathologic lesion is a c a t a r r h a l i n f l a m m a t i o n of the respiratory t r a c t with a generalized lymphoid h y p e r p l a s i a Y T h e r e also m a y be inclusions a n d b r o n c h i a l necrosis in the lung. Relatively few fatal cases of p n e u m o n i a , d o c u m e n t e d b y isolation of adenovirus f r o m
From the Department o/ Virology and Epidemiology; the Departments o[ Pathology and Pediatrics, Baylor University College of Medicine; and the Texas Children's Hospital, Houston, Texas. Aided by a research grant, CA-04600, from the National Cancer Institute. *Address, Department of Virology and Epidemiology, BayIor University College o[ Medicine, Houston, Texas 77025.
the p o s t m o r t e m material, have been described thus far in the literature. 2-4, s, 9 O f these only one has been described in the U n i t e d States. ~ W e recently h a d the o p p o r t u n i t y to study the m o r p h o l o g y of an i n f a n t with dissemin a t e d disease f r o m w h o m p o s t m o r t e m viral cultures revealed adenovirus t y p e 7. Because of the relative p a u c i t y of p r o v e d e x a m p l e s of fatal infections due to adenovirus, this r e p o r t is m a d e .
CASE REPORT The patient, a 5~-month-01d white female, was admitted to .the Texas Children's Hospital for evaluation of persistent feVer. She had been consistently ill from the time of birth with episodes of diarrhea from the age of 1 week until several weeks prior to admission. At about 2 months of age, she developed rhinorrhea and a't 4 months she developed bilateral otitis media. A skin infection occurred at the age of 4 months which had cleared by the time of admission with the exception of localized ulcerated areas on the right labia majora and to the right of the anus. The infant had been treated with sulfa, peni-
84
Benyesh-Melnick and Rosenberg
cillin, kanamycin, and chloramphenicol. A blood count several days prior to admission revealed a white count of 5,000 cells per cubic millimeter. Physical examination on admission revealed a pale, irritable infant with a temperature of 104; pulse rate, 140, respiratory rate, 40; blood pressure by the flush technique, 100; weight, 13 pounds, 14 ounces. The tympanic membranes were slightly injected bilaterally. The lungs were clear. The spleen was barely palpable. The liver was palpable 4 cm. below the right costal margin. Neurologic examination was normal. Laboratory data included a hemoglobin of 9.4 Gm. per 100 ml. with a hematocrit of 32 per cent. T h e white count was 1,200 with 32 polymorphonuclear leukocytes, 2 bands, 52 lymphocytes, and 14 monocytes. One nucleated red cell was seen per 100 white cells. The platelet count was 145,000, and the reticuIoeyte count was 0A per Cent. The red ceIts were hypochromic. A urinalysis revealed 100 mg. of protein per 100 ml. Serum pr~eins were 4.9 Gin. per 100 with albumin, 2.4 Gm.; alpha-1 globulin, 0.4 Gm.; alpha-2 globulin, 1 Gm.; beta globulin, 1 Gm.; and gamma globulin, 0.1 Gm. Other laboratory data included electrolytes which were normal. The cerebrospinal fluid revealed no abnormalities. Two blood cultures were negative.-A bone marrow biopsy was interpreted as histiocytic hyperplasia with maturation arrest of the granulocytes. Serum agglutinations for typhoid, paratyphoid, Brucellosis, and Proteus O X 19 were negative. X-rays of the skull and mastoids were interpreted as normal. A chest x-ray revealed in-
Fig. 1. An oval, intranuclear inclusion is present within a histiocyte (center of field) in an alveolar space of the lung. (I-Iematoxylin and eosin stain. Original magnificatior~ x500,)
January
1964
creased markings in the right perihilar region suggestive of mild bronchopneumonia. In the hospital she had persistent fever of between 101 and 104 ~. O n the fourth hospital day she developed an erythematous, elevated, multiform rash in a generalized distribution with a few vesicles. Some of the lesions were 2 to 2.5 cm. in diameter with an indurated center. Smear of the vesicular fluid revealed no organisms. On the night of the fourth hospital day, in the absence of an etiologic agent and with persistence of fever, the infant was started on penicillin and colistemethate sodium. The disease had gradually deteriorated through the hospital course and the patient died on the fifth hospital day. At postmortem examination there was a generalized erythernatous macular rash. Microscopically there was intra-epidermal edema with collections of bacteria but no inflammatory cell response. The lungs were poorly aerated with multiple areas of induration throughout both lungs. The trachea and bronchi were filled with a viscid mucoid secretion. There was extensive destruction of the pulmonary parenchyma with a dense intra-alveolar inflammatory response and focal areas of parenchymal necrosis. There was extensive destruction of the epithelium of the bronchi with an inflammatory infiltrate into the wall of these spaces. T h e lumens of the bronchi were filled with an eosinophilic debris, The pleura was thickened, and there was a diffuse mononuclear infiltrate with focal areas of hemorrhage. In the epithelial cells of the bronchi and within the large histiocytes in the air spaces, there were numerous intranuclear inclusions (Fig. 1). These were generally lightly basophilic and Feulgen positive. No inclusions were present in the cytoplasm. The liver was large and mottled with irregular congested areas. Microscopically, there were multiple areas of necrosis with a minimal mononuclear inflammatory response in these areas. Large numbers of intranuclear inclusions were encountered in the parenchymal cells. These inclusions were identical to those encountered in the lung. In the adrenal glands there were multiple focal areas of necrosis with large numbers of bacteria but no inflammatory cells; no inclusions were identified in the adrenal glands. No structural abnormalities or inclusions were present in the kidney. In the spleen there were vascular congestion and depletion of lymphoid follicles. The thymus was considerably depleted of lymphoid tissue and Hassall's corpuscles were
Volume 64 Number 1
not identified. There was a small amount of mucopurulent material within the middle ears bilaterally. There were no abnormalities in the central nervous system. A single isolated area of inflammation was present in the thyroid with a few mononuclear cells and a few bacteria, but no inclusions. VIRAL ISOLATIONS A serum sample ' obtained 2 days before the child died as well as portions of the kidney, lung, spIeen, and liver removed at autopsy were taken for viral isoIations. Part of the kidney was propagated in tissue culture, employing the techniques we use for detection of Intent viruses in postmortem organs of children,r' The remaining part of the kidney, the other organs, and serum sample were kept at -20 ~ C. until further tested. On the seventh day after seeding the kidney cells, when almost complete monolayers were present, all the cultures spontaneously showed cytopathic degeneration similar in appearance to that observed with adenoviruses. The cytopathic effect progressed very rapidly. On the eighth day the culture fluids were harvested. The agent was subcultured in human embryo kidney cultures and subsequently identified as adenovirus type 7 by means of hemagglutination and neutralization tests. The virus caused complete hemagglutination of rhesus monkey erythrocytes but failed to agglutinate rat erythrocytes, the pattern established for adenovirus types 3, 7, 11, 14, 16, 20, 21, 25, and 28. In neutralization tests in which pools of adenovirus xabbit antisera (types 1-1 I) were employed, the virus was completely neutralized by a pool containing adenovirus type 7 antiserum and failed to be neutralized by the pools which did not contain it. The virus was further neutralized by adenovirus type 7 antiserum alone.~ The same virus was success~ully isolated in human embryo kidney cuttures from 10 per cent suspensions of the frozen kidney, lung, spleen, and liver as well as from the serum. Upon electron microscopic examiuationt the serum revealed the presence of 81 m# adenovirus particles. Particles of the same type were also found in the virus-containing material obtained ~The identification of virus was perforhaed by Dr. Rosb,n Q. Robinson, Respi~ovirus Unit and International Influenza Center for the Americas, CDC, Atlanta, Ga., and confirmed by us. tPerformed by Dr. K. O. Smith, Department of Virology and Epldenfiology.
Fatal injection with adenovirus type 7
85
T a b l e I. Results of titrations of o r g a n suspensions a n d serum, with the e m p l o y m e n t of h u m a n embryo kidney cell cultures
Specimen
I TCDso per gram tissue
Kidney Lung Spleen Liver Serum *TCDro per milliliterof undilutedserum.
4.8 6.2 6.2 7.5 1.86
by passing the patient's specimens in tissue culture: The quantity of virus present in the different organs and the serum were determined by titrations in human embryo kidney cultures. Tenfold dilutions prepared from the 10 per cent organ suspensions and the serum were inoculated in 0.1 ml. amounts into each of four human embryo kidney cultures. The cultures were observed for eytopathic changes for 19 days. Maximum TCD~0 titers were obtained on the thirteenth day. From the results presented in Table I it is obvious that all the organs contained large quantities of virus, especially the liver which revealed 107-s TCDs0 per gram of tissue. The concentration of virus in the blood was relatively low. Unfortunately a postmortem blood sample was not available for viral examination. It is possible that part of the virus in the serum might have been neutralized by circulating antibody. Because of the lack of sufficient quantity of serum no attempts were made to determine the presence of antibodies. DISCUSSION I n m a n y viral diseases the establishment of a n etiologic relationship b e t w e e n the virus isolated a n d the illness is of considerable difficulty, is I n the case reported here the large quantities of adenovirus type 7 f o u n d in the iung, kidney, spleen, a n d liver ( T a b l e I ) , t a k e n together with the presence of i n t r a n u c l e a r inclusions in the l u n g a n d liver, show that active virus infection was present in a disseminated form at the time of death. T h e clinical course was consistent with t h a t described by other investigators for epidemic s, s-10 o r sporadic 1, 2, 4 cases of adenovirus-associated p n e u m o n i a in children. T h e histopathologic findings also corresponded to those described for adenovirusassociated fatal pneumonias2-4, s, 9, 14 with
86
Benyesh-Melnick and Rosenberg
the exception of the extensive involvement of the liver in our case. This was also the tissue yielding the highest titer of virus (107.5 TCDs0 per gram). It is of interest that adenovirus type 5 has been recently found to cause fatal disease when inoculated into newborn hamsters. T h e only pathologic changes found were in the liver where there was nuclear pyknosis with clumping of the chromatin into basophilic masses surrounded by vesicular spaces. 1~ T h e viremia found 2 days prior to death in the case presented is in keeping with the rash and the disseminated character of the pathologic and virologic findings. Adenovirus types 4 and 7 viremia has been recently reported in this country in naval recruits with rubelliform illness during the acute stage of the disease. 1~ I t is difficult to say that th~ virus isolated in this case was responsible for the entire course of the disease from the time of infancy. It is possible that with the depletion of maternal antibodies, by the time the child had reached the age of 5 months, the initially depressed infection underwent a rapid dissemination. The possibility of a basic defect in immunity could not be completely discarded for there was a borderline level of serum g a m m a globulin (0.1 Gm. per 100) as well as a generalized lymphoid depletion. The absence of Hassall's bodies in the thymus of this girl is hard to interpret. This phenomenon has thus far been described only in sex-linked congenital agammaglobulinemia of the male. Morphologically, there are several pulmonary disorders associated with the presence of intranuclear inclusions. T h e most common is cytomegalic inclusion disease in its disseminated form or as an isolated pneumonia. Pulmonary necrosis and exudation are uncommon in cytomegalic inclusion disease although both m a y occur. T h e inclusion-bearing cells are usually free within the alveolar spaces but m a y be in the epithelial lining of the terminal air ducts. Large intranuclear, usually basophilic inclusions are encountered. Frequently, basophilic
January 1964
cytoplasmic inclusions are also encountered. Varicella may involve the lung when dis. seminated in the face of altered immunity or steroid therapy. Herpes simplex may result in disseminated inclusion disease in t h e neonate. From an architectural standpoint, there are several differences. Varicella pneumonitis occurs as an extension of the primary skin lesion. Herpes simpIex characteristically "involves tissues in a generalized distribution, particularly in those infants under 1 year of age. Temporal lobe encephalitis, hepatic necrosis with inclusions, and adrenal necrosis with inclusions are frequently encountered. In the present case there was no central nervous system involvernent. Hepatic necrosis with inclusions was present in this case. There were also localized areas of adrenal necrosis but without inclusions. On a cytologic basis it is difficult, if not impossible, to distinguish between the inclusions resulting from herpes simplex, from varicella, and from adenovirus. Isolation of the virus has been the only means of positive identification. SUMMARY
Adenovirus type 7 was isolated from a fatal case of pneumonia and disseminated disease. The virus was isolated in high titers from the kidney (104's TCDs0 per gram), lung (106"2), spleen (10~'2), and liver (10 ~'5) removed at autopsy as well as from a serum sample (10 ls per milliliter) obtained 2 days before the child died. Postmortem examination revealed extensive involvement of the lung and liver with Feulgen-positive intranuclear inclusions in both organs. No such inclusions were found in other organs..
REFERENCES
1. Sterner, G.: Atypical pneumonia in an infant, associated with APC virus infection, Acta paediat. 45: 449, 1956. 2. Pereira, H. G., and Kelly, B.: Studies on natural and experimental infections by adenovirus, Proc. Roy. Soc. 50: 755, 1957. 3. Chany, C., Lepine, T., Lelong, N., Le-TanVinh, Satge P., and Virat, J.: Severe and
Volume 64
4.
5. 6.
7.
8.
9.
Number 1
fatM pneumonia in infants and young children associated with adenovirus infections, Am. J. Hyg. 67: 367, 1958. Deinhardt, F., May, R. D., Calhoun, H. H., and Sullivan, H. E.: The isolation of adenovirus type 1 from a fatal case of viral "pneumonitis," A. M. A~ Arch. Int. Med. 102: 816, 1958. Kjellen, L., Sterner, G., and Svedmyr, A.: On the occurrence of adenoviruses in Sweden, Acta paediat. 46: 164, 1957. Sterner, G. L.: Infections w~th adenovirus type 7 in children and their relationship to acute respiratory disease, Acta paediat. 48: 287, 1959. TyrreI1, D. A. J., Balducci, D., and Zaiman, T. E.: Acute infections of the respiratory tract and the adenoviruses, Lancet 2: 1326, 1956. Jen, K. F.,. Tai, Y., Lin, Y. C., and Wang, H. Y.: The role of adenovirus in the etiology of infantile pneumonia and pneumonia compIicating measles, Chinese M. J. 81: 14t, 1962. Dreizin, R. S., Boldyreva, A. S., Isachenko, V. A., and Kniazeva, L. D.: Outbreaks 6f
Fatal injection with adenovirus type 7
10.
l l.
12. 13. 14.
15.
16.
87
adenovirus infections in Gorki, Problems of Virology 5: 196, 1960. Van Zaane, D. J., and Van der Veen, J.: Quelques symptomes cliniques particuliers chez les enfants atteints d'une infection a adenovirus, la Presse m6d. 70: t021, 1962. Rowe, W. T., and Huebner, R. J.: Present knowledge of the clinical significance of the adenoidal-pharyngeaI-conjunctival group of viruses, Am. J. Trop. Med. 5: 453-460, 1956. Benyesh-NIelnick, M., and Rosenberg, H.: (in preparation). Huebner, R. J.: The virologists' dilemma, Ann. New York Acad. Sc. 67: 430, 1957. Goodpasture, E. W., Auerbaeh, S. H., Swanson, H. S., and Cotter, E. F.: Virus pneumonia of infants secondary to epidemic infections, Am. J. Dis. Child. 57: 997, 1939. Pereira, H. G., Allison, A. C., and Niven, J. S. F. L.: Fatal infection of newborn hamsters by an adenovirus of human origin, Nature 196: 244, 1962. Gutekunst, R. R., and Heggie, A. D.: Virem!a and viruria in adenovirus infections, New England J. Med. 264: 374, 1961.
83
The isolation of adenovirus type 7from a fatal case ofpneumonia and disseminated disease The isolation o[ adenovirus type 7 in high titers from postmortem organs and serum from a 5-month-old girl with pneumonia and disseminated disease is reported. The clinical history and bacteriologic, pathologic, and virologic findings are presented. The morphologie differential diagnosis is discussed.
Matilda Benyesh-Melnick, M.D.,* and Harvey S. Rosenberg, M.D. HOUSTON,
TEXAS
T H E association of adenoviruses a n d atypical p n e u m o n i a of infancy a n d c h i l d h o o d has been d e m o n s t r a t e d in recent years. 1-1~ T h e clinical p a t t e r n in infants is suggestive of disseminated disease with localization in the lung. T h e r e m a y / be paroxysmal respiratory distress, central nervous system findings, gastrointestinal symptoms, acute nephritis, and a m o r b i l l i f o r m rash. T h e basic pathologic lesion is a c a t a r r h a l i n f l a m m a t i o n of the respiratory t r a c t with a generalized lymphoid h y p e r p l a s i a Y T h e r e also m a y be inclusions a n d b r o n c h i a l necrosis in the lung. Relatively few fatal cases of p n e u m o n i a , d o c u m e n t e d b y isolation of adenovirus f r o m
From the Department o/ Virology and Epidemiology; the Departments o[ Pathology and Pediatrics, Baylor University College of Medicine; and the Texas Children's Hospital, Houston, Texas. Aided by a research grant, CA-04600, from the National Cancer Institute. *Address, Department of Virology and Epidemiology, BayIor University College o[ Medicine, Houston, Texas 77025.
the p o s t m o r t e m material, have been described thus far in the literature. 2-4, s, 9 O f these only one has been described in the U n i t e d States. ~ W e recently h a d the o p p o r t u n i t y to study the m o r p h o l o g y of an i n f a n t with dissemin a t e d disease f r o m w h o m p o s t m o r t e m viral cultures revealed adenovirus t y p e 7. Because of the relative p a u c i t y of p r o v e d e x a m p l e s of fatal infections due to adenovirus, this r e p o r t is m a d e .
CASE REPORT The patient, a 5~-month-01d white female, was admitted to .the Texas Children's Hospital for evaluation of persistent feVer. She had been consistently ill from the time of birth with episodes of diarrhea from the age of 1 week until several weeks prior to admission. At about 2 months of age, she developed rhinorrhea and a't 4 months she developed bilateral otitis media. A skin infection occurred at the age of 4 months which had cleared by the time of admission with the exception of localized ulcerated areas on the right labia majora and to the right of the anus. The infant had been treated with sulfa, peni-
84
Benyesh-Melnick and Rosenberg
cillin, kanamycin, and chloramphenicol. A blood count several days prior to admission revealed a white count of 5,000 cells per cubic millimeter. Physical examination on admission revealed a pale, irritable infant with a temperature of 104; pulse rate, 140, respiratory rate, 40; blood pressure by the flush technique, 100; weight, 13 pounds, 14 ounces. The tympanic membranes were slightly injected bilaterally. The lungs were clear. The spleen was barely palpable. The liver was palpable 4 cm. below the right costal margin. Neurologic examination was normal. Laboratory data included a hemoglobin of 9.4 Gm. per 100 ml. with a hematocrit of 32 per cent. T h e white count was 1,200 with 32 polymorphonuclear leukocytes, 2 bands, 52 lymphocytes, and 14 monocytes. One nucleated red cell was seen per 100 white cells. The platelet count was 145,000, and the reticuIoeyte count was 0A per Cent. The red ceIts were hypochromic. A urinalysis revealed 100 mg. of protein per 100 ml. Serum pr~eins were 4.9 Gin. per 100 with albumin, 2.4 Gm.; alpha-1 globulin, 0.4 Gm.; alpha-2 globulin, 1 Gm.; beta globulin, 1 Gm.; and gamma globulin, 0.1 Gm. Other laboratory data included electrolytes which were normal. The cerebrospinal fluid revealed no abnormalities. Two blood cultures were negative.-A bone marrow biopsy was interpreted as histiocytic hyperplasia with maturation arrest of the granulocytes. Serum agglutinations for typhoid, paratyphoid, Brucellosis, and Proteus O X 19 were negative. X-rays of the skull and mastoids were interpreted as normal. A chest x-ray revealed in-
Fig. 1. An oval, intranuclear inclusion is present within a histiocyte (center of field) in an alveolar space of the lung. (I-Iematoxylin and eosin stain. Original magnificatior~ x500,)
January
1964
creased markings in the right perihilar region suggestive of mild bronchopneumonia. In the hospital she had persistent fever of between 101 and 104 ~. O n the fourth hospital day she developed an erythematous, elevated, multiform rash in a generalized distribution with a few vesicles. Some of the lesions were 2 to 2.5 cm. in diameter with an indurated center. Smear of the vesicular fluid revealed no organisms. On the night of the fourth hospital day, in the absence of an etiologic agent and with persistence of fever, the infant was started on penicillin and colistemethate sodium. The disease had gradually deteriorated through the hospital course and the patient died on the fifth hospital day. At postmortem examination there was a generalized erythernatous macular rash. Microscopically there was intra-epidermal edema with collections of bacteria but no inflammatory cell response. The lungs were poorly aerated with multiple areas of induration throughout both lungs. The trachea and bronchi were filled with a viscid mucoid secretion. There was extensive destruction of the pulmonary parenchyma with a dense intra-alveolar inflammatory response and focal areas of parenchymal necrosis. There was extensive destruction of the epithelium of the bronchi with an inflammatory infiltrate into the wall of these spaces. T h e lumens of the bronchi were filled with an eosinophilic debris, The pleura was thickened, and there was a diffuse mononuclear infiltrate with focal areas of hemorrhage. In the epithelial cells of the bronchi and within the large histiocytes in the air spaces, there were numerous intranuclear inclusions (Fig. 1). These were generally lightly basophilic and Feulgen positive. No inclusions were present in the cytoplasm. The liver was large and mottled with irregular congested areas. Microscopically, there were multiple areas of necrosis with a minimal mononuclear inflammatory response in these areas. Large numbers of intranuclear inclusions were encountered in the parenchymal cells. These inclusions were identical to those encountered in the lung. In the adrenal glands there were multiple focal areas of necrosis with large numbers of bacteria but no inflammatory cells; no inclusions were identified in the adrenal glands. No structural abnormalities or inclusions were present in the kidney. In the spleen there were vascular congestion and depletion of lymphoid follicles. The thymus was considerably depleted of lymphoid tissue and Hassall's corpuscles were
Volume 64 Number 1
not identified. There was a small amount of mucopurulent material within the middle ears bilaterally. There were no abnormalities in the central nervous system. A single isolated area of inflammation was present in the thyroid with a few mononuclear cells and a few bacteria, but no inclusions. VIRAL ISOLATIONS A serum sample ' obtained 2 days before the child died as well as portions of the kidney, lung, spIeen, and liver removed at autopsy were taken for viral isoIations. Part of the kidney was propagated in tissue culture, employing the techniques we use for detection of Intent viruses in postmortem organs of children,r' The remaining part of the kidney, the other organs, and serum sample were kept at -20 ~ C. until further tested. On the seventh day after seeding the kidney cells, when almost complete monolayers were present, all the cultures spontaneously showed cytopathic degeneration similar in appearance to that observed with adenoviruses. The cytopathic effect progressed very rapidly. On the eighth day the culture fluids were harvested. The agent was subcultured in human embryo kidney cultures and subsequently identified as adenovirus type 7 by means of hemagglutination and neutralization tests. The virus caused complete hemagglutination of rhesus monkey erythrocytes but failed to agglutinate rat erythrocytes, the pattern established for adenovirus types 3, 7, 11, 14, 16, 20, 21, 25, and 28. In neutralization tests in which pools of adenovirus xabbit antisera (types 1-1 I) were employed, the virus was completely neutralized by a pool containing adenovirus type 7 antiserum and failed to be neutralized by the pools which did not contain it. The virus was further neutralized by adenovirus type 7 antiserum alone.~ The same virus was success~ully isolated in human embryo kidney cuttures from 10 per cent suspensions of the frozen kidney, lung, spleen, and liver as well as from the serum. Upon electron microscopic examiuationt the serum revealed the presence of 81 m# adenovirus particles. Particles of the same type were also found in the virus-containing material obtained ~The identification of virus was perforhaed by Dr. Rosb,n Q. Robinson, Respi~ovirus Unit and International Influenza Center for the Americas, CDC, Atlanta, Ga., and confirmed by us. tPerformed by Dr. K. O. Smith, Department of Virology and Epldenfiology.
Fatal injection with adenovirus type 7
85
T a b l e I. Results of titrations of o r g a n suspensions a n d serum, with the e m p l o y m e n t of h u m a n embryo kidney cell cultures
Specimen
I TCDso per gram tissue
Kidney Lung Spleen Liver Serum *TCDro per milliliterof undilutedserum.
4.8 6.2 6.2 7.5 1.86
by passing the patient's specimens in tissue culture: The quantity of virus present in the different organs and the serum were determined by titrations in human embryo kidney cultures. Tenfold dilutions prepared from the 10 per cent organ suspensions and the serum were inoculated in 0.1 ml. amounts into each of four human embryo kidney cultures. The cultures were observed for eytopathic changes for 19 days. Maximum TCD~0 titers were obtained on the thirteenth day. From the results presented in Table I it is obvious that all the organs contained large quantities of virus, especially the liver which revealed 107-s TCDs0 per gram of tissue. The concentration of virus in the blood was relatively low. Unfortunately a postmortem blood sample was not available for viral examination. It is possible that part of the virus in the serum might have been neutralized by circulating antibody. Because of the lack of sufficient quantity of serum no attempts were made to determine the presence of antibodies. DISCUSSION I n m a n y viral diseases the establishment of a n etiologic relationship b e t w e e n the virus isolated a n d the illness is of considerable difficulty, is I n the case reported here the large quantities of adenovirus type 7 f o u n d in the iung, kidney, spleen, a n d liver ( T a b l e I ) , t a k e n together with the presence of i n t r a n u c l e a r inclusions in the l u n g a n d liver, show that active virus infection was present in a disseminated form at the time of death. T h e clinical course was consistent with t h a t described by other investigators for epidemic s, s-10 o r sporadic 1, 2, 4 cases of adenovirus-associated p n e u m o n i a in children. T h e histopathologic findings also corresponded to those described for adenovirusassociated fatal pneumonias2-4, s, 9, 14 with
86
Benyesh-Melnick and Rosenberg
the exception of the extensive involvement of the liver in our case. This was also the tissue yielding the highest titer of virus (107.5 TCDs0 per gram). It is of interest that adenovirus type 5 has been recently found to cause fatal disease when inoculated into newborn hamsters. T h e only pathologic changes found were in the liver where there was nuclear pyknosis with clumping of the chromatin into basophilic masses surrounded by vesicular spaces. 1~ T h e viremia found 2 days prior to death in the case presented is in keeping with the rash and the disseminated character of the pathologic and virologic findings. Adenovirus types 4 and 7 viremia has been recently reported in this country in naval recruits with rubelliform illness during the acute stage of the disease. 1~ I t is difficult to say that th~ virus isolated in this case was responsible for the entire course of the disease from the time of infancy. It is possible that with the depletion of maternal antibodies, by the time the child had reached the age of 5 months, the initially depressed infection underwent a rapid dissemination. The possibility of a basic defect in immunity could not be completely discarded for there was a borderline level of serum g a m m a globulin (0.1 Gm. per 100) as well as a generalized lymphoid depletion. The absence of Hassall's bodies in the thymus of this girl is hard to interpret. This phenomenon has thus far been described only in sex-linked congenital agammaglobulinemia of the male. Morphologically, there are several pulmonary disorders associated with the presence of intranuclear inclusions. T h e most common is cytomegalic inclusion disease in its disseminated form or as an isolated pneumonia. Pulmonary necrosis and exudation are uncommon in cytomegalic inclusion disease although both m a y occur. T h e inclusion-bearing cells are usually free within the alveolar spaces but m a y be in the epithelial lining of the terminal air ducts. Large intranuclear, usually basophilic inclusions are encountered. Frequently, basophilic
January 1964
cytoplasmic inclusions are also encountered. Varicella may involve the lung when dis. seminated in the face of altered immunity or steroid therapy. Herpes simplex may result in disseminated inclusion disease in t h e neonate. From an architectural standpoint, there are several differences. Varicella pneumonitis occurs as an extension of the primary skin lesion. Herpes simpIex characteristically "involves tissues in a generalized distribution, particularly in those infants under 1 year of age. Temporal lobe encephalitis, hepatic necrosis with inclusions, and adrenal necrosis with inclusions are frequently encountered. In the present case there was no central nervous system involvernent. Hepatic necrosis with inclusions was present in this case. There were also localized areas of adrenal necrosis but without inclusions. On a cytologic basis it is difficult, if not impossible, to distinguish between the inclusions resulting from herpes simplex, from varicella, and from adenovirus. Isolation of the virus has been the only means of positive identification. SUMMARY
Adenovirus type 7 was isolated from a fatal case of pneumonia and disseminated disease. The virus was isolated in high titers from the kidney (104's TCDs0 per gram), lung (106"2), spleen (10~'2), and liver (10 ~'5) removed at autopsy as well as from a serum sample (10 ls per milliliter) obtained 2 days before the child died. Postmortem examination revealed extensive involvement of the lung and liver with Feulgen-positive intranuclear inclusions in both organs. No such inclusions were found in other organs..
REFERENCES
1. Sterner, G.: Atypical pneumonia in an infant, associated with APC virus infection, Acta paediat. 45: 449, 1956. 2. Pereira, H. G., and Kelly, B.: Studies on natural and experimental infections by adenovirus, Proc. Roy. Soc. 50: 755, 1957. 3. Chany, C., Lepine, T., Lelong, N., Le-TanVinh, Satge P., and Virat, J.: Severe and
Volume 64
4.
5. 6.
7.
8.
9.
Number 1
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Fatal injection with adenovirus type 7
10.
l l.
12. 13. 14.
15.
16.
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