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Epithelial giant cells in measles as an aid in diagnosis
July, 197o T h e J o u r n a l of P E D I A T R I C S
59
Epithelial giant cells in measles as an aid in diagnosis Ascertainment of the presence of epithelial multinucleate giant cel'~s, a relatively specific feature in the cytopathology of measles, is a simple and useful diagnostic tool. During the prodromal stage and the first day of the rash, these cells are recoverable from accessible epithelial surfaces, such as the pharynx, the lining cells of nasal and buecal cavities, and eonjunctiva. In the next few days they disappear progressively. They may also be seen in sputum and in urinary deposits. Although their identification is of value mainly in the prodromal stage, it may also be useful in the differential diagnosis of a rash when measles is a questionable possibility. Epithelial giant cell formation was not observed in children vaccinated with Sehwarz strain, live, attenuated measles virus.
Reginald Lightwood, M.D. (London), F.R.C.P. (London), D.P.H. (England), and Regina Nolan,* with the assistance of M. Franco ~ and A. J. S. White ~ S A L I S B U R Y , RHODESIA~ AND LOS A N G E L E S , ~ A L I F .
I ~ M E A S L E S , a highly characteristic cellular phenomenon is the formation of multinucleated giant cells within reactive foci. These giant cells develop early in the infection and i n fatal cases are often observed in the prodromal stage. However, when death occurs after the appearance of the rash, these cells have usually disappeared from the tissues. Two types of measles giant cells have been distinguished; the reticulo-
From the Department of Paediatrie3, The University College of Rhodesia, and the Department of Pediatrics, The Unive'rsit~ of California at Los Angeles. ~Senlor Medical Technologlst. ~Medlcal students in the Faculty o] Medletne, ~ e University College of Rhodesia, reee~wng Student Researoh Grants from the University College of Rhodesia. Reprint address: John M. Adams, M.D., Det)artment ot Pediatrics, School of Medicine, University of Gall]ornia, Los Angeles, Calif. 90024.
endothelial giant cell and the epithelial giant cell?, 2 The reticuloendothelial type as described was originally found in histologic sections of the lungs in fatal cases?, 4 This type has since been observed and described by many workers, in lymph nodes, tonsils, adenoids, spleen, thymus, lungs, and the lymphoid tissue of the intestinal tract, including the appendix? -11 The reticuloendothelial giant cells are found in the prodromal stage and usually disappear soon after the rash appears at about the time when circulating antibodies become detectable? 2, 1~ The epithelial type o f measles giant cell forms in the prodromal stage, and for the same reason, usually disappears soon after the rash is fully out. Epithelial giant cells have been observed with the microscope in sections of Vol. 77, No. 1, pp. 59-64
60
Lightwood and NoIan
lung, bronchi, trachea, the epithelial linings of the u p p e r respiratory tract and the mouth,l, 14-~9 and also in the mucosal lining of the urinary b l a d d e r ? I n the clinical setting, sputum, 14-17 nasal secretions, 17-2~ and urinary cellular deposit 22 are sources of material for the identification of measles epithelial giant cells. I n addition to these sources, we have used stained phat~cngeal, buccal, and conjunctival smears obtained by means of ordinary cotton-wool swabs. This paper presents data concerning the occurrence of multinucleate giant cells observed in the material obtained during attacks of measles from these readily accessible epithelial surfaces. T h e patients on w h o m the observations were made were African children seen in the pediatric department of the teaching hospital of the University College of Rhodesia in Salisbury or at a nearby African isolation hospital. Epithelial giant cells said to be "specific" for measles ~, 20 consist of syncytial masses
Fig. 1. A portion of a group of multinucleate giant cells (4 to 8 nuclei per celt_) is illustrated in which nuclear (arrows) and cytoplasmic inclusion bodies were seen within a surrounding clear zone. Pharyngeal smear stained with Mayer's hematoxylin alum and eosin. •
The Journal o[ Pediatrics July 1970
with m a n y nuclei in which nuclear inclusions occur and sometimes cytoplasmic inclusions are seen. These multinucleate cells vary greatly in size and nuclear n u m b e r ; the nuclei m a y be as few as 4 to 6 or as m a n y as 70 to 100, not then being countable with any degree of accuracy. I n Rhodesia we had no facilities for the application of fluorescein antibody-staining or specific serologic methods, because these techniques were at the time not available in the territory. Therefore, the measles giant cells were identified on their cytologic character, including the presence of inclusion bodies in the less degenerate cells (see Discussion). METHODS
Pharyngeal and conjunctival swabs were taken daily from 50 African children, aged 7 months to 7 years, from the day they were first admitted with measles. Swabs f r o m 50 control subjects without measles were also taken. I n order to avoiding drying out of the material before reaching the laboratory, cytologic preparations were m a d e on the spot. S m e a r s from the pharynx and conjunctiva were made on slides and fixed by
Fig. 2. Illustrated here is a small part of an extensive mass of closely approximated, multinucleate giant cells. The nuclei are seen crowded together and nuclear inclusion bodies (arrows), surrounded by a clear zone, are present. Pharyngeal smear, Mayer's hematoxylin and eosin. •
Volume 77 Number 1
Epithelial giant cells in measles
immersion in 95 per cent alcohol. The slides were stained with Mayer's hematoxylin-alum and eosin, then dehydrated, mounted, and examined microscopically. The slides were scanned under low-power (x 125 magnification) and then examined under x 500 magnification. Specimens were also examined at higher magnifications, and photomicrographs were taken in order to illustrate the nuclear and cytoplasmic inclusions (Figs. 1 and 2). In some instances, buccal smears, nasal mucus, and urinary cell deposits were similarly examined. RESULTS
Measles patients. Pharyngeal smears were obtained from 10 patients in the prodromal stage (Koplik's spots and without rash). The findings in these 10 patients are shown in a proportional bar diagram together with the results in patients first presenting with a measles rash (see diagram demonstrating the day by day percentage prevalence of multinucleate giant cells in pharyngeal specimens, Fig. 3). Fifty patients with measles, all had multinucleate giant cells in the pharyngeal material. The number of giant cells diminished day by day, and they had invariably disappeared before the seventh day after the appearance of rash. In all of 7 examinations of the buccal mucosa overlying Koplik's spots, multinucleate giant cells were also found. In 22 of the 50 patients, multinucleate giant cells were also present
in conjunctival specimens. After finding few cells in the conjunctival smears at first, we decided to take these swabs before "eye toilet," because cleaning procedures seemed to wash away many epithelial cells in the process of shedding. As with the pharyngeal recoveries, all multinucleate giant cells in the conjunctival specimens had disappeared before the seventh day after the first appearance of rash. In one child suspected of having measles and swabbed 3 days before a measles rash appeared (not included in the diagram, Fig. 3), multinucleate giant cells were observed in daily pharyngeal and conjunctival specimens. After the rash appeared, they rapidly diminished in number and in this particular case disappeared by the third day after the rash. Nasal mucus was aspirated in the early stage of measles from 6 children, the material being collected by the method proposed by Auger. ~1 For these collections we attached a polyethylene tube to a 90 ml. capacity syringe. The tube was inserted into the posterior part of the nasal cavity beneath the inferior turbinate and suction was applied. In all of these 6 patients, multinucleate giant cells were observed in the nasal mucus as well as in the pharyngeal and conjunctival swabs. Examination of material taken from inside the anterior nares often shows leukocytes, and fewer intact epithelial giant cells are likely to be present. We examined the cellular deposits in the
DAYS AFTER APPEARANCE OF RASH 1st 2rid 3pd 4th 5th 6th 7thiSth 9th lOth 11th 12tP '" 87.5'~ ,gl I
g I-~Z
}2,
-
4C 'v:
SPECIMENS T,,K~.
1.0 11
16
2 4 31
61
37 34
2 2 13
8
7
6
5
3
Fig. 3. Proportional bar diagram demonstrating the day by day percentage prevalence of multinucleate giant cells present in pharyngeal specimens.
62
Lightwood and NoIan
urine of 7 children in the early stage of measles and found epithelial-type multinucleate giant cells in 2 of them. These cells contained eosinophilic cytoplasmic inclusions, the appearances of which closely resembled those illustrated by Bolande. 22 Control subjects. Fifty well-nourished African c h i l d r e n w i t h i n the measles age group who were not ill were similarly examined by means of pharyngeal and conjunctival swabs; these controls were selected at random. No multinucleate giant cells were found in the pharyngeal or conjunctival smears. E P I T H E L I A L G I A N T CELLS N O T FOUND
AFTER
VACCINATION
One of us (R. L . ) * examined smears taken from a consecutive series of 22 Children, aged 9 months to 5 years, who received a first and standard dose of Schwarz strain, live attenuated measles vaccine propagated in chick embryo (Pitman-Moore Co., Division of the Dow Chemical Co., Indianapolis, Ind.). Pharyngeal and buccal, and sometimes c0njunctlval swabs were taken immediately before the vaccine was administered (control swabs) a n d subsequently on the sixth and eighth days or the seventh and ninth days after vaccination. Fixation and staining methods were the same as those used in the study on measles patients. No multinucleate giant cells Were seen in any of the control smears or in any of the postvaccination smears. Specific measles immunofluorescent staining was negative in the cells of serial smears made from 2 randomly chosen vaccination subjects. DISCUSSION
As "specific" multinuclear giant cells are present in epithelial tissues in the early and acute stages of measles, procedures for demonstrating them in material obtainable from mucosal surfaces deserve to be more widely used. Sources of material suitable for examination are pharyngeal, buccal, and con*Done in the ViroIogy Research Laboratory, the Department of Pediatrics, University of California at Los Angeles.
The Journal o[ Pediatrics July 1970
junctival swabs and nasal mucus and urinary cellular deposit; also sputum can be examined if obtainable. The finding of measles giant ceils in any of these specimens is particularly helpful under the following circumstances : (1) In patients before the rash appears, especially in that small proportion in whom Koplik's spots are not manifest. Furthermore, lesions in the buccal mucosa, almost indistinguishable from Koplik's spots, may be present in a minority of patients with Coxsackie A9 virus infection. 2 (2) In children with dark pigmented skins in whom it is often impossible to recognize the early macular stage of the measles rash and perhaps even the maculopapular stage. When the observer is not certain about the nature of the rash, diagnosis must depend on a history of possible contact, general symptoms, the appearance of the patient, and the subsequent course of the illness, bearing in mind that certain cases of measles are atypical. (3) When the rashes of other exanthematous disorders, such as rubella, scarlet fever, roseola, typhus, and drug sensitivity are considered in the differential diagnosis. Knowledge regarding the forms and varying appearances of measles giant cells is needed for their identification. It has been established that they are formed as the result of fusion of cells and not by atypical nuclear division. 23 Thus, in monkey kidney cell cultures infected wiIdh measles virus, syncytial formation was seen to begin about 40 hours after infection of the cell culture. 2~ At first, cells in contact with each other fused to form a single cell with 2 or 3 nuclei, and these nuclei congregated in the central part of the cell. These small, early, multinucleate cells then fused with others to form large giant cells containing as many as 100 nuclei. During this process, cytoplasmic inclusion bodies appeared, followed by intranuclearoinclusions. In the human infection the probable evolution of measles giant cells has been suggested by Sherman and Ruckle? They considered it reasonable to judge the successive stages in the evolution of giant cells by the
Volume 77 Number 1
degree of degenerative change in cells of the bronchial epithelium observed in a postmortem which began one hour after death. According to their description, the initial change appeared as a fine granularity of the cytoplasm followed by dissolution of the cell walls to produce a syncytium. Meanwhile, nuclear changes began and inclusions formed in the nuclei. Typically, a clear zone separated the inclusion material from the nuclear membrane beneath which most of the chromatin was concentrated. Subsequent changes were those of increasingly severe cellular damage. There was nuclear basophilia with eventual shrinking and clumping of the many nuclei into a dark mass of chromatin, and the cytoplasm of the giant cell sometimes disappeared. By this stage it is impossible to identify individual nuclei, and the cell is no longer suitable for observing inclusion bodies. Allowing for the differences in histologic appearances between cut sections and stained smears, our observations are consistent with this description of the probable sequence of changes in measles giant cells. We found that it was usually difficult or impossible to recognize inclusions in cells showing advanced degenerative changes; often it was easier to see them in the smaller giant cells with fewer nuclei which, presumably, are at an earlier stage of formation (Fig. 1). A point of interest is the reason for the disappearance of measles giant cells soon after the rash develops. Complement-fixing and neutralizing antibodies are detectable in the blood from about the second day of the rash, and perhaps it may be assumed that from that time new giant cells are not formed. Meanwhile, existing giant cells, already degenerating, would disintegrate and some would be shed from the epithelial surfaces. Persistence of giant cells, however, have been recorded in fatal cases of measles pneumonia 1, 2s, 2G (Hecht's disease), just as there are reports of prolonged proliferation of virus in patients who fail to form neutralizing antibodies. 2 Perhaps the question should be considered
Epithelial giant cells in measles
63
as to whether giant cells, as they are shed from epithelial surfaces, play a part in dissemination of virus, acting, as it were, like seed pods. U n d e r the conditions of cell culture, it has been found that the release of virus into the fluid phase appears to be exceedingly slowY a Another point is that the time of persistence of giant cells in the epithelium of the upper respiratory tract, after appearance of rash, is not greatly different from the time after the rash during which it is thought that a patient may still spread infection. O n the other hand, by the second day of the rash, coincident with the detection of circulating antibody, it becomes difficult to recover measles virus f r o m the pharynx and nasal secretions. 2' 2~ But, interestingly, viruria has been reported as persisting for a longer period of time, 2~ and Sherman and Ruckle 1 observed giant cells "specific" for measles in sections of the mucosa of the urinary bladder in a child whose death took place on the ninth day of her illness and the seventh day after the rash of measles appeared. According to the observations of Bolande 22 inclusion-bearing cells may be found in the urine as early as 8 days after exposure; they then increased in n u m ber until the stage of Koplik's spots and rash, diminishing and disappearing thereafter. We are indebted to the following persons for advice and help in this study: Dr. A. J. Beale and Dr. David Morley in London; Dr. W. M. Buchanan, Prof. J. S. Cmickshank, and Dr. D. W. Shennan at the University College of Rhodesia; Dr. John M. Adams and Mrs. Natalie Stein in the Department of Pediatrics, University of California at Los Angeles. REFERENCES
1. Sherman, F. E., and Ruckle, G.: In vivo and in vitro cellular changes specific for measles, Arch. Path. 65: 587, 1958. 2. Katz, S. L., and Enders, J. F.: Measles virus, in Horsfall, F. L., and Tamm, I., editors: Viral and rickettsial infections of man, ed. 4, Philadelphia, 1965, J. B. Lippincott Company. 3. Kromayer, E.: l~ber die Sogenannte Katarrhalpneumonie nach Masern und Keuchhusten, Virchow. Arch. Path. Anat. 117: 452, 1889. 4. Hecht, V.: Die Riesensellenpneumonie hn
64
5. 6.
7.
8. 9. 10.
11. 12.
13.
14.
Lightwood and NoIan
Kindesalter, ein Historich-Experimentelle Studie, Beitr. Path. Anat. 48: 263, 1910. Denton, J.: The pathology of fatal measles, Amer. J. Med. Sci. 169: 531, 1925. Warthin, A. S,: Occurrence of numerous large giant cells in tonsils and pharyngeal mucosa in the prodromal stage of measles, Arch. Path. 11: 864, 1931. Finkeldey, W.: t,)ber Riesenzellbefunde in den Gaumenmandeln, Zugleich ein Beitrag zur Histopathologie der Mandelveranderungen im Maserninkubatlonsstadium, Arch. Path. Anat. 281: 323, 1931. Herzberg, M.: Giant cells in the lymphoid tissue of the appendix in the prodromal stage of measles, J. A. M. A. 98: 139, 1932. Hathaway, B. M.: Generalized dissemination of giant cells in lymphoid tissue in prodromal stage of measles, Arch. Path. 19: 819, 1935. Semsroth, K. H.: Multinucleate epithelial giant cells with inclusion bodies in prodromal measles: Report of autopsy, Arch. Path. 28: 386, 1939. Roberts, G. B. S., and Bain, A. D.: The pathology of measles, J. Path. Bact. 76: 111, 1958. Ruckle, G.: Studies with measles virus. II. Isolation of virus and immunologic studies in persons who have had the natural disease, J. Immun. 78: 341, 1957. Enders, J. F., Peebles, T. C., McCarthy, K., Milovanovie, M., Miltus, A., and Holloway, A.: Measles virus: A summary of experiments concerned with isolation, properties, and behavior, Amer. J. Public Health 47- 275, 1957. Broadhurst, J., MacLean, M. E., and Saurino, V.: Inclusion bodies in measles, J. Infect. Dis. 61: 201, 1937.
The Journal o/ Pediatrics July 1970
15. Semsroth, K. H.: Multinucleate epithelial giant ceils with inclusion bodies in prodromal measles, Arch. Path. 28: 386, 1939. 16. Stryker, W. A.: Disseminated giant cell reaction: A possible prodrome of measles, Amer. J. Dis. Child. 59: 468, 1940. 17. Tompkins, Y., and Macaulay, J. C.: A characteristic cell in nasal secretions during prodromal measles, J. A. M. A. 157: 711, 1955. 18. Roberts, G. B. S., and Bain, A. D.: The pathology of measles, J. Path. Bact. 76: 111, 1958. 19. Beale, A. J., and Campbell, W.: A rapid cytological method for the diagnosis of measles, J. Clin. Path. 12: 335, 1959. 20. Mottet, N. K., and Szanton, V.: Exfoliated measles giant cells in nasal secretions, Arch. Path. 72: 434, 1961. 21. Auger, W. J.: An original method of obtaining sputum from infants and children, J. PED~AT. 15: 640, 1939. 22. Bolande, R. P.: Inclusion-bearing cells in the urine, Pediatrics 24: 7, 1959. 23. Matumoto, M.: Multiplication of measles virus in cell cultures, Bact. Rev. 30: 152, 1966. 24. Aoyama, Y.: Changes of cultured cells infected with measles virus, Jap. J. Exp. Med. 29: 535, 1959. 25. Milles, G.: Measles pneumonia (with a note on the giant cells of measles), Amer. J. Clin. Path. 15: 334, 1945. 26. Adams, J. M., Baird, C., and Filloy, L.: Inclusion bodies in measles encephalitis, J. A. M. A. 195: 150, 1966. 27. Gresser, I., and Katz, S. L.: Isolation of measles virus from urine, New Eng. J. Med. 263: 452, 1960.
59
Epithelial giant cells in measles as an aid in diagnosis Ascertainment of the presence of epithelial multinucleate giant cel'~s, a relatively specific feature in the cytopathology of measles, is a simple and useful diagnostic tool. During the prodromal stage and the first day of the rash, these cells are recoverable from accessible epithelial surfaces, such as the pharynx, the lining cells of nasal and buecal cavities, and eonjunctiva. In the next few days they disappear progressively. They may also be seen in sputum and in urinary deposits. Although their identification is of value mainly in the prodromal stage, it may also be useful in the differential diagnosis of a rash when measles is a questionable possibility. Epithelial giant cell formation was not observed in children vaccinated with Sehwarz strain, live, attenuated measles virus.
Reginald Lightwood, M.D. (London), F.R.C.P. (London), D.P.H. (England), and Regina Nolan,* with the assistance of M. Franco ~ and A. J. S. White ~ S A L I S B U R Y , RHODESIA~ AND LOS A N G E L E S , ~ A L I F .
I ~ M E A S L E S , a highly characteristic cellular phenomenon is the formation of multinucleated giant cells within reactive foci. These giant cells develop early in the infection and i n fatal cases are often observed in the prodromal stage. However, when death occurs after the appearance of the rash, these cells have usually disappeared from the tissues. Two types of measles giant cells have been distinguished; the reticulo-
From the Department of Paediatrie3, The University College of Rhodesia, and the Department of Pediatrics, The Unive'rsit~ of California at Los Angeles. ~Senlor Medical Technologlst. ~Medlcal students in the Faculty o] Medletne, ~ e University College of Rhodesia, reee~wng Student Researoh Grants from the University College of Rhodesia. Reprint address: John M. Adams, M.D., Det)artment ot Pediatrics, School of Medicine, University of Gall]ornia, Los Angeles, Calif. 90024.
endothelial giant cell and the epithelial giant cell?, 2 The reticuloendothelial type as described was originally found in histologic sections of the lungs in fatal cases?, 4 This type has since been observed and described by many workers, in lymph nodes, tonsils, adenoids, spleen, thymus, lungs, and the lymphoid tissue of the intestinal tract, including the appendix? -11 The reticuloendothelial giant cells are found in the prodromal stage and usually disappear soon after the rash appears at about the time when circulating antibodies become detectable? 2, 1~ The epithelial type o f measles giant cell forms in the prodromal stage, and for the same reason, usually disappears soon after the rash is fully out. Epithelial giant cells have been observed with the microscope in sections of Vol. 77, No. 1, pp. 59-64
60
Lightwood and NoIan
lung, bronchi, trachea, the epithelial linings of the u p p e r respiratory tract and the mouth,l, 14-~9 and also in the mucosal lining of the urinary b l a d d e r ? I n the clinical setting, sputum, 14-17 nasal secretions, 17-2~ and urinary cellular deposit 22 are sources of material for the identification of measles epithelial giant cells. I n addition to these sources, we have used stained phat~cngeal, buccal, and conjunctival smears obtained by means of ordinary cotton-wool swabs. This paper presents data concerning the occurrence of multinucleate giant cells observed in the material obtained during attacks of measles from these readily accessible epithelial surfaces. T h e patients on w h o m the observations were made were African children seen in the pediatric department of the teaching hospital of the University College of Rhodesia in Salisbury or at a nearby African isolation hospital. Epithelial giant cells said to be "specific" for measles ~, 20 consist of syncytial masses
Fig. 1. A portion of a group of multinucleate giant cells (4 to 8 nuclei per celt_) is illustrated in which nuclear (arrows) and cytoplasmic inclusion bodies were seen within a surrounding clear zone. Pharyngeal smear stained with Mayer's hematoxylin alum and eosin. •
The Journal o[ Pediatrics July 1970
with m a n y nuclei in which nuclear inclusions occur and sometimes cytoplasmic inclusions are seen. These multinucleate cells vary greatly in size and nuclear n u m b e r ; the nuclei m a y be as few as 4 to 6 or as m a n y as 70 to 100, not then being countable with any degree of accuracy. I n Rhodesia we had no facilities for the application of fluorescein antibody-staining or specific serologic methods, because these techniques were at the time not available in the territory. Therefore, the measles giant cells were identified on their cytologic character, including the presence of inclusion bodies in the less degenerate cells (see Discussion). METHODS
Pharyngeal and conjunctival swabs were taken daily from 50 African children, aged 7 months to 7 years, from the day they were first admitted with measles. Swabs f r o m 50 control subjects without measles were also taken. I n order to avoiding drying out of the material before reaching the laboratory, cytologic preparations were m a d e on the spot. S m e a r s from the pharynx and conjunctiva were made on slides and fixed by
Fig. 2. Illustrated here is a small part of an extensive mass of closely approximated, multinucleate giant cells. The nuclei are seen crowded together and nuclear inclusion bodies (arrows), surrounded by a clear zone, are present. Pharyngeal smear, Mayer's hematoxylin and eosin. •
Volume 77 Number 1
Epithelial giant cells in measles
immersion in 95 per cent alcohol. The slides were stained with Mayer's hematoxylin-alum and eosin, then dehydrated, mounted, and examined microscopically. The slides were scanned under low-power (x 125 magnification) and then examined under x 500 magnification. Specimens were also examined at higher magnifications, and photomicrographs were taken in order to illustrate the nuclear and cytoplasmic inclusions (Figs. 1 and 2). In some instances, buccal smears, nasal mucus, and urinary cell deposits were similarly examined. RESULTS
Measles patients. Pharyngeal smears were obtained from 10 patients in the prodromal stage (Koplik's spots and without rash). The findings in these 10 patients are shown in a proportional bar diagram together with the results in patients first presenting with a measles rash (see diagram demonstrating the day by day percentage prevalence of multinucleate giant cells in pharyngeal specimens, Fig. 3). Fifty patients with measles, all had multinucleate giant cells in the pharyngeal material. The number of giant cells diminished day by day, and they had invariably disappeared before the seventh day after the appearance of rash. In all of 7 examinations of the buccal mucosa overlying Koplik's spots, multinucleate giant cells were also found. In 22 of the 50 patients, multinucleate giant cells were also present
in conjunctival specimens. After finding few cells in the conjunctival smears at first, we decided to take these swabs before "eye toilet," because cleaning procedures seemed to wash away many epithelial cells in the process of shedding. As with the pharyngeal recoveries, all multinucleate giant cells in the conjunctival specimens had disappeared before the seventh day after the first appearance of rash. In one child suspected of having measles and swabbed 3 days before a measles rash appeared (not included in the diagram, Fig. 3), multinucleate giant cells were observed in daily pharyngeal and conjunctival specimens. After the rash appeared, they rapidly diminished in number and in this particular case disappeared by the third day after the rash. Nasal mucus was aspirated in the early stage of measles from 6 children, the material being collected by the method proposed by Auger. ~1 For these collections we attached a polyethylene tube to a 90 ml. capacity syringe. The tube was inserted into the posterior part of the nasal cavity beneath the inferior turbinate and suction was applied. In all of these 6 patients, multinucleate giant cells were observed in the nasal mucus as well as in the pharyngeal and conjunctival swabs. Examination of material taken from inside the anterior nares often shows leukocytes, and fewer intact epithelial giant cells are likely to be present. We examined the cellular deposits in the
DAYS AFTER APPEARANCE OF RASH 1st 2rid 3pd 4th 5th 6th 7thiSth 9th lOth 11th 12tP '" 87.5'~ ,gl I
g I-~Z
}2,
-
4C 'v:
SPECIMENS T,,K~.
1.0 11
16
2 4 31
61
37 34
2 2 13
8
7
6
5
3
Fig. 3. Proportional bar diagram demonstrating the day by day percentage prevalence of multinucleate giant cells present in pharyngeal specimens.
62
Lightwood and NoIan
urine of 7 children in the early stage of measles and found epithelial-type multinucleate giant cells in 2 of them. These cells contained eosinophilic cytoplasmic inclusions, the appearances of which closely resembled those illustrated by Bolande. 22 Control subjects. Fifty well-nourished African c h i l d r e n w i t h i n the measles age group who were not ill were similarly examined by means of pharyngeal and conjunctival swabs; these controls were selected at random. No multinucleate giant cells were found in the pharyngeal or conjunctival smears. E P I T H E L I A L G I A N T CELLS N O T FOUND
AFTER
VACCINATION
One of us (R. L . ) * examined smears taken from a consecutive series of 22 Children, aged 9 months to 5 years, who received a first and standard dose of Schwarz strain, live attenuated measles vaccine propagated in chick embryo (Pitman-Moore Co., Division of the Dow Chemical Co., Indianapolis, Ind.). Pharyngeal and buccal, and sometimes c0njunctlval swabs were taken immediately before the vaccine was administered (control swabs) a n d subsequently on the sixth and eighth days or the seventh and ninth days after vaccination. Fixation and staining methods were the same as those used in the study on measles patients. No multinucleate giant cells Were seen in any of the control smears or in any of the postvaccination smears. Specific measles immunofluorescent staining was negative in the cells of serial smears made from 2 randomly chosen vaccination subjects. DISCUSSION
As "specific" multinuclear giant cells are present in epithelial tissues in the early and acute stages of measles, procedures for demonstrating them in material obtainable from mucosal surfaces deserve to be more widely used. Sources of material suitable for examination are pharyngeal, buccal, and con*Done in the ViroIogy Research Laboratory, the Department of Pediatrics, University of California at Los Angeles.
The Journal o[ Pediatrics July 1970
junctival swabs and nasal mucus and urinary cellular deposit; also sputum can be examined if obtainable. The finding of measles giant ceils in any of these specimens is particularly helpful under the following circumstances : (1) In patients before the rash appears, especially in that small proportion in whom Koplik's spots are not manifest. Furthermore, lesions in the buccal mucosa, almost indistinguishable from Koplik's spots, may be present in a minority of patients with Coxsackie A9 virus infection. 2 (2) In children with dark pigmented skins in whom it is often impossible to recognize the early macular stage of the measles rash and perhaps even the maculopapular stage. When the observer is not certain about the nature of the rash, diagnosis must depend on a history of possible contact, general symptoms, the appearance of the patient, and the subsequent course of the illness, bearing in mind that certain cases of measles are atypical. (3) When the rashes of other exanthematous disorders, such as rubella, scarlet fever, roseola, typhus, and drug sensitivity are considered in the differential diagnosis. Knowledge regarding the forms and varying appearances of measles giant cells is needed for their identification. It has been established that they are formed as the result of fusion of cells and not by atypical nuclear division. 23 Thus, in monkey kidney cell cultures infected wiIdh measles virus, syncytial formation was seen to begin about 40 hours after infection of the cell culture. 2~ At first, cells in contact with each other fused to form a single cell with 2 or 3 nuclei, and these nuclei congregated in the central part of the cell. These small, early, multinucleate cells then fused with others to form large giant cells containing as many as 100 nuclei. During this process, cytoplasmic inclusion bodies appeared, followed by intranuclearoinclusions. In the human infection the probable evolution of measles giant cells has been suggested by Sherman and Ruckle? They considered it reasonable to judge the successive stages in the evolution of giant cells by the
Volume 77 Number 1
degree of degenerative change in cells of the bronchial epithelium observed in a postmortem which began one hour after death. According to their description, the initial change appeared as a fine granularity of the cytoplasm followed by dissolution of the cell walls to produce a syncytium. Meanwhile, nuclear changes began and inclusions formed in the nuclei. Typically, a clear zone separated the inclusion material from the nuclear membrane beneath which most of the chromatin was concentrated. Subsequent changes were those of increasingly severe cellular damage. There was nuclear basophilia with eventual shrinking and clumping of the many nuclei into a dark mass of chromatin, and the cytoplasm of the giant cell sometimes disappeared. By this stage it is impossible to identify individual nuclei, and the cell is no longer suitable for observing inclusion bodies. Allowing for the differences in histologic appearances between cut sections and stained smears, our observations are consistent with this description of the probable sequence of changes in measles giant cells. We found that it was usually difficult or impossible to recognize inclusions in cells showing advanced degenerative changes; often it was easier to see them in the smaller giant cells with fewer nuclei which, presumably, are at an earlier stage of formation (Fig. 1). A point of interest is the reason for the disappearance of measles giant cells soon after the rash develops. Complement-fixing and neutralizing antibodies are detectable in the blood from about the second day of the rash, and perhaps it may be assumed that from that time new giant cells are not formed. Meanwhile, existing giant cells, already degenerating, would disintegrate and some would be shed from the epithelial surfaces. Persistence of giant cells, however, have been recorded in fatal cases of measles pneumonia 1, 2s, 2G (Hecht's disease), just as there are reports of prolonged proliferation of virus in patients who fail to form neutralizing antibodies. 2 Perhaps the question should be considered
Epithelial giant cells in measles
63
as to whether giant cells, as they are shed from epithelial surfaces, play a part in dissemination of virus, acting, as it were, like seed pods. U n d e r the conditions of cell culture, it has been found that the release of virus into the fluid phase appears to be exceedingly slowY a Another point is that the time of persistence of giant cells in the epithelium of the upper respiratory tract, after appearance of rash, is not greatly different from the time after the rash during which it is thought that a patient may still spread infection. O n the other hand, by the second day of the rash, coincident with the detection of circulating antibody, it becomes difficult to recover measles virus f r o m the pharynx and nasal secretions. 2' 2~ But, interestingly, viruria has been reported as persisting for a longer period of time, 2~ and Sherman and Ruckle 1 observed giant cells "specific" for measles in sections of the mucosa of the urinary bladder in a child whose death took place on the ninth day of her illness and the seventh day after the rash of measles appeared. According to the observations of Bolande 22 inclusion-bearing cells may be found in the urine as early as 8 days after exposure; they then increased in n u m ber until the stage of Koplik's spots and rash, diminishing and disappearing thereafter. We are indebted to the following persons for advice and help in this study: Dr. A. J. Beale and Dr. David Morley in London; Dr. W. M. Buchanan, Prof. J. S. Cmickshank, and Dr. D. W. Shennan at the University College of Rhodesia; Dr. John M. Adams and Mrs. Natalie Stein in the Department of Pediatrics, University of California at Los Angeles. REFERENCES
1. Sherman, F. E., and Ruckle, G.: In vivo and in vitro cellular changes specific for measles, Arch. Path. 65: 587, 1958. 2. Katz, S. L., and Enders, J. F.: Measles virus, in Horsfall, F. L., and Tamm, I., editors: Viral and rickettsial infections of man, ed. 4, Philadelphia, 1965, J. B. Lippincott Company. 3. Kromayer, E.: l~ber die Sogenannte Katarrhalpneumonie nach Masern und Keuchhusten, Virchow. Arch. Path. Anat. 117: 452, 1889. 4. Hecht, V.: Die Riesensellenpneumonie hn
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Kindesalter, ein Historich-Experimentelle Studie, Beitr. Path. Anat. 48: 263, 1910. Denton, J.: The pathology of fatal measles, Amer. J. Med. Sci. 169: 531, 1925. Warthin, A. S,: Occurrence of numerous large giant cells in tonsils and pharyngeal mucosa in the prodromal stage of measles, Arch. Path. 11: 864, 1931. Finkeldey, W.: t,)ber Riesenzellbefunde in den Gaumenmandeln, Zugleich ein Beitrag zur Histopathologie der Mandelveranderungen im Maserninkubatlonsstadium, Arch. Path. Anat. 281: 323, 1931. Herzberg, M.: Giant cells in the lymphoid tissue of the appendix in the prodromal stage of measles, J. A. M. A. 98: 139, 1932. Hathaway, B. M.: Generalized dissemination of giant cells in lymphoid tissue in prodromal stage of measles, Arch. Path. 19: 819, 1935. Semsroth, K. H.: Multinucleate epithelial giant cells with inclusion bodies in prodromal measles: Report of autopsy, Arch. Path. 28: 386, 1939. Roberts, G. B. S., and Bain, A. D.: The pathology of measles, J. Path. Bact. 76: 111, 1958. Ruckle, G.: Studies with measles virus. II. Isolation of virus and immunologic studies in persons who have had the natural disease, J. Immun. 78: 341, 1957. Enders, J. F., Peebles, T. C., McCarthy, K., Milovanovie, M., Miltus, A., and Holloway, A.: Measles virus: A summary of experiments concerned with isolation, properties, and behavior, Amer. J. Public Health 47- 275, 1957. Broadhurst, J., MacLean, M. E., and Saurino, V.: Inclusion bodies in measles, J. Infect. Dis. 61: 201, 1937.
The Journal o/ Pediatrics July 1970
15. Semsroth, K. H.: Multinucleate epithelial giant ceils with inclusion bodies in prodromal measles, Arch. Path. 28: 386, 1939. 16. Stryker, W. A.: Disseminated giant cell reaction: A possible prodrome of measles, Amer. J. Dis. Child. 59: 468, 1940. 17. Tompkins, Y., and Macaulay, J. C.: A characteristic cell in nasal secretions during prodromal measles, J. A. M. A. 157: 711, 1955. 18. Roberts, G. B. S., and Bain, A. D.: The pathology of measles, J. Path. Bact. 76: 111, 1958. 19. Beale, A. J., and Campbell, W.: A rapid cytological method for the diagnosis of measles, J. Clin. Path. 12: 335, 1959. 20. Mottet, N. K., and Szanton, V.: Exfoliated measles giant cells in nasal secretions, Arch. Path. 72: 434, 1961. 21. Auger, W. J.: An original method of obtaining sputum from infants and children, J. PED~AT. 15: 640, 1939. 22. Bolande, R. P.: Inclusion-bearing cells in the urine, Pediatrics 24: 7, 1959. 23. Matumoto, M.: Multiplication of measles virus in cell cultures, Bact. Rev. 30: 152, 1966. 24. Aoyama, Y.: Changes of cultured cells infected with measles virus, Jap. J. Exp. Med. 29: 535, 1959. 25. Milles, G.: Measles pneumonia (with a note on the giant cells of measles), Amer. J. Clin. Path. 15: 334, 1945. 26. Adams, J. M., Baird, C., and Filloy, L.: Inclusion bodies in measles encephalitis, J. A. M. A. 195: 150, 1966. 27. Gresser, I., and Katz, S. L.: Isolation of measles virus from urine, New Eng. J. Med. 263: 452, 1960.