TRANSACTIONSOF THEROYALSOCIETYOFTROPICALMEDICINEAND H~~m1~(1992)86,298-300

Concomitant intestinal adenovirus infection and pulmonary cytomegalovirus infection in children causing fatal enteritis and pneumonia

Thomas Butler*, Dale Dunn and Jane Colmer Departments of Internal Medicine and Pathology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA and the International Centre for Diarrhoeal Disease Research, G.P.O. Box 128, Dhaka, Bangladesh

Abstract Three children in Bangladesh who presented with diarrhoea, cough! dyspnoea, fever, and signs of malnutri- tion and died in the hosoital were shown at nest-mortem examination to have both adenovirus infection of the intestine (by immunofluorescence) and cytomegalovirus infection of the lung (by immunoperoxidase staining). This finding of dual viral infections of the intestine and lung in patients with concomitant enteritis and pneumonia provides a basis for symptoms emanating simultaneously from these two organ systems.

Introduction The leading causes of childhood death in developing

countries are pneumonia (DENNY & LODA, 1986) and diarrhoea (SNYDER & MERSON, 1982), and some natients present with both conditions simultaneously (BUTLER et al., 1987). The occurrence of distinct groups of infec- tious pathogens in the respiratory tract (BERMAN & MCINTOSH, 1985) and intestinal tract (GUERRANT et al., 1983) has been invoked to exnlain diseases of the two or- gans: The viral pathogens, cytomegalovirus and adeno- virus, may infect both the lung and the intestine (HO, 1982; SHIELDS et al., 1985). Since both viruses produce giant cells with intranuclear inclusions, their differentia- tion in tissues sometimes requires immunological stain- ing.

Materials and Methods Patients admitted to the Dhaka hospital of the Interna-

tional Centre for Diarrhoeal Disease Research, Ban- gladesh with histories of diarrhoea and/or pneumonia who died were eligible for inclusion in an autopsy proto- col after informed consent was obtained. The 3 patients described in this naner were included in a nrevious de- scription of 140 p&i&its in an autopsy protocol (BUTLER et al.. 1987) and a histoloaical analvsis of 93 uatients with pneumonia (TOMASHEFS~Y et al.,” 1989). Inpatient care consisted of rehydration with intravenous fluid (contain- ing sodium 133 mmolilitre, potassium 13 mmolilitre, chloride 98 mmolilitre, and acetate 48 mmol/litre) and liberal antibiotic usage. Autopsies were done using stand- ard procedures. In 3 cases which showed intranuclear in- clusions in both lung and intestine, sections were depar- affinized, treated with 1% bovine serum albumin in phosphate-buffered saline, and stained with the indirect immunofluorescent stain for adenovirus using Bartel’s viral respiratory screening and identification kit (Baxter Microscan, West Sacremento, California, USA) with an affinity purified mouse monoclonal antibody directed against types 1-3, 5-8, 14, 18, 31, 40 and 41 (GARY et al., 1979). Cytomegalovirus was detected by immunoper- oxidase staining (Str Ari Gen@, Biogenex Laboratories, San Ramon, California, USA) (WILCOX et al., 1990).

Results Patients

The 3 children presented with histories of diarrhoea and respiratory complaints and all appeared poorly nourished.

Case I. A 4 month-old female nresented with a historv of watery stools 5 times a day for 2 d and dyspnoea for one dav. Her mother had died in childbirth. and the na- tient had failed to gain weight on soya milk feeding since birth. Her temperature was 38.9’C and chest radio- graphy showed infiltrates in upper lung fields. Stool examination showed 3-12 leucocytes per high power field, no ova or parasites, and negative culture for bac- terial pathogens. A blood culture was negative. Blood leucocyte count was 8200/mm3 with 40% lymphocytes. “Author for offprinr requests.

The blood CO2 content was reduced to 10.7 mmolilitre. The patient was treated with ampicillin for suspected bacterial nneumonia but died on the fifth dav in hosnital.

Case 2: A one month-old male was brought &I the clinic with a history of watery stools 10 times a day for one day and development of cyanosis and obtund&on for one dav. He had been fed on a combination of breast milk and goat’s milk. His temperature was 38.3”C. Stool showed 8 leucocytes per high power field, no ova or para- sites, and negative culture for bacterial pathogens. The blood CO2 content was reduced to less than 7 mmolilitre and serum glucose was reduced to 10 mgilO0 ml. The patient received glucose infusion but died on the day of admission before further diagnostic tests could be carried out.

Case 3. A 10 year-old female presented with a history of loose stools, sometimes blood-stained, for 30 d and a drv cough for 20 d. About 20 d before admission she noticed ‘ihe onset of weight loss and pedal oedema. Her temperature was normal but subsequently in hospital she showed temperature spikes. Chest radiography showed interstitial infiltrates. Stool examination showed 50 leu- cocytes per high power field and ova of Ascaris Zumbri- coides and Trichuris tt-ichiura. but negative culture for bac- terial pathogens. Blood leucocyte count was raised at 26 800 /mm3 with 37% lvmnhocvtes. Blood orotein concen- tration was reduced to ‘3.3 g/l00 ml. A blood culture was negative. The patient received ampicillin for sus- pected bacterial pneumonia but died on the tenth day in hospital.

Gross autopsy findings In case 1, the serosal surface of the distal ileum was

purple and the ileal mucosa was friable with areas of ul- ceration and darkened discolouration. In cases 2 and 3, the mucosa of the distal ileum and colon showed areas of erythema and superficial ulceration. The lungs of all cases showed scattered areas of congestion, purple col- ouration, and induration indicative of inflammation and consolidation.

Microscopical findings In all 3 cases, the lamina propria of the ileum and

colon showed areas of infiltration with nredominantlv mononuclear cells and the presence of occasional large cells with intranuclear inclusions. In areas of mucosal ul- ceration, there were polymorphonuclear leucocytes. The lungs in all 3 cases showed infiltration of interstitial areas with mononuclear leucocytes and the presence of cyto- megalovirus inclusion cells. Other organs that were af- fected by giant cells with intranuclear inclusions were the kidney in case 1, liver in cases 2 and 3, and nancreas in case 3. Indirect -immunofluorescent staining-for adeno- virus gave positive results on inclusions in the colons of all 3 cases -(Figure) and gave negative results when ap- plied to the lunas. Immunoperoxidase staining for cvto- megalovirus gave negative results when app&d to col- onic lesions but positive results for the lungs of all 3 cases.

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Figure. Colon of case 1. A. The lamina propria showing an infiltrate of pn elusions (arrow). Haematoxylin and eosin (X 100). B. Intranuclear inclusion

Discussion Only one case of diarrhoea caused by enteric adeno-

virus has been previously reported as fatal (MADELEY, 1986). Adenovirus and cytomegalovirus have not hereto- fore been considered important pathogens in tropical de- veloping countries for either diarrhoeal syndromes or pneumonia. The viruses that have been best studied in developing countries are rotavirus in the intestine (GUER- RANT et al., 1983) and respiratory syncytial virus and adenoviruses in the lung (BERMAN & MCINTOSH, 1985). A prevalence of 100% for cytomegalovirus infection has been reported in serosurveys in the Philippines, Uganda, and Tanzania (HO, 1982), but the prevalence of sympto- matic disease is unknown. In India, children with acute diarrhoea were shown to be excreting non-enteric or en- teric adenoviruses in about 2% and 13% of 2 samples of patients (BHAN et al., 1988), and in Brazil about 5% of chidlren with diarrhoea excreted adenoviruses that were enteric types in 36% of the cases (LEITE et al., 1985). Serological testing of children for adenoviruses of the en- teric types 40 and 41 in both developing and developed countries showed prevalences of infection ranging from 0 to 60% (KIDD et al., 1983).

Our finding of inflammation and ulceration in the colon are consistent with other reports of pathology of the gastrointestinal tract in immunocompromised pa- tients with adenovirus infection. OKANO et al. (1988) re- ported fatal intestinal bleeding in a patient with chronic Epstein-Barr infection who had colonic ulcers that were demonstrated by immunological staining to contain both adenovirus and Epstein-Barr virus.

Immunodeficiency is frequently present in sympto- matic infection due to adenovirus or cytomegalovirus, as is recognized now in patients with cancer treated with chemotherapy, acquired immune deficiency syndrome (AIDS), and after renal and bone marrow transplants (PETERSON et al., 1980; REED et al., 1990; SHIELDS et al., 1985; WILCOX et al., 1990). The malnutrition that af- fects children in developing countries is characterized by defective cell-mediated immunity (CHANDRA, 1983). The normal peripheral blood lymphocyte counts in our pa- tients suggest that the immune deficiency predisposing them to adenovirus and cytomegalovirus infections was qualitatively different from that of AIDS.

Acknowledgements We thank Dr Moyenul Islam and Dr A. K. Azad for their as-

sistance.

References Berman, S. & McIntosh, K. (1985). Selective primary health

care: strategies for control of disease in the developing world. XXI. Acute respiratory infections. Reviews of Infectious Dis- eases, 7,674-691.

ominantly mononuclear cells with enlarged cells demonstrating intranuclear in- in crypts positive for adenovirus (arrow). Immunofluorescent stain (x400).

Bhan, M. K., Raj, I’., Bhandari, N., Svensson, L., Stintzing, G., Prasad, A. K., Jayashree, S. & Srivastava, R. (1988). Role of enteric adenoviruses and rotaviruses in mild and severe acute enteritis. Pediatric Infectious Disease Journal, 7, 320.323.

Butler, T., Islam, M., Azad, A. K., Islam, M. R. & Speelman, P. (1987). Causes of death in diarrhoeal diseases after rehy- dration therapy: an autopsy study of 140 patients in Ban- gladesh. Bulletin of the World Health Organization, 65, 317-321.

Chandra, R. K. (1983). Nutrition, immunity, and infection: present knowledge and future directions. Lancet, i, 688-691.

Denny, F. W. & Loda, F. A. (1986). Acute respiratory infec- tions are the leading cause of death in children in developing countries. American Journal of Tropical Medicine and Hygiene, 35, l-2.

Gary, G. W., jr, Hierholzer, J. C. & Black, R. E. (1979). Char- acteristics of noncultivable adenoviruses associated with diar- rhea in ‘infants: a new subgroup of human adenoviruses. Journal of Clinical Microbiology, 10,96103.

Guerrant, R. L., Kirchhoff, L. V., Shields, D. S., Nations, M. K., Leslie, J., de Sousa, M. A., Araujo, J. G:, Correia, L. L., Sauer, K. T., McClelland, K. E., Trowbridge, F. L. & Hughes, J. M. (1983). Prospective study of diarrhea1 illnesses in north-eastern Brazil: patterns of disease, nutritional im- pact, etiologies, and risk factors.Journal of Infectious Diseases, 14,986-997.

Ho, M. (1982). Cytomegalovirus. Biology and Infection. New York: Plenum Medical Book Company, pp. 79-104.

Kidd, A. H., Banatvala, J. E. & de Jong, J. C. (1983). Anti- bodies to fastidious faecal adenoviruses (species 40 and 41) in sera from children.Joumal of Medical Virology, 11,333-341.

Leite, J. I’. G., Pereira, H. G., Azeredo, R. S. & Schatzmayr, H. G. (1985). Adenoviruses in faeces of children with acute gastroenteritis in Rio de Janiero, Brazil. 3ournal of Medical Virology, 15,203-209.

Madeley, C. R. (1986). The emerging role of adenoviruses as in: ducers of gastroenteritis. Pediatric Infectious Disease, 5, S63- Sh4.

Okano, M., Thiele, G. M., Davis, J. R., Nauseef, W. M., Mi- tros, F. & Purtilo, D. T. (1988). Adenovirus type-2 in a pa- tient with lethal hemorrhagic colonic ulcers and chronic active Epstein-Barr virus infection. Annals of Internal Medicine, 108, 693-699.

Peterson, P. K., Balfour, N. H., Marker, S. C., Fryd, D. S., Howard, R. J. & Simmons, R. L. (1980). Cytomegalovirus disease in renal allograft recipients: a prospective study of the clinical features, risk factors and impact on renal transplanta- tion. Medicine, 59, 283-300.

Reed, E. C., Wolford, J. L., Kopecky, K. J., Lilleby, K. E., Dandliker, P. S., Todaro, J. L., McDonald, G. B. & Meyers, G. D. (1990). Ganciclovir for the treatment of cytomegalo- virus gastroenteritis in bone marrow transplant patients. An- nals of Internal Medicine, 112,505-5 10.

Shields, A. F., Hackman, R. C., Fife, K. H., Corey, L. & Meyers, J. D. (1985). Adenovirus infections in patients undergoing bone-marrow transplantation. New EnglundJour- nal ofMedicine, 312,529-533.

Snyder, J. D. & Merson, M. H. (1982). The magnitude of the

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global problem of acute diarrhoeal disease: a review of active surveillance data. Bulletin of the World Health Organization, 60,605-613.

Tomashefsky, J. F., Butler, T. &Islam, M. (1989). Histopatho- logy and etiology of childhood pneumonia: an autopsy study of 93 patients in Bangladesh. Pathology, Z&71-78.

Wilcox, C. M., Diehl, D. L., Cello, J. P., Margaretten, W. &

Jacobson, M. A. (1990). Cytomegalovirus esophagitis in pa- tients with AIDS. A clinical, endoscopic, and pathological correlation. Annalsof Internal Medicine, 113,589-593.

Received I7 June 1991; revised 2 September 1991; accepted for publication I3 September 1991

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1992) 86,30&30 1

1 Short Report 1

Tropical spastic paraparesis associated with HTLV-1 in Egypt*

Niel T. Constantine’, Daniel A. Scott’, Mohamed Kamalz, Chester R. Robert+, Arndt Rolfs4, H. C. Schumacher4 and Peter Marx4 IUS Naval Medical Research Unit No. 3, Cairo, Egypt; 2Abbassia Fever Hospital, Cairo, Egypt; 3Walter Reed Army Institute of Research, Washington, DC, USA; “Free University of Berlin, Berlin, Germany

Human T-lymphotrophic virus type 1 (HTLV-1) is as- sociated with a form of malignancy, adult T-cell leukae- mia (BLATTNER et al., 1982), a-chronic degenerative neurological disease. HTLV-l-associated mvelonathv, and tropical spastic ‘paraparesis (TSP) (GE&N et ai.; 1985; RODGERS-JOHNSON et al., 1988). This report de- scribes the first documented case of HTLV-l-associated TSP in a patient from Egypt. The patient was a 45 years old man who appeared healthy, but complained of weak- ness in his legs and inability to run. Neurological symp- toms first appeared in 1976, one year following a 6-litre blood transfusion for a bleeding ulcer. At that time, the patient experienced a gradual onset of urinary difficulty, constipation, and impotency. Over the next 12 years, he gradually developed weakness in his left, then right, leg, without discrete remissions or exacerbations. There was no history of paresthaesias, visual disturbances, or a fam- ily history of neurological disease.

Neurological examination revealed hyper-reflexia in both lower extremities, bilateral Babinski signs, and a scissors gait. There was slight spasticity in the lower ex- tremities without wasting or fasciculations. Cranial nerves and the upper extremities were normal, as well as a normal sensory examination, a negative Romberg sign, and normal fine motor co-ordination. Cranial and spinal magnetic resonance imagery did not reveal mass lesions or white matter plaque. Cerebral spinal fluid (CSF) ana- lysis revealed 34 mg/dl protein, 50 mgidl glucose, and 20 lymphocytesidl. Blood chemistries and a complete blood count were normal, and a serum rapid plasma re- agin test and a fluorescent treponemal antibody test were negative for Treponema pallidurn.

Serological screening for HTLV-1 by enzyme immu- noassay (EIA) was repeatedly reactive and the presence of antibodv to HTLV-1 was confirmed bv Western blot (strong reactivity to all major antigens including gp46) and radio-immunonrecinitation (RIPA). The CSF was

*This study was supported by the Naval Medical Research and Development Command, Naval Medical Command, National Capital Region, Bethesda, MD 20814, Work Unit No. 3M463105.H29.AA.335. The opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the view of the Navy Department, the Department of Defense, the Government of the United States or the World Health Organization.

Gessain, A., Barin, F., \Je&ant; J. C., Gout, O., Maurs, L., Calender, A. & de The, G, (1985). Antibodies to human T- lymphotropic virus type-l in patients with tropical spastic paraparesii. Lancet, ii, 407-410.

Correspondence and requests for Gffprints should be addressed to: Research Publications Branch, NAVMEDRSCHU

Khalifa, A. S., Khalil, R., Gawad, A. A., Constantine, N. T. & Woodv, 1. N. (19901. Surveillance for exoosure to HTLV-1

THREE, code: lOlA, PSC 452 Box 5000, FPO AE 09835-0007, USA.

in E&titian iniants grid children with vaiious malignancies. Journal of Infectious Diseases, 162,995-996.

also reactive for antibodies to HTLV-1 by EIA, and was confirmed as positive by Western blot and RIPA. Per- ipheral lymphocytes were positive for HTLV-1 deoxyri- bonucleic acid bv the Dolvmerase chain reaction. Several env, gag, pol, and LFR legions were detected and, by comnarison to the MT-2 reference cell line. there was one HTLV-1 gene per 15 000 mononuclea; cells. At- tempts to culture the virus from either the CSF or per- ipheral lymphocytes were unsuccessful. Sera collected from the natient’s wife. 13 vear old daughter. mother. and broth& were all negativi for antibodks to’HTLV-i by EIA. His father was deceased.

The patient’s symptoms and neurological examination were compatible with TSP, and the laboratory data sug- gested an association with HTLV-1 infection. The epi- demiological association of TSP with HTLV-1 has been well documented (GESSAIN et al., 1985) and HTLV-1 has been isolated from the DeriDheral 1vmDhocvtes and CSF of patients with TSI? (GCKHANN et-al.; 1985).

The most likely source of HTLV-1 was the blood transfusion received by the patient in 1975. Initial obser- vations in Japan suggested that TSP was contracted by blood tranif&ion a;d more recent data confirmed an ai- sociation between TSP and HTLV-1 infection acauired by transfusions (OSAME et al., 1986; OSAME & ~~ATA, 1989). Patients in Japan had an onset of symptoms within 12 months of their transfusion, as described for the patient in this report.

The prevalence of HTLV-1 infection is low in north- east Africa and the Middle East (FOX et al., 1988; OSAME & IGATA, 1989; SCOTT etal., 1989; CONSTANTINE etal., 1989: KHALIFA et al.. 1990) and the onlv other case of TSP’reported from tLe region was an Eihiopian immi- grant to Sweden (RYBERG et al., 1987). This report is of the first case of HTLV-l-associated TSP in a resident of Egypt which was apparently contracted by blood transfu- sion.

We thank Dr Zoheir Farid, Prof. Dr Mamdouh Salama and Dr N. I. Girgis for their assistance with the evaluation of this patient, and Dr Douglas M. Watts for reviewing the manu- script.

References Blattner, W. A., Kayanaraman, V. S., Robert-Guroff, M., Lis-

ter, T. A., Galton, D. A., Sarin, I’. S., Crawford, M. H., Catovsky, D., Greaves, M. & Gallo, R. C. (1982). The human type-C retrovirus, HTLV, in Blacks from the Carri- bean region, and relationships to adult T-cell leukemiailym- phoma. InternationalJournal of Cancer, 30,257-264.

Constantine, N. T., Corwin, A., Scott, D. A., Mohamed, M. A., Osman, N. M. & Watts, D. M. (1989). HTLV-1 infec- tion among blood donors, children, and high risk groups in north-east Africa. 38th Annual Meeting of the American So- ciety of Tropical Medicine and Hygiene, Honolulu, December 1989. Abstract no. 333.

Fox, E., Constantine, N. T., Abbatte, E. A., Said-Salah & Ro- dier, G. (1988). Low prevalence of infection by human T- lymphotropic virus type 1 in populations at risk for human immunodeiiciency virus in Djibouti, East Africa. Annales de I’lnstitut PasteurlViroloeie. 139.443-447.