684847 (lin Ilathol 1992;45:684--688 Adenovirus infection of the large bowel in HIV positive patients J Clin Pathol: first published as 10.1136/jcp.45.8.684 on 1 August 1992. Downloaded from A Maddox, N Francis, J Moss, C Blanshard, B Gazzard

Abstract Recently Janoff et al showed the presence of Aims: To describe the microscopic adenovirus infection of colonic epithelial cells appearance of adenovirus infection in the in five HIV positive patients with diarrhoea, large bowel of human immunodeficiency two cases of which were confirmed by electron virus (HIV) positive patients with diar- microscopy and two by immunofluorescence rhoea. of cultured colonic epithelial cells.5 We now Methods: Large bowel biopsy specimens report the histopathological, immunocyto- from 10 HIV positive patients, eight of chemical, and electron microscopic findings in whom were also infected with other gas- 10 patients with colonic epithelial adenovirus trointestinal pathogens, with diarrhoea infection. were examined, together with six small bowel biopsy specimens from the same group of patients. Eight of the patients Methods had AIDS. The biopsy specimens were Details of patients and their clinical histories examined by light microscopy performed are shown in the table. All were homosexual on haematoxylin and eosin stained and men and all had mild to moderate proctitis on immunoperoxidase preparations, the lat- sigmoidoscopy. Two (cases 3 and 8) had ter using a commercially available anti- contact bleeding. Nine of 10 had a noticeable body (Serotec MCA 489). Confirmation reduction in CD4+ cells to less than 55 mm3 was obtained with electron microscopy. and eight had other gastrointestinal infections Results: The morphological appearance of which might cause diarrhoea. cells infected with adenovirus showed Tissue sections were prepared after standard characteristic nuclear and cellular chan- fixation in neutral buffered formalin and pro- ges, although the inflammatory reaction cessing to paraffin wax. Sections 4 um thick was non-specific. Immunoperoxidase were stained with haematoxylin and eosin, staining for adenovirus was sensitive and periodic acid-Schiffand alcian blue, and exam- specific, and the presence of viral inclu- ined by light microscopy. Control cases were http://jcp.bmj.com/ sions consistent with adenovirus was con- taken from three HIV positive patients in firmed by electron microscopy. whom no infection was found, one patient with Conclusions: The light microscopic fea- CMV colitis, four patients with cryptospor- tures of adenovirus infection are distinc- idiosis and one patient with graft versus host- tive and immunocytochemistry with a like changes in their colorectal biopsy speci- commercially available antibody is a sen- mens. sitive and specific means of confirming Immunocytochemical studies were per- on October 2, 2021 by guest. Protected copyright. the diagnosis. Further studies of the role formed with adenovirus primary antibody of adenovirus in causing diarrhoea in (Serotec No MCA 489) at a dilution of 1 in these patients are indicated. 1000 on all test and control cases. Sections were mounted on poly-l-lysine coated slides, subjected to 15 minutes protease digestion and Diarrhoea and gastrointestinal symptoms are incubated with primary antibody for one hour. common in HIV positive patients. Almost all Secondary antibody and avidin-biotin complex patients with AIDS report diarrhoea at some were incubated for one hour each and after time during the course of their disease. Poten- washing developed with diaminobenzidine for Department of tially causative infectious agents are recovered five minutes. Histopathology, Charing Cross and from 49%1 to 85%2 of these patients. There is Two other control antibodies were applied to Westminister Medical a higher rate of detection in patients with selected test and control sections: primary School, 17 Horseferry AIDS. The association between symptoms and anti-CMV antibody (Dakopatts) using a pre- Road, London SWlP 2AR some of the organisms is unclear. viously described method4; and primary anti- A Maddox Adenovirus infection is a known cause of herpes simplex type 1 antibody (Dakopatts N Francis diarrhoea in children. It has also been isolated B 1 4 polyclonal) at a dilution of 1 in 100 with J Moss from HIV positive patients. The first sugges- incubation and development times as above Department of tion of an association between adenovirus and Gastroenterology, Staining procedures were run with known Westminster Hospital diarrhoea or colitis in AIDS was reported in CMV and herpes simplex positive controls and C Blanshard two patients in whom adenovirus was isolated negative controls of test cases without primary B Gazzard from a culture ofbiopsy tissue.3 However, both antibody. Correspondence to: had histological evidence of cytomegalovirus Paraffin wax sections were dewaxed, Dr N Francis confirmed electron to in 2% aqueous Accepted for publication (CMV) infection, by rehydrated water, postfixed 31 January 1992 microscopy and immunocytochemistry.4 osmium tetroxide and then dehydrated in Adenovirus infection of the large bowel in HIV positive patients 685

Clinical histories ofpatients studied Initial presentation Pathology Centers for Other Other Disease Weight Adenovirus gastrointestinal gastrointestinal Case T,I control Diarrhoea loss Biopsy cell/20 infections infections J Clin Pathol: first published as 10.1136/jcp.45.8.684 on 1 August 1992. Downloaded from No Age mm' classification Duration per day (kg) site crypt (biopsy) (stool) Treatment Follow up 1 32 12 4C1 AIDS 3/12 6-7 x 0 (a) SI 0 liquid/soft R 40 AZT Ongoing (b) R 10 ? CMV diarrhoea (c) R 7 (d) Ca 1 CMV AZT Diarrhoea TC 4 stopped resolved R 35 2 32 38 4C1 AIDS 5/12 Constipated 3 (a) R 5 CMV Foscarnet haemato- A 0 CMV+HPV AZT Died chaezia (b) R 25 Acyclovir 3 49 10 4C1 AIDS 4/12 5-6 x 4 (a) SI 0 Campylobacter Ganciclovir stool vol R 160 CMV Foscarnet 2L Ciprofloxacin Resolved (b) R 0 CMV Clear, then recurred (c) R 10 Resolved 4 35 54 4C2 ARC 4/12 2-3 x soft 0 (a) R 10 Spiro Giardia Acyclovir Resolved Metronidazole (b) R 7 AZT 5 37 46 4C1 AIDS 2/12 6-7 x liquid 4 (a) Oe 0 CMV Crypto Acyclovir SC 1 Crypto AZT Improved 6 42 23 4C1 AIDS 6/12 6-7 x liquid 10 (a) R 1 (b) SI 0 Micro AZT Ongoing (c) R 1 diarrhoea 7 35 29 4C1 AIDS 1/12 5-8 x soft 3 (a) R 5 DDI Improved SI 0 Acyclovir after 2/12 8 22 976 Asymp- 2/12 4-6 x soft/ 6 (a) R 1 tomatic liquid (b) R 0 Ongoing DC 0 Spiro diarrhoea SI 0 9 36 34 4C1 AIDS 3/12 2-3 x soft 2 (a) R 1 Spiro. GvHd (b) R 1 Spiro (c) AC 1 CMV Acyclovir TC 1 Spiro Foscarnet Resolved SC 0 10 49 49 4C2 ARC 7/52 6 x soft/ 5 (a) R 5 liquid SI 0 Micro DDI Ongoing Oe 0 diarrhoea Key Oe: Oesophagus, SI: Small intestine, Ca: Caecum, AC: Ascending colon, TC: Transverse colon, DC: Descending colon, SC: Sigmoid colon, R: Rectum, A: Anus, HPV: Human papillomavirus, GvHd: Graft versus host disease, AZT: Azidothymidine (Zidovudine), DDI: Dideoxyinosine, Spiro: Spirochaetosis, Crypto: Cryptosporidiosis, Micro: Microsporidiosis.

ascending grades of ethanol. The tissue was having abnormal cells in the superficial epithe- embedded by inverting a capsule of Spurr's lial layer at the time of light microscopic resin over the selected area of the section examination. One case was discovered in a http://jcp.bmj.com/ (chosen by light microscopy), and polymerised patient with graft-versus-host-like changes in overnight at 60°C. The embedded material was the control group after adenovirus antibody removed from the glass slide by repeated immunocytochemical staining, but on review application of steam and a rod cooled in liquid of the haematoxylin and eosin stained sections nitrogen to the underside of the slide. Ultra- abnormal cells were recognisable. thin sections were stained with aqueous uranyl The morphology of the infected cells and the acetate and Reynold's lead citrate and then associated histological changes in the large on October 2, 2021 by guest. Protected copyright. examined by transmission electron micro- bowel mucosa are illustrated in figs 1 and 2. scopy. The infected cells were almost always in the surface epithelium or crypt mouth and the nuclei often located immediately adjacent to Results (table) the basement membrane. In one case we found LIGHT MICROSCOPY an infected cell in the base of a crypt. We found All but one of the cases were identified as no infected endothelial or stromal cells, in

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Figure 1 Cell infected with adenovirus with irregular Figure 2 Six infected cells displaying crescentic amphophilic nucleus (note basal position) (haematoxylin nuclei in vacuolated cytoplasm (haematoxylin and and eosin). eosin). 686 Maddox, Francis, Moss, Blanshard, Gazzard

vacuolar areas of the cytoplasm were positive for acid sialomucins (normal for the site). The associated mucosal appearances were rather non-specific and involved a mild to moderate chronic inflammatory infiltrate. J Clin Pathol: first published as 10.1136/jcp.45.8.684 on 1 August 1992. Downloaded from Neutrophils were only occasionally present. The epithelium showed some disarray, with an irregular, serrated epithelial surface, nuclear

*; pseudostratification, and blurring of the boundary between epithelial cell base and lamina propria. There was a variable degree of oedema and apoptotic nuclear debris. These changes did not seem to correlate with virus load, which we estimated by counting the number of positive cells in a length of mucosa roughly equal to 20 crypt widths. In our patients this varied from 1 to 160. Patients at either end of this scale had similar epithelial and inflammatory changes. Interestingly, and perhaps not surprisingly, the two patients with the greatest numbers of infected cells had the lowest CD4 counts. In case 1 who had colonoscopic survey with multiple biopsy Figure 3 Multiple infected cells showing strongly positive nuclear staining with specimens, the number of infected cells adenovirus monoclonal antibody (immunoperoxidase). increased with distance from the ileo-caecal valve.

contrast to CMV. Because the cytoplasm is IMMUNOCYTOCHEMISTRY (table) usually filled with mucin and vacuolated, the Optimal protease digestion, antibody dilu- low power appearance is that of a goblet cell tions, and incubation times were established lying below the level of epithelial nuclei in that using sections from case 1 in which electron area. The nuclear changes range from that of a microscopy had shown the presence of intra- crescent or sickle-shaped amphophilic nucleus nuclear viral particles of 60-80 nm with within a vacuolated cytoplasm to a moderately morphology consistent with adenovirus. The enlarged nucleus with an irregular outline cells with cytopathic changes showed strong which appears amphophilic at low power, but blackish-brown nuclear staining with the which at high power often resolves into a adenovirus antibody (fig 3), which often number of variably sized small intranuclear delineated individual intranuclear inclusions inclusions; these may coalesce and fill the (fig 4) and revealed cells, particularly with a http://jcp.bmj.com/ nucleus. These two extremes of nuclear mor- sickled nuclear morphology, that it would not phology probably represent the same changes be possible to ascertain were infected with seen in different planes. With mucin stains, the adenovirus on haematoxylin and eosin staining alone. There was no positive staining in mor- phologically normal cells and there was no false positive staining of the CMV or herpes control cases. Furthermore, in six patients on October 2, 2021 by guest. Protected copyright. duodenal biopsy specimens, obtained at the same time as the rectal ones, were consistently negative.

ELECTRON MICROSCOPY In three cases with relatively few infected cells the cells could not be found after lifting off parts of the paraffin wax sections. In one case (case 1) infected cells were successfully obtained for electron microscopy and showed closely packed intranuclear crystalline arrays of viral particles of a uniform morphology and hexagonal/rectangular pattern, 60-80 nm in size (fig 5). In case 1, with large numbers of infected cells, a greater number showed intra- nuclear viral particles than were detected by light microscopy. Although the size is margin- ally smaller than adenovirus in culture or from stool isolates (70-80 nm), the size difference is compatible with shrinkage artefact due to recovery from processed and fixed tissue. The particle size is too large for polyoma (40-50 Figure 4 Three infected cels showng individual nuclear inclusions positive with nm) or parvovirus (30-45 nm), and too small adenovirus antibody (immunoperoxidase). for cytomegalovirus. Adenovirus infection of the large bowel in HIV positive patients 687 J Clin Pathol: first published as 10.1136/jcp.45.8.684 on 1 August 1992. Downloaded from http://jcp.bmj.com/ on October 2, 2021 by guest. Protected copyright. .. AdO ` - f Figure 5 Electron micrograph showing intranuclear viral particles and (inset) regular pattern of arrangement. Bar = 5 pm, inset bar = 200 nm; L = lumen.

Discussion patients with HIV disease and some of the Diarrhoea is a common and debilitating prob- earliest reports of AIDS mentioned this.'3 14 lem in patients infected with HIV, particularly Many different types are found, urine isolates those who have AIDS. The organisms com- often being those of subgenus B,'5 16 while monly found include cryptosporidia, micro- stool isolates are mainly subgenus D."6 Several sporidia, salmonella, Mycobacterium avium workers have investigated the problem of diar- intracellulare (MAI) and cytomegalovirus rhoea in HIV disease to determine the propor- (CMV). The role of adenovirus as an enteric tion of patients in whom pathogens are found pathogen in HIV is uncertain, although it has and to characterise these pathogens. Some been implicated in fatal hepatic infection in have included adenovirus in their assessment. HIV67 and in other immunodeficiency Of those that have not, Connolly et al found an states.6 8 It is known to be associated with a infective cause in 33% of patients with AIDS frequently fatal gastroenteritis in bone marrow related complex and 87% of patients with transplant recipients9 '0 and is a common AIDS with non-cryptosporidial diarrhoea cause of respiratory tract infections and gastro- investigated as inpatients. 17 Together with pos- enteritis in otherwise healthy children." 12 In itive cryptosporidia cases reported separate- these groups with gastroenteritis adenovirus ly,"8 this represents 77 (72%) of a consecutive types 40 and 41 (subgenus F) are the usual series of 107 patients with diarrhoea. Similarly, isolates. Smith et al found an infective pathogen in 17 of Adenovirus can be isolated from many 20 (85%) patients with AIDS with diarrhoea 688M6laddox, Frac,i.s, Mo ss, BI3lonhard, (Gazzard

on histological and microbiological investiga- relative contribution, if any, of any one of a set tion. of potential pathogens. However, the epithelial By contrast, Rolsten et al found 38% of HIV damage and disarray in areas containing cells patients had an identifiable intestinal patho- infected with adenovirus demonstrates a gen. " Intestinal biopsy specimens showed cytopathic effect and therefore a possible J Clin Pathol: first published as 10.1136/jcp.45.8.684 on 1 August 1992. Downloaded from non-specific inflammation. Dryden et al exam- pathogenic role on histological grounds. ined 441 stool samples from 179 consecutive HIV positive patients with diarrhoea.' An We thank Pina Sannino for immunocvtochemistry wsork, Ron infective aetiology was found in 49% of Barnett for photography, and Ian Shore for clcctron micros- patients. copy. The percentage of patients with AIDS and diarrhoea in whom an infective cause is not 1 Dryden MS, Shanson DC. The microbial causes of diar- found thus ranges from 15% to 51%. These rhoea in patients infected with the human immunodefi- differences be due to several ciency virus. _lInfect 1988; 17:107- 14. may factors, 2 Smith PD, Clifford Lane H, Gill VJ, et al. Intestinal including the type and frequency of sampling, infections in patients with the acquired immunodefi- the local ciency syndrome (AIDS). Ann Intern Med pathogen sought, variations in indi- 1988;108:328-33. vidual pathogens, or other unknown causes. A 3 Parkin J, Tvms S, Roberts A, Burnell R, Jeffries D, Pinching recent found of HIV A. "Cvtomegalovirus" colitis. Can it be caused by prospective study 30% adenovirus? III International Conference on AIDS. positive patients with diarrhoea had no identi- 1987;1: 159. fiable infective cause. 4 Francis ND, Boylston AW, Roberts AHG, Parkin JM, Pinching AJ. Cytomegalovirus infection in gastrointesti- Of those investigators who have tested for nal tracts of patients infected with HIV- I or AIDS. 7 Clin adenovirus in studies of diarrhoea in Pathol 1989;42:1055-64. HIV 5 Janoff EN, Orenstein JM, Manischewitz JF, Smith PD. disease, some have found no correlation. Adenovirus colitis in the acquired immunodeficiency et ELISA tests on stool syndrome. Gastroenterologv 1991;100:976 9. Laughon al, using 6 Krilov LR, Rubin LG, Frogel M, et al. Disseminated samples, found no cases of adenovirus in 49 adenovirus infection with hepatic necrosis in patients with with while et human immunodeficiency virus infection and other patients AIDS,2' Kaljot al, using immunodeficiency states. Rev Infect Dis 1990;12:303-7. similar techniques, found four of 77 patients 7 Janner D, Petru AM, Belchis D, Azimi P. Fatal adenovirus adenovirus in their In con- infection in a child with acquired immunodeficiencv secreting stool.22 syndrome. Paediatr Itnfect Dis _7 1990;9:434-6. trast, Cunningham et al, using electron micros- 8 Varki NM, Bhuta S, Drake T, Porter DD. Adenovirus and culture on one or more hepatitis in two successive liver transplants in a child. Arch copy stool samples, Pathol Lab Med 1990,114:106-9. investigated 68 symptomatic HIV positive 9 Yolken RH, Bishop CA, Townsend TR, et al. Infectious with diarrhoea and gastroenteritis in bone marrow transplant recipients. N patients recovered adeno- EnglJ Med 1982,306:1009-12. virus from 18.23 10 Landry ML, Fong CKY, Nedderman K, Solomon L, The of intestinal adenovirus Hsiung GD. Disseminated adenovirus infection in an morphology immunocompromised host. Am I Med 1987,83:555-9. infection is poorly described. The largest affec- 11 Flewett TH, Bryden AS, Davies H, Morris CA. Epidemic ted are viral enteritis in a long-stay children's ward. lancet group otherwise healthy children, when 1975;i:4-5. the site of infection is the small bowel and 12 Uhnoo I, Wadell G, Svensson L, Johansson ME. Impor- therefore infrequently biopsied.24 Reports of tance of enteric adenoviruses 40 and 41 in acute gastroenteritis in infants and young children. Y C/lin http://jcp.bmj.com/ intestinal adenovirus infection in immunocom- Microbiol 1984;20:365-72. to the 13 Gottlieb MS, Schroff R, Schanker HM, et al.Pneumocystis promised patients, however, point large carinii pneumonia and mucosal candidiasis in previously bowel as the site of infection. Recently, Janoff healthy homosexual men. N Engl Y Med et al have described the of 1981;305:1425-31. morphology large 14 Siegal FP, Lopez C, Hammer GS, et al. Severe acquired bowel adenovirus infection in five of 67 homo- immunodeficiencv in male homosexuals, manifested by sexual HIV with 51 chronic perianal ulcerative herpes simplex lesions. N Etng positive patients diarrhoea, Y Med 1981,305:1439-44. of whom had AIDS.' Adenovirus was identi- 15 de Jong PJ, Valderrama G, Spigland I, Horwitz MS. fied transmission electron or Adenovirus isolates from urine of patients with acquired on October 2, 2021 by guest. Protected copyright. by microscopy immunodeficiencv syndrome. Lancet 1983;i:1293-6. culture of the biopsy specimen with sub- 16 Hierholzer JC, Wigand R, Anderson LJ, Adrian T, Gold immunofluorescence. In JWM. Adenoviruses from patients with AIDS: a plethora sequent retrospect, of serotvpes and a description of five new serotypes of three of the patients at light microscopy were subgenus D (types 43-47). y Infect Dis found to have inclusions in colonic 1988;158:804-13. epithelial 17 Connolly GM, Shanson D, Hawkins DA, Harcourt Webster cells which are similar to those we have JN, Gazzard BG. Non-cryptosporidial diarrhoea in described here. human immunodeficiency virus (HIV) infected patients. Gut 1989;30:195-200. The light microscopic appearances of colo- 18 Connolly GM, Dryden MS, Shanson DC, Gazzard BG. nic cells infected with are Cryptosporidial diarrhoea in AIDS and its treatment. Gut adenovirus distinc- 1988;29:593-7. tive and can be appreciated at low power. In 19 Rolston KVI, Rodriquez S, Hernandez M, Bodey GP. the basal of the often Diarrhoea in patients infected with the human immuno- particular, position deficiency virus. AmJ Med 1989;86:137-8. vacuolated cells is helpful and can alert one to 20 Blanshard C, Gazzard BG. An algorithm for the investiga- a more detailed examination of the tion of diarrhoea in HIV infection. Gut 1991;32:A1225. nuclear 21 Laughon BE, Druckman DA, Vernon A, et al. Prevalence of morphology. Furthermore, we have shown that enteric pathogens in homosexual men with and without a available monoclonal acquired immunodeficiency syndrome. Gastroenterology commercially antibody 1988;94:984-93. will react with infected cells in formalin fixed, 22 Kaljot KT, Ling JP, Gold JWM, et al. Prevalence of acute wax-embedded tissue and has a enteric viral pathogens in acquired immunodeficiency paraffin high syndrome patients with diarrhoea. Gastroenterology degree of sensitivity and specificity. 1989;97:1031-42. The role of adenovirus is still 23 Cunningham AL, Grohmann GS, Harkness J, Law C, pathogenic Marriott D, Tindall B. Gastrointestinal viral infections in uncertain. In contrast to Janoff, 80% of our men who were symptomatic and seropositive for human had another enteric immunodeficiency virus. 7 Infect Dis 1988;158:386-91. patients recognised patho- 24 Philips AD. Mechanisms of mucosal injury: human studies. gen. All were severely immunocompromised In: Farthing MJG, ed. Viruses and the gut. Proceedings of and to a of infections. Under the Ninth International Workshop 1988. London: Smith, susceptible range Kline & French, British Society of Gastroenterology, these circumstances it is difficult to elicit the 1988.