Non-opportunistic causes of diarrhoea in HIV infection

Non-opportunistic causesof diarrhoea in HIV infection E. 0. RIECKEN M. ZEITZ R. ULLRICH

Gastrointestinal symptoms are leading complaints in acquired immunodeficiency syndrome (AIDS), and they have been reported to occur in 50 to 90% of patients (Malebranche et al, 1983; Quinn et al, 1986). Among these, diarrhoea is one of the most prominent and in well-conducted prospective clinical studies it has been observed in as many as 48 out of 98 cases with gastrointestinal symptoms (Heise et al, 1988) and in 68 out of 132 patients with overt human immunodeficiency virus (HIV) induced immunodeficiency (RenC et al, 1989). Apart from diarrhoea, weight loss was the most important clinical finding in these studies, and malabsorption has been reported in a number of investigations (Gillin et al, 1985; Kotler et al, 1984; Dworkin et al, 1985; Miller et al, 1988; Ullrich et al, 1989). Furthermore, enteritis and proctitis are well known in these patients (Connolly et al, 1989; Leading article, 1989). Opportunistic diseases have been repeatedly and increasingly recognized as a major cause of these disturbances, and, as part of the typical spectrum of AIDS, were included in the definition of AIDS by the Centers for Disease Control (CDC, 1986) on clinical grounds. This definition has been taken over by the World Health Organization. According to this definition, an opportunistic disorder is an infectious or non-infectious disease which indicates the presence of cellular immunodeficiency in HIV-infected patients in the absence of other causes for cellular immunodeficiency. In HIV infection the degree of immunodeficiency is reflected by a reduction in the number of CDCpositive T lymphocytes to below 400/@ and a decrease in the CD4/CD8 ratio to below 1. On the basis of increasing experience, the definition of AIDS has recently been modified by the inclusion of the following four diseases: extrapulmonary tuberculosis, two or more episodes of Salmonella septicaemia, HIV-associated progressive subacute encephalitis, and the ‘wasting syndrome’ (CDC, 1987). The latter is defined as an unintended weight loss of more than 10% of the body weight and diarrhoea of more than one month’s duration or fever in the absence of a detectable infective agent. Throughout this article HIV denotes HIV-1 BailliPre’s Clinical GastroenterologyVol. 4, No. 2, June 1990 ISBN &7020-146%9

385 Copyright 0 1990, by Baillitre Tindall All rights of reproduction in any form reserved

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In this context, however, it is important to note that the proportion of missing opportunistic and other detectable pathogens in patients with diarrhoea and the 'wasting syndrome' is considerably lower when special consideration is given to the mode of detection. In this respect, the importance of using fresh biopsy material from the intestinal and colonic mucosa for histology and electron microscopy as well as for cultural investigations has been stressed (Heise et al, 1988; Smith et al, 1988; Ren6 et al, 1989) in addition to multiple examinations of stool specimens (Connolly et al, 1989). However, the fact remains that, in spite of thorough and repeated laboratory investigations, there is a fraction of patients in all these studies in whom neither pathogens nor other opportunistic causes can be found. The percentage of patients with diarrhoea (which is unfortunately not consistently defined) without an identifiable intestinal pathogen varies in the different studies between 15% (Smith et al, 1988) and 50% (Colebunders et al, 1988); in our own study it was 27% (Ullrich et al, 1989). Furthermore, the causative relevance of several agents detected remains questionable (Laughon et al, 1988). It has been suggested that in homosexuals with AIDS, non-opportunistic organisms such as Giardia lamblia, chlamydia, gonococcus and Entamoeba (Ouinn et al, 1983) might be acquired venereally, and these might cause diarrhoea either due to enteritis or proctitis. However, Ren6 et al (1989) reported no significant differences in infectious agents recovered from homosexual and non-homosexual patients, and conclusive studies comparing the different risk groups are needed. In this chapter, findings indicative of HIV-associated enteropathy will be reviewed and the significance of non-opportunistic infections in patients having acquired the AIDS virus will be discussed. HIV-ASSOCIATED ENTEROPATHY The often negative result of a thorough search for an intestinal pathogen in HIV-infected patients with diarrhoea and/or the 'wasting syndrome' gave early rise to the hypothesis that HIV itself might cause enteropathy (Kotler et al, 1984). In fact, evidence has been accumulated which suggests that HIV enteropathy might occur. Presence o f H I V in the intestinal m u c o s a

First, the presence of HIV in the intestinal mucosa has been reported in several studies (Nelson et al, 1988; Fox et al, 1989; Ullrich et al, 1989), which are summarized in Table 1. HIV infection of mononuclear cells in the lamina propria was found in all studies (including preliminary reports by Mathijs et al, 1988; Jarry et al, 1988), but the infection of epithelial cells is controversial. The detection of HIV RNA in basal crypt cells by Nelson et al (1988) was confirmed by Mathijs et al (1988) in three out of three rectal biopsies of patients with AIDS and diarrhoea, while in the remaining studies HIV-infected cells were either completely absent in the epithelial layer

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NON-OPPORTUNISTIC CAUSES OF DIARRHOEA Table 1. Detection of HIV-infected cells in the intestinal mucosa.

Patients/CDC stage GI symptoms

Nelson et al (1988)

Ullrich et al (1989, 1990)

Fox et al (1989)

10/IV

11/II or III, 40/IV

25/IV

9/10

All

All

2 duodenum, 3 colon, 5 rectum

Duodenum

5 jejunum, 24 rectum

Method

In situ hybridization

Immunohistology

In situ hybridization

HIV detection

5/10 (2/2 duodenum, 0/3 colon, 3/5 rectum)

20/51

7/24 rectum, 0/5 jejunum

Location

Cryptbase Lamina propria

Lamina propria

Lamina propria

Infected cells

Argentaffin cells Enterocytes Goblet cells Lymphocytes Macrophages

Lymphocytes Macrophages

Lymphocytes Macrophages

None

Lactase deficiency Hypoproliferation

None

Part of intestine investigated

Correlation

(Jarry et al, 1988; Fox et al, 1989) or very rarely observed and assessed rather as intraepithelial lymphocytes (Ullrich et al, 1989). It remains to be answered whether the differences in the localization of viral R N A and viral proteins reported are due to the limited number of patients studied and reflect true biological differences, or if they are due to technical reasons, e.g. artefacts such as non-specific binding of probes, as demonstrated for eosinophils in the mucosa (Fox et al, 1989), or binding of DNA nick-translated probes (Mathijs et al, 1988; Nelson et al, 1988) to integrated proviral DNA. Another question which awaits further studies is the unequivocal identification of the HIV-infected cells, both in the lamina propria and the epithelial layer. Nelson et al (1988) used an argentaffin stain and morphology to identify HIV RNA-positive cells as enterochromaffin cells, enterocytes and goblet cells (see also Levy et al, 1989); the HIV-infeeted mononuclear cells found in the studies cited were morphologically assessed as lymphocytes or macrophages. The development of double-labelling methods combining in situ hybridization and immunohistology will clarify this question. In connection with the demonstration of HIV in the intestinal mucosa, results of in vitro studies have to be noted. Studies on epithelial cells have been confined to tumour cell lines. Adachi et al (1987) demonstrated a productive infection of three out of five colorectal cancer cell lines with HIV, and showed that susceptible cell lines contained CD4 RNA, but not uninfectible cell lines. In addition, there is a preliminary report by Kagnoff et al (1989) characterizing the HIV-susceptible human colon cancer cell line SW-620, which revealed expression of surface CD4, while it lacked other T cell markers but contained cytokeratin P18 and chromogranin A mRNA, findings that are most consistent with an enterochromaffin origin for this cell line.

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Thus, intestinal epithelial cells might be infected with HIV, probably via CD4, but the relevance of this finding remains to be studied, since its demonstration in vivo is controversial; furthermore, whether infection of these cells would lead to cell death or functional impairment is questionable, as no cytopathic alterations were documented by Adachi et al (1987) or Kagnoff et al (1989). In contrast, intestinal mononuclear cells are unequivocally reported as targets of HIV infection in vivo, and functional impairment or cell death of lymphocytes and macrophages subsequent to HIV infection is well documented (reviewed in Rosenberg and Fauci, 1989). Abnormalities of the mucosal immune system in HIV infection

A second line of evidence in support of the possible existence of HIV enteropathy stems from immunological studies of the lamina propria. A reduction in the number of CD4-positive mononuclear cells, the known target cells of the AIDS virus, in the intestinal lamina propria has been described by several investigators (Budhraja et al, 1985; Rodgers et al, 1986; Ellakany et al, 1987). In our own studies, only a minor reduction in the number of CD4-positive cells has been found, with an increase in the number of CD8-positive cells (Ullrich et al, 1990; Zeitz et al, 1990). Thus, as in the circulation, the CD4/CD8 ratio is decreased in the lamina propria of the intestine in AIDS. However, it is important to note that the reduction in the number of CD4-positive cells in the intestinal lamina propria is considerably less pronounced compared with the peripheral blood. The reason for this finding is not completely understood. It is known that replication of HIV and its cytopathic effect depend on cellular activation and proliferation (Margolick et al, 1987; Nabel and Baltimore, 1987). Recent studies demonstrate that intestinal lamina propria T cells do not proliferate after stimulation with antigen under normal conditions (Zeitz et al, 1988b). HIV infection of intestinal CD4-positive cells therefore may cause an impairment of the function of these regulatory cells rather than leading to cell death. An additional finding supporting this hypothesis is a decrease in the number of CD25-positive cells (cells expressing the a-chain of the interleukin 2 receptor) in the intestinal lamina propria in HIV infection (Ullrich et al, 1990; Zeitz et al, 1990). The ~t-chain of the interleukin 2 receptor (CD25) is a marker of T cell activation, and CD25-positive cells are present in the intestinal lamina propria under normal conditions (Zeitz et al, 1988a; Schieferdecker et al, 1990). A loss of these activated cells in HIV infection indicates a functional impairment of mucosal T cells. Comparison of mucosal CD4/CD8 ratios and the distribution of mucosal mononuclear cells in HIV-infected patients and healthy homosexual men revealed them to be normal in the latter (Rodgers et al, 1986). This may indicate that enteric infections in this population are the result of sexual practices--as one would expect--rather than immunological disturbances. Another aspect that may be relevant in HIV-infected patients is intestinal secretory immunity. Kotler et al (1987) were able to demonstrate that the density of plasma cells containing immunoglobulin (Ig)A in small intestinal and colonic biopsies from patients with AIDS were reduced as compared

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389

with controls. In addition, IgM-containing plasma cells were increased, while IgG-containing cells were found to be normal. IgA is the major secretory immunoglobulin and its deficiency has been associated with recurrent intestinal infections as well as with systemic complications such as circulating immune complexes due to increased antigen absorption (Buckley and Dees, 1969; Cunningham-Rundles et al, 1979). Further studies are needed to clarify the relevance of secretory mucosal immunity in AIDS in the disturbance of mucosal barrier function leading to recurrent infections and its relation to mucosal HIV infection. Mucosal structure and function in HIV infection

Third, support for HIV-induced enteropathy comes from recent studies in which clinical investigations on intestinal function have been combined with refined morphological methods, such as morphometry, microdissection and quantitative enzyme cytochemistry (Miller et al, 1988; Batman et al, 1989; Ullrich et al, 1989; Cummins et al, 1990). In early descriptive studies of the small intestine in HIV-infected patients, partial villous atrophy with crypt hyperplasia has been found independent of intestinal pathogens (Malebranche et al, 1983; Kotler et al, 1984). Measurement of mucosal architecture on microscopic sections (Francisco and Kotler, 1988; Miller et al, 1988; Batman et al, 1989) confirmed these findings, and it was suggested that this crypt hyperplastic villous atrophy is inflicted by an immune reaction against HIV-infected cells similar to graft-versus-host disease (Batman et al, 1989). However, this type of mucosal transformation should be accompanied by an increase in activated mucosal T cells, which has not been described in HIV infection; in contrast, a decreased expression of CD25 in the intestinal lamina propria of HIV-infected patients has been found (Ullrich et al, 1990; Zeitz et al, 1990). Furthermore, using three-dimensional morphometry on microdissected specimens, a method known to yield more refined and reliable information on mucosal transformation (Leading article, 1989), we found that villous atrophy with moderate crypt hyperplasia was confined to patients with intestinal pathogens (Ullrich et al, 1989; Zeitz et al, 1990). This has been assumed to be secondary to enteric infections in which this type of mucosal transformation has been observed and has been interpreted to be the result of a hyper-regenerative adaptational response (Riecken et al, 1989). When intestinal infection was absent in HIV-infected patients, slight villous atrophy, normal crypt depth and decreased mitotic cell counts were seen (Ullrich et al, 1989; Zeitz et al, 1990), indicating low-grade mucosal atrophy in the small intestine with hyporegeneration. Recently, Cummins et al (1990) reported similar findings of villous atrophy with impaired crypt hyperplasia in microdissected specimens of eight HIV-infected patients. In our studies, epithelial hyporegeneration was associated with disturbed maturation of enterocytes as indicated by decreased or absent lactase activity (Ullrich et al, 1989; Zeitz et al, 1990). The changes were most pronounced in patients with mucosal HIV infection but without intestinal infection, suggesting that HIV infection was indeed the cause of these mucosal changes. This interpretation is supported by the immunological

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findings described above and by data that are strongly in favour of a functional relationship between mucosal architecture and the mucosal immune system. MacDonald and Spencer (1988) recently induced villous atrophy and crypt cell hyperplasia in organ cultures of human fetal small intestine by T cell activation. In addition, lymphokine-mediated crypt cell hyperplasia associated with activated T cells has been observed in mice (Ferguson and Mowat, 1982). Furthermore, the existence of specialized activated T cells in the normal intestinal mucosa which may be involved in the maintenance of normal mucosal architecture has been demonstrated (Zeitz et al, 1988a, b). Therefore, depletion or functional impairment of these cells (Rodgers et al, 1986; Ellakany et al, 1987; Ullrich et al, 1990; Zeitz et al, 1990) might lead to mucosal atrophy with hyporegeneration. It is relevant in this respect that the clinical and laboratory findings in this study (Ullrich et al, 1989) but also in others (Kotler et al, 1984; Gillin et al, 1985) suggest malabsorption in HIV-infected patients in the absence of enteric pathogens, except for HIV itself, even at early stages of the disease (Ullrich etal, 1989). The lacking correlation between gastrointestinal symptoms and malabsorption on the one hand and infectious diseases of the gastrointestinal tract on the other (Kotler et al, 1984; Ullrich et al, 1989) is also in favour of an HIV-associated enteropathy. It has to be stated, however, that it is not clear whether the differences in the mucosal structure of HIV-infected patients reported so far are entirely due to technical reasons; therefore, the type of intestinal mucosal transformation in HIV infection requires further exploration. With respect to the often dominating watery diarrhoea seen in HIV infection, it is also likely that a number of different mechanisms are involved in the manifestation of enteric symptoms. In this context, it is relevant that extensive axon degeneration in jejunal intrinsic autonomic nerves has been described in all the biopsies from 11 HIV-infected patients without any evidence of infective pathogens (Griffin et al, 1988). While two of these patients were asymptomatic, all others are reported to have had diarrhoea and some of them did not reveal villous atrophy. However, viral particles were not detected in the nerve tissue of these patients, therefore the attribution of the changes ascribed to HIV remains speculative. When discussing HIV enteropathy as a possible consequence directly related to the virus, several factors must also be taken into consideration. First, malnutrition is an important feature of HIV infection (Chlebowski et al, 1989). Second, chemo- and radiotherapy as well as strong oral medication like antibiotics may produce mucosal alteration (Ecknauer and Rommel, 1978; Leading article, 1989) with either hypo- or hyperregenerative intestinal mucosal transformation and damage to the surface cells. Third, overgrowth of the small intestine with anaerobic bacteria, although not demonstrated as yet in HIV-infected patients (Budhraja et al, 1987; Batman et al, 1989), may induce diarrhoea. Finally, it is possible that alterations are due to unidentified pathogens, e.g. microsporidia (see below). In the studies cited care had been taken to avoid interference from some of these factors: nutrition or medication were unlikely causes of the

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391

alterations observed by Ullrich et al (1989), and Batman et al (1989) failed to detect any enteropathogens, and in particular microsporidia, by electron microscopy. However, further systematic studies are necessary to investigate the role of the various factors discussed in the structure and function of the intestinal mucosa, and to clarify whether specific structural characteristics can be identified to differentiate HIV enteropathy. In summary, the hypothesis that HIV-associated enteropathy may be caused by the virus itself is based on the demonstration of the virus in mononuclear cells of the intestinal mucosa and, possibly, in surface epithelium, which is associated with mucosal transformation and maturational defects of the epithelium. Damage to autonomic nerve tissue is also seen. These structural alterations may be associated with functional disturbances and gastrointestinal symptoms, i.e. diarrhoea and malabsorption, but it remains to be seen whether they represent specific structural characteristics of HIV enteropathy. NON-OPPORTUNISTIC INFECTIVE AGENTS IN HIV-INFECTED PATIENTS Diarrhoea in AIDS may not only be 'idiopathic' or due to opportunistic infectious and non-infectious diseases; it may also be the result of a variety of non-opportunistic pathogens (Table 2). These pathogens consist of a spectrum of bacterial, protozoal, viral, fungal and helminthic agents, some of which produce enteritis while others induce rather localized disease in the gastrointestinal tract with proctitis or proctocolitis (Laughon et al, 1988). In a substantial proportion, however, infective agents are harboured in the gastrointestinal tract of HIV-infected patients without producing symptoms. Regarding the pattern and frequency of pathogens producing diarrhoea in HIV-infected patients, personal conduct and the geographical area have been recognized to be of relevance (Blaser and Cohn, 1986; Janoff and Smith, 1988). Thus, in industrialized countries, Neisseria gonorrhoeae and Treponema pallidum as well as herpes simplex virus and hepatitis A and B virus transmission is facilitated by unprotected anal intercourse in homosexuals. Similarly, enteric pathogen transmission of Shigella species, Salmonella species and Campylobacter ]ejuni as well as protozoal infections (Giardia lamblia, Entamoeba histolytica) are eased by promiscuous anallingual contacts. Furthermore, it has been recognized that diarrhoea in patients with AIDS is much more frequent in developing countries, such as Zaire and Haiti (Malebranche et al, 1983; Quinn et al, 1986), ranging from 50% up to more than 90%, while its frequency in the US and Central Europe does not usually exceed 50% (Kotler et al, 1984; Archer and Glinsman, 1985; Heise et al, 1988). However, special reference to the frequency and types of enteric pathogens in AIDS using a prospective protocol that includes extensive microbiological investigations of numerous stool specimens as well as of biopsy material from the upper and lower gastrointestinal tract is rare (Colebunders et al, 1988; Heise et al, 1988; Smith et al, 1988; Connolly et al,

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1989; Ren6 et al, 1989) (see also Table 2). The most elaborate of these studies largely fulfilling the above criteria is that of Ren6 and coworkers (1989), who reported one or more enteric pathogens in 58 out of 132 AIDS patients, 60% of whom were monoinfected and 40% were polyinfected: 43 opportunistic pathogens and 45 non-opportunistic pathogens were detected. In 12 patients, both opportunistic and non-opportunistic pathogens were present. It is noteworthy that Salmonella typhimurium was the most common non-opportunistic bacterial pathogen, with an occurrence rate of 5%, while that of the other bacteria was much lower (for discussion of Salmonella species as non-opportunistic organisms, see section on salmonellosis below). Nonetheless, the most common agent associated with diarrhoea in this study, in agreement with other investigations, was Cryptosporidium, an opportunist. This study demonstrated, however, an equal frequency of non-opportunistic and opportunistic pathogens; therefore, a thorough search for non-opportunistic pathogens in addition to opportunistic agents is needed. When comparing these results with other studies (Tables 2 and 3), a potential sampling bias has to be taken into account (Laughon et al, 1988). This is particularly true for the recruitment of patients, the mode of which depends largely on the type of the referral centre. Thus, clinics for sexually Table 3. Agents recovered from symptomatic homosexual or bisexual patients with and without A I D S at rates significantly different from the asymptomatic carrier rate.

Number of patients H I V serospositive A n y agent of enteric disease A n y agent of diarrhoea

Without

Without

AIDS,

A I D S , with

AIDS, with diarrhoea

AIDS, no diarrhoea

asymptomatic

diarrhoea or proctitis

234 28% 11.9%

68 64% 63.2%*

49 100% 54.9%*

28 100% 39.3%*

6.3%

48.5%*

40.8%*

10.7%

2.6% 0.4% 0% 0% 2.1% 0%

19.1% * 8.8%* 4.4% * 1.5% 10.3% * 1.5%

10.2% * 4.1% 6.1% * 14.3%* 4.1% 4.1%*

7.1% 0.0% 0.0% 0.0% 3.6% 0.0%

5.8 %

41.2 % *

24.5 % *

35.7 % *

0.4% 2.9% 2.5%

13.2%* 8.8% * 19.1%*

0.0% 10.2% * 14.3%*

0.0% 7.1% 28.6%*

33.8% 19.1% 2.9%

14.3%*,t 40.8%* 18.4% *

10.7% 14.3% 7.1% *

Specific agents of diarrhoea:

Campylobacter species Shigella species Clostridium difficile toxin Cryptosporidium Giardia lamblia Vibrio parahaernolyticus A n y agent of proctitis

Specific agents of proctitis:

Neisseria gonorrhoeae Chlamydia trachomatis Herpes simplex virus

Agents of uncertain significance:

Blastocystis hominis Candida albicans Cytomegalovirus

35.3% 10.3% 0.9 %

Modified from Laughon et al (1988). * P < 0 . 0 5 by ×2 or Fisher's exact test compared with asymptomatic patients. t Negative association.

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E . O . RIECKEN ET AL

transmitted diseases have often been used as a reservoir for the study of patients (Quinn et al, 1983; Laughon et al, 1988). Similarly, there is a great variation in the investigation protocols, which may account for differences in the results obtained. Thus, the first comprehensive study on the prevalence of enteric pathogens in homosexual men was carried out from 1978 to 1981 by Quinn et al (1983) at a sexually transmitted disease clinic. The study protocol was designed to detect up to 30 types of enteric pathogens in a large group of patients and to correlate these with the clinical status, but no biopsy material was obtained on anoscopy for microbiological investigations. Following this protocol, another important study was carried out in 1984 and 1985 by Laughon et al (1988) in Baltimore after AIDS had been recognized. This time, the study was conducted at an infectious disease referral clinic rather than at a sexually transmitted disease clinic. Again, no biopsy material was gained on sigmoidoscopy for microbiological work-up. One of the relevant results of this study in homosexuals was a high yield of enteric pathogens, with considerable variations in prevalence rates that correlate with the presence or absence of diarrhoea (Table 3). The prevalence rates, however, were substantially lower than in the preceding studies by Quinn et al (1983) and by Philipps et al (1981), which is assumed to be due to changes in sexual practices and differences in the patient populations studied. With respect to opportunistic agents, it was apparent that AIDS was associated with Cryptosporidium and cytomegalovirus but not herpes simplex virus infection, and only Cryptosporidiuminfection was associated with diarrhoea. In summary, the different studies show that the spectrum and frequency of non-opportunistic enteric pathogens in HIV infection associated with diarrhoea vary to a considerable degree, depending largely on the recruitment of patients. Main factors of influence are personal conduct, sexual behaviour, geographical area and study design, which are reflected in the results of the different risk groups under investigation. Specific enteric non-opportunistic organisms associated with diarrhoea in HIV infection The clinical response to a specific enteric organism needs special consideraTable 4. Non-opportunistic infectious agents as causes of diarrhoea in HIV infection. Bacteria:

Shigella species Salmonella species restricted to the gut Campylobacter species Viruses: Adenovirus Herpes simplex virus Protozoa:

Giardia lamblia Entamoeba histolytica Microsporidia

NON-OPPORTUNISTIC CAUSES OF DIARRHOEA

395

tion since it is influenced not only by the virulence of the organism and the number of inoculated agents, but also by the particular immunological status of the HIV-infected host. The main enteric organisms associated with diarrhoea to be considered are listed in Table 4. Those pathogens classically seen in proctitis and proctocolitis in homosexual men in the framework of the sexually transmitted disease syndrome will be omitted in this context (Philipps et al, 1981; Quinn et al, 1983), since they give rise to localized disease and inflammatory discharge rather than enteritic diarrhoea (Quinn et al, 1983; Laughon et al, 1988).

Shigellosis Shigellosis is well documented in homosexually active men. As demonstrated on the occasion of a flare-up of this infection, it may develop as the result of oral-anal and orogenital contact in homosexual men and in people adopting 'alternative life-styles' (Dritz et al, 1977). This epidemic was associated with a dramatic change in the Shigella species from Shigella sonnei to Shigellaflexneri 2a and then to 3a serotype (Bader et al, 1977; Dritz et al, 1977), with the emergence of strains resistant to antibiotics. Many of the infections in men with oral-anal contacts did not have a common food source. Symptoms in this population may be acute disease with fever, colicky diarrhoea and general sickness. Shigella infection in patients with AIDS may also be associated with diarrhoea, tenderness of the abdomen, fevers and general illness; in addition, it seems to have a tendency towards bacteraemia with signs of generalized infection (Whimbey et al, 1986; Baskin et al, 1987), which is hardly seen at all in immunocompetent patients (Struelens et al, 1985). The clinical importance of Shigella septicaemia is still under debate, but it has been associated with a high mortality rate (Duncan et al, 1981) especially in AIDS and is assumed to reflect the underlying immunological defect in this condition (Baskin et al, 1987). Since shigellaemia is a potentially fatal but treatable disease in AIDS, the pathogen should be specifically tested for and aggressively treated (Baskin et al, 1987), but further information is required.

Salmonellosis Infections with Salmonella species are more frequent in AIDS than shigellosis. Its increased incidence in AIDS has been well documented, as is the case in other conditions with cellular immunodeficiency (Jacobs et al, 1985; Smith et al, 1985; Bodey et al, 1986) and there are estimates that it is 20 times more frequent than in the general population (Bodey et al, 1986). The clinical picture of Salmonella infections in AIDS patients differs from that seen in other individuals. The usual presentation in AIDS is acute enteritis with diarrhoea and fever, but its course is more severe, often persistent and prone to relapses. There is a strong preponderance of Salmonella typhimurium of unknown origin. The most striking differences,

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however, are a high incidence of 'bacteraemia' (Glaser et al, 1985; Jacobs et al, 1985) complicating gastroenteritis, and occurrence prior to the diagnosis of AIDS (Glaser et al, 1985). Since the presence of Salmonella in the blood is usually associated with fever and other signs of a generalized infection, Salmonella 'bacteraemia' has to be regarded as septicaemia. The source of infection in these cases often cannot be identified and recurrence of septicaemia in spite of appropriate antimicrobial treatment is common (Nadelman et al, 1985). Therefore, early long-term treatment following initial antimicrobial therapy has been recommended and is necessary (Jacobs et al, 1985). On the basis of these characteristics, unexplained Salmonella septicaemia in populations known to be at high risk for the development of AIDS has been regarded since 1985 as an opportunistic pathogen. These observations and subsequent experience have led to the inclusion of recurrent Salmonella septicaemia into the definition of AIDS as an opportunistic disease by the CDC (1987). Thus, Salmonella infection can no longer be regarded as a simple specific bacterial infection in AIDS; recurrent Salmonella septicaemia is a true opportunistic disease.

Campylobacter species This group of Gram-negative micro-organisms comprises a number of pathogens that are associated with diarrhoea in humans, the most frequent agent of which is Campylobacterjejuni. It has been seen in homosexuals with gastrointestinal symptoms without AIDS in ten out of 158 men and in two out of 75 without symptoms (Quinn et al, 1984). The symptoms in these patients included various combinations of diarrhoea, abdominal cramps, tenesmus and haematochezia. However, acute illness also occurs commonly in the general population and results of a national surveillance of Campylobacter infections in the US showed that several outbreaks have occurred, of which one in Florida caused illness in over 1000 persons (Riley and Finch, 1985). Campylobacterspecies have also been reported to be a major cause of illness in the UK and the frequency of these pathogens was found to increase steadily after Campylobactersurveillance began in 1977, surpassing those of Salmonella species in this country in 1981 (Skirrow, 1987). The reports on Campylobacterinfections in AIDS are sparse (Nachamkin et al, 1984; Dworkin et al, 1985; Cimolai et al, 1987). The cases involved strains of Campylobacter laridis, a rare pathogen belonging to the group of nalidixic-resistant thermophilic Campylobacter species, a multiple antibiotic-resistant strain of Campylobacter jejuni, and Campylobacter cinaedi, all belonging to new genetic groups that are different from common Campylobacter species. All cases were associated with bacteraemia, as was the case in other immunocompromised patients (Dworkin et al, 1986), but only one of the three cases presented with diarrhoea. However, bacteraemia was also commonly observed in non-AIDS patients preferentially in those infected with Campylobacterfetus (Guerrant et al, 1978). The paucity of the cases reported shows a clear need for more information on the pathogenicity and clinical course of Campylobacter infections in AIDS.

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Herpes simplex virus Herpes simplex virus infection is commonly associated with perianal disease and proctitis in homosexual men (Goodell et al, 1983; Surawicz et al, 1986), producing perianal lesions, discrete vesiculopustular alterations and diffuse or focal friability of the rectal mucosa. Clinically, this infection produces symptoms such as anorectal pain, tenesmus and rectal discharge, with pruritus in addition to neurological symptoms, i.e. difficulties in micturition, sacral paraesthesias and thigh pain. Constipation and haematochezia may also be seen, but diarrhoea due to extended colitic disease has not been documented (Bodey et al, 1986) and extension of the lesions into the lower sigmoid colon is rare (Goodell et al, 1983). Herpes simplex virus infection in AIDS is also common; extended mucocutaneous ulceration of long duration is diagnostic of AIDS and is included as an opportunistic disease in the CDC definition (CDC, 1987). Infections are typically severe and recurrent episodes are characteristic (Siegal et al, 1981; Quinnan et al, 1984). However, discharge results, as in infection of homosexual men, predominantly from rectal disease and true diarrhoea from this infection or more generalized disease is missing. The diagnosis is made by demonstration of the virus electron microscopically or in cell cultures from material from the ulcerated lesions. Treatment with acyclovir is effective and long-term therapy is required. Enteritic disturbances from other viruses that are not producing opportunistic disease may occur in AIDS and have been observed with adenoviruses (Horwitz et al, 1984; Heise et al, 1988; Smith et al, 1988). This virus may induce diarrhoea. Systematic work-up of these viral pathogens and their significance in AIDS is lacking, however, and further investigations are required.

Protozoa Giardia lamblia and Entamoeba histolytica infestation in the general population is pronounced but variable, depending on the geographical region (Quinn, 1988). Its prevalence has been estimated to be below 5% in the US. Its often much higher frequency in the urban male homosexual population is also well documented (Table 5) and known in Europe, the US and other regions of the world (Quinn, 1988). However, with the advent of Table 5. Protozoan infections in AIDS patients. Study location US (Smith) US (Dworkin) US (Laughon) Haiti (DeHovitz) Zaire (Henry) Zaire (Colebunders) Uganda (Sewankambo) From Quinn (1989).

Number of patients 20 22 49 110 46 106 23

Entamoeba histolytica 25% 0% 0% 0% 8% 5% 9%

Giardia lamblia 15% 5% 4% 3% 0% 0% 4%

Cryptosporidium

lsospora

15% 14% 16% 59% 8% 22% 48%

0% 0% 2% 18% 19% 7% 13%

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AIDS and changes in sexual practices due to better health education, the prevalence of these protozoa has decreased decisively (Laughon et al, 1988) and is reported to be less than 3.5% in homosexual men (Quinn, 1988). The association of these micro-organisms with male homosexuals suffering from HIV infection and AIDS is also well documented (Colebunders et al, 1988; Laughon et al, 1988; Connolly et al, 1989; Ren6 et al, 1989) (see also Table 2), but their role in the aetiology of diarrhoea is often unclear due to an often asymptomatic carrier state and also because of coinfection with other diarrhoeic pathogens (Laughon et al, 1988). In the majority of cases of symptomatic giardiasis in non-immunocompromised patients, persistent diarrhoea is a characteristic feature which may be accompanied by flatulence and abdominal colicky pain (Black et al, 1977). In AIDS, a variable clinical response to infection with Giardia lamblia is observed. Immunological studies indicate that patients with previous contact with G. lamblia and antibodies to this pathogen have no or minor symptoms when reinfected (Janoff and Smith, 1988). These findings support the concept that, at least for some intestinal pathogens, prior exposure may lead to less severe illness in AIDS patients. The role of Entamoeba histolytica infection in AIDS with respect to the induction of diarrhoea and gastrointestinal symptoms is still more debatable. Comparison of the clinical characteristics of E. histolytica infested homosexuals with those who did not have the organism revealed a similar pattern of symptoms, suggesting this agent to be a common commensal in the homosexual population (Allason-Jones et al, 1986). There were no significant differences between the two groups with respect to age, recent travel abroad or previous sexually transmitted disease. Furthermore, strikingly no true invasiveness of this potential pathogen has been observed in AIDS (Janoff and Smith, 1988). This has been assumed to be related either to pre-existing immunity to the parasite or, more likely, to the lack of the appropriate local intestinal conditions that are thought to induce invasive mechanisms of the agent in the gut (Janoff and Smith, 1988). Treatment of infested homosexuals without symptoms therefore does not have a very rational base (Allason-Jones et al, 1986), but is recommended on clinical grounds (Sullam, 1987).

Findings of unknown significance When discussing unexplained diarrhoea in HIV infection it is necessary to note that microsporidia (Dobbins and Weinstein, 1985; Modigliani et al, 1985; Connolly et al, 1989) and tubuloreticular inclusions (Dobbins and Weinstein 1985; Sidhu et al, 1985, Connolly et al, 1989) have been found electron microscopically in the gut mucosa of these patients, but their significance as the cause of diarrhoea is not known due to lack of systematic studies.

Microsporidia Microsporidia are protozoan organisms that are intracellularly localized, spores as well as sporonts, which cannot be recognized with certainty in the

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intestinal mucosa with any stain, even when using semi-thin sections (Dobbins and Weinstein, 1985; Owen, 1989). This is in contradistinction to their stainability by PAS and acid-fast stain in the muscle. Electron microscopy is therefore needed (Dobbins and Weinstein, 1985; Sidhu et al, 1985) to detect these organisms with certainty. In the small intestine they have been localized in epithelial cells without producing apparent damage (Dobbins and Weinstein, 1985; Modigliani et al, 1985). Macrophages as well as plasma cells were slightly increased, and villus atrophy has been described in two cases (Modigliani et al, 1985; Cali and Owen, 1988). Approximately 10% of the epithelial cells were found to be infected in the patient studied by Dobbins and Weinstein (1985); Rijpstra et al (1988) reported a wide variation in the number of parasites between sites of infection in three patients. Although the organism is shed with the epithelial cells into the intestine, it has not been identified in the stools. One case of microsporidian peritonitis (Zender et al, 1989) has been reported, and infestation of the liver in one patient was associated with granulomatous hepatitis and necrosis predominantly localized to the portal region (Terada et al, 1987). Systematic electron microscopical and clinical studies must be awaited to determine the frequency of microsporidian infestation in HIV infection, as well as its clinical significance. Tubuloreticular inclusions

Tubuloreticular inclusions have been identified in the intestinal and colonic mucosa of HIV-infected patients more frequently than microsporidia, thus they were seen in seven of the eight patients studied by Dobbins and Weinstein (1985) and in four of the ten patients investigated by ConnoUy et al (1989). Tubuloreticular structures were found in endothelial cells and in lymphocytes, monocytes and macrophages of the lamina propria (Kostianovsky et al, 1983; Dobbins and Weinstein, 1985; Orenstein et al, 1985). On two occasions they were also seen within a capillary lumen (Dobbins and Weinstein 1985). Tubuloreticular structures are thought to be modified endoplasmic reticulum and are known to occur in a variety of diseases and viral infections (Kostianovsky et al, 1983), where they are thought to be non-specific products of viral infection. In AIDS they have been identified electron microscopically to easily that they have been considered by Dobbins and Weinstein (1985) to be a non-specific marker for AIDS. In this respect, the report by Ozick et al (1989) is of interest, in which tubuloreticular structures were described in a homosexual man who presented with diarrhoea of unknown cause in whom the diagnosis of HIV infection was suggested by these structures and made prior to any clinical manifestation of the immunodeficiency syndrome. Future studies will have to be awaited to define more precisely the significance of these structures in diarrhoea associated with HIV infection.

Acknowledgement The studies were supported by the Bundeminister fiir Forschung und Technologie (FKZ 11-048-88).

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