Journal of Antimicrobial Chemotherapy (1996) 37, Suppl. B, 61-70
Recently recognised microbial enteropathies and HIV infection
Michael J. G. Farthing, M. Paul Kelly and Andrew M. Veitch
Digestive Diseases Research Centre, Medical College of St Bartholomew's
Charterhouse Square, London EC1M 6BQ, UK
Hospital,
Persistent diarrhoea and small bowel enteropathy are important features of HIV
infection. At least 80% of cases of persistent diarrhoea in patients with HIV/AIDS
can be attributed to a specific enteropathogen. The coccidian parasites
Cryptosporidium parvum, Isospora belli and Cyclospora and the Microsporidia
account for at least 50% of cases of persistent diarrhoea in the industrialised and
developing world with major contributions from Mycobacteriwn avium complex and
other bacteria and cytomeglovirus. The intracellular protozoa can be detected on
faecal microscopy although confirmation may be required by intestinal mucosal
biopsies. Although up to 20% of cases of persistent diarrhoea can not be attributed
to a specific infection, the question as to whether HIV infection itself can produce
enteropathy remains uncertain. Recent developments in the treatment of persistent
diarrhoea in HIV include the use of albendazole for microsporidial diarrhoea and
co-trimoxazole for the treatment and eradication of Cyclospora.
Introduction
Persistent diarrhoea occurs in up to 75% of patients with HIV/AIDS in Western
populations (Dworkin et al., 1985; Antony et al., 1988; Smith et al., 1988; Rene et al.,
1989; Rolston et al., 1989; May et al., 1993) and in up to 100% of those infected in
some developing countries (Quinn et al., 1987) and has a high morbidity and mortality.
At least 80% of cases of chronic diarrhoea can be attributed to a specific enteropathogen
of which the coccidial parasites are among the most important. Infective diarrhoea in
HIV/AIDS can involve both the small intestine and the colon and the pan-enteric
involvement probably accounts for the high volume of faeces lost that is characteristic
of cryptosporidial and microsporidial diarrhoea. Disruption of the villous architecture
of the small intestine commonly accompanies infective diarrhoea in HIV infection but
the specific determinants of this enteropathy and the mechanisms by which it occurs
are poorly understood. Further work is required to define the complex interactions
between microbial enteropathogens, intestinal immune responses within the mucosa to
these infective agents and their interactions with HIV itself. In advanced HIV infection,
nutritional factors may also play a part.
Definition of enteropathy
Enteropathy is characterised by a variable reduction in villous height usually associated
with a hyperplastic response in the crypts. There is therefore a decrease in the villous
61
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£, 1996 The British Society for Antimicrobial Chemotherapy
62
M. J. G. Farthing et at.
crypt ratio which inevitably is accompanied by a decrease in the surface area of the small
intestine. There may also be evidence of damage to the surface epithelial cells
(enterocytes) with a reduction in the height of the cell changing it from a columnar
to a cuboidal shape. The changes in villous morphology and enterocyte height are
almost invariably associated with inflammatory cell infiltrates in the lamina propria
and within the epithelium itself (intraepithelial lymphocytes). These morphological
changes in the small intestine are often accompanied by diarrhoea but, if only the
proximal small intestine is involved, the compensatory effects of the ileum and
colon in retrieving water and sodium may render the individual symptom-free. This
is well recognised in the classic enteropathy of coeliac disease which is due to the
wheat protein, gliadin, in which, the total thickness of the mucosa is generally
maintained or maybe increased when crypt hyperplasia is marked. Thus, the term
'villous atrophy' is not strictly accurate. However, in rotavirus infection in which
there is primary loss of enterocytes in the mid-villous region there is a 'true' villous
atrophy initially and only subsequently does crypt hyperplasia occur during the recovery
phase.
Mechanisms of enteropathy
The enteropathy of coeliac disease is thought to be immune mediated. There is
compelling evidence to suggest that T cell activation within the lamina propria may be
directly associated with the changes in villous morphology although the precise
mechanisms by which this occurs have not been clearly delineated (MacDonald &
Spencer, 1988; da Cunha et al., 1990; Livnett et al., 1993). Thus, mucosal inflammation
may be one of the key factors in the enteropathy associated with intestinal infection
and is commonly associated with cryptosporidiosis, microsporidiosis, giardiasis
and infection with Cyclospora. Despite the presence of T lymphocytes within the
mucosa in patients with HIV and opportunistic infection in the gut, there is little
evidence that these T cells are activated (Lim et al., 1993, 1994), a factor which does
seem to be important in the production of villous atrophy in coeliac disease and
experimental enteropathy in vitro (MacDonald & Spencer, 1988). In a recent study by
Snijders et al. (1955) the cytokine profile in jejunal mucosal cultures was studied in
patients with HIV and cryptosporidiosis, microsporidiosis and in HIV infected
individuals without diarrhoea. In addition, Snijders et al. studied control tissue
from subjects who were not injected with HIV. There was, however, no evidence of
increases of IL-1/?, IL6, IL8 or IL10 production in the HIV-infected individuals nor
was there evidence of increased tumour necrosis factor (TNF-a) or soluble tumour
necrosis factor receptor (sTNFR) expression. These findings would support
immunohistochemical studies which suggest that mucosal immune activation is not a
major feature of cryptosporidiosis and microsporidiosis in HIV. Our own studies in
Africans infected with HIV conform with these observations although we did find slight
but significantly increased expression of the T cell activation marker, HML-1 (Veitch
et al., 1995).
As stated previously, there is evidence that certain enteropathogens such as rotavirus
can damage enterocytes directly and produce true villous atrophy. It is possible that,
coccidia, particularly the intracellular organisms, Microsporidia and Cyclospora could
act in this way although there is no direct evidence to support this finding. It has been
Microbial enteropathy and HIV infection
63
proposed that giardia may disturb villous morphology not only by activating T cells
through classic antigen stimulated pathways but also by the direct effect of its mannosebinding lectin, taglin, which shares some features with the plant lectin concavanalin A
which is a mitogen.
Enteropathy occurs in HIV infection, sometimes in the absence of intestinal infection
(Kotter et al., 1984; Ullrich et al., 1989). HIV has been detected within the intestinal
mucosa (Fox et al., 1989) and it has been proposed that the virus itself may be
responsible, at least in part, for the enteropathy of HIV/AIDS. In-vitro infection has
been shown to reduce villous height and induce crypt hyperplasia in fetal small intestinal
explants supporting this proposal (Batman et al., 1994). However, no direct evidence
that this is the case in vivo has been forthcoming and sceptics favour the suggestion that
the so-called 'pathogen negative diarrhoeas' in HIV are actually due to opportunistic
infection which has failed to be detected or is due to an, as yet, unrecognised pathogen.
It is well established, however, that there is evidence of generalised immune activation
in HIV/AIDS as indicated by increased levels of sTNFR and neopterin (Zangole et al.,
1993). We have shown a close correlation in African's with AIDS of these markers with
villous height and the degree of mucosal inflammation which does not, of course,
establish a causal link (Kelly et al., 1994). There is, however, a remarkably good
relationship between the intensity of mucosal inflammation and the intra-epithelial
lymphocyte count and villous height but it is unclear, how this inflammatory response
relates to the abnormalities of villous architecture when the evidence for T cell
activation is relatively minor and the cytokine profile is not significantly different from
that of normal small intestinal mucosa.
Finally, there is some evidence that nutritional factors may play a part in producing
enteropathy. In experimental models, low calorie and low protein diets can produce
abnormalities of villous architecture although the small intestinal epithelium is relatively
resistant to the effects of nutritional deficits. In addition, deficiencies in micronutrients, e.g. folic acid and vitamin A, can affect intestinal epithelial integrity. In advanced
AIDS, the body mass is often dramatically reduced and, in our own recent studies in
Africa, we found a weak, but nevertheless significant, correlation between villous height
and triceps skin fold thickness (Kelly et al., 1994), a marker of the size of the body
fat compartment. Again, one may merely be observing a correlation between two
dependent variables rather than any causal relationship.
Microbial diarrhoea and enteropathy
A variety of microbial enteropathogens have been linked aetiologically with persistent
diarrhoea in patients with HIV infection (Table I). Mycobacterium avium complex
(MAC) is one of the major bacterial enteropathogens causing chronic diarrhoea and
enteropathy, producing appearances in the small intestine reminiscent of Whipple's
disease (Roth et al., 1985; Rolston et al., 1989) due to Tropheryma whippleii. It is the
protozoa however, particularly the coccidial enteropathogens such as Cryptosporidium
parvum, the microsporidia and Isospora belli, that are responsible for the majority of
cases of persistent diarrhoea. The role of Blastocystis hominis remains uncertain
although it is isolated relatively frequently from HIV-infected individuals with
diarrhoea.
The relative importance of these enteropathogens is, to some extent, determined by
geographic location. /. belli for example, is uncommon in AIDS patients in the USA
64
M. J. G. Farthing et al.
Table I. Microbial aetiology of HIV-related
enteropathy
Bacteria
Protozoa
Helminths
Viruses
M. avium complex
C. parvum
Microsporidia
Cyclospora
/. belli
G. intestinalis
Blastocystis hominis?
Slrongyloides stercoralis
Adenovirus (types 40 & 41)
CMV
Rotavirus
HIV?
and Europe where it accounts only for approximately 1% of patients with chronic
diarrhoea, but in tropical and sub-tropical countries such as Haiti and parts of Africa
it is more common, accounting for between 15-28% of cases (De Hovitz et al., 1986).
Currently in the UK, C. parvum and microsporidia account for almost 50% of cases
with chronic diarrhoea followed by cytomegalovirus, Giardia intestinalis and MAC
(Table II) Gazzard & Blanchard (1993). The apparent distribution of infective agents
has also changed with time as is well demonstrated by two studies from Zambia reported
in 1990 and 1995 (Conlon et al., 1990; Drobniewski et al., 1995). In the earlier study,
43% of cases of chronic diarrhoea with enteropathy were considered pathogen negative
(Conlon et al., 1990). C. parvum and /. belli accounted for more than half of the cases
in which a pathogen was identified. However, in the study reported four years later
(Drobniewski et al., 1995), only 19% of individuals were considered pathogen negative
due, principally, to the identification of microsporidia including Enterocytozoon bieneusi
and the related member of the sporozoa, Septata intestinalis, which was recently
identified for the first time in AIDS patients in Southern Africa (Kelly et al., 1994;
Drobniewski et al., 1995). As in the previous study, C. parvum and /. belli were
considered to be the major aetiologic agents in the HIV patients with persistent
diarrhoea (Figure).
Table II. Proven causes of persistent
diarrhoea in HIV/AIDS in the United
Kingdom (cf. Gazzard & Blanchard,
1993)
n (%)
C. parvum
Microsporidium
G. intestinalis
M. avium complex
Bacteria
/. belli
Cytomegalovirus
Others
29 (25)
24 (21)
11 (10)
6(5)
7(6)
1 (<D
14(12)
18 (16)
65
Microbial enteropathy and HIV infection
45
40
36
30
+#•
S 25
-
5 20
15
10
1
5
0
1990
1994
Figure. Microbial aetiology ( • , none; D, Microsporidium; • , Cryptospondium; • , Isopora; D,
Strongyloides) pcrsistant HIV-related diarrhoea in Zambia for 44 cases in 1990 (Conlon et al., 1990) and 71
cases in 1994 (Kelly et al., 1994).
Newly recognised enteropathogens
Two new protozoan pathogens, microsporidia and cyclospora have been identified
during the past few years which produce clinically important diarrhoeal disease and
enteropathy. These organisms are found in patients with HIV in many geographic
locations and there is now effective or, at least, partially effective, therapy available.
Microsporidia
Microsporidia belong to the phylum microspora which consists of approximately 80
genera and over 700 species. The microsporidia are intracellular spore-forming protozoa
which have been found in most animal groups (Bryan et al., 1991). They were first
identified in 1857 and have been known to cause disease in fish, rabbits and a variety
of other mammals including laboratory rodents and also primates. Their identification
as a human pathogen has occurred relatively recently.
The organism. Microsporidia form spores which are the infective form of the parasite
and enable the organism to survive in the environment outside its animal host. Spores
vary in size from 1-20 fim in diameter although those of the species infecting mammals
are generally small with diameters of 1-2 /im. Within the spore, there is a coiled polar
tube which acts as the conduit by which the microsporidium is transferred into the
intestinal epithelial cell of the host. Once in the host cell the organism divides and
produces sporoblasts which in intestinal microsporidiosis pass into the environment via
the faeces. Spores remain viable and infective in the environment for up to 4 months.
Although the basic structure of the microsporidia is similar among the various genera
and species there are some important differences which allow species identification. E.
bieneusi and 5. intestinalis can be distinguished on the appearances of the polar tube
which in the former is present as a double coil but in the latter as a single coil. In
addition, 5. intestinalis takes up an intracellular location within a parasitophorous
66
M. J. G. Farthing et al.
vacuole, whereas E. bieneusi is free within the enterocyte cytoplasm. Microsporidia are
considered to be primitive eukaryotes being devoid of mitochondria and containing
ribosomes which resemble those of bacteria.
Epidemiology. Infection is transmitted by the faecal-oral route. Although data
on the world-wide prevalence of human intestinal microsporidia is far from
complete, it is evident from studies in the last few years that it is becoming
increasingly recognised an an important enteric infection in AIDS patients, perhaps
accounting for up to 35% of cases of persistent diarrhoea (Drobniewski et al.,
1995). Symptomatic infection in immunocompetent individuals would appear to be
rare.
Pathogenesis. The mechanism by which this parasite produces partial villous
atrophy is unknown. However, the intracellular location would suggest that the
organism may have a direct effect on enterocyte function and thus, it seems likely that
villous atrophy is due at least in part to a direct effect of the parasite by increasing
enterocyte loss. Crypt hyperplasia occurs in microsporidial infection in the small
intestine and is usually associated with increased chronic inflammatory cells in the
lamina propria (Modigliani et al., 1985; Drobniewski et al., 1995). The main focus of
infection appears to be the small intestine with the majority of organisms found in the
jejunum. Organisms are either present in very low numbers or absent in stomach, colon
or rectum.
Clinical features. Clinically, microsporidiosis in HIV patients is indistinguishable
from cryptosporidiosis. The cardinal feature is persistent diarrhoea with increased
faecal volumes often up to and excess of 1-2 L/24 h. In addition, there may be
fever, abdominal discomfort and objective evidence of intestinal malabsorption
such as increased faecal fat. When infection is prolonged, anorexia and profound
weight loss become increasingly evident. There are no obvious macroscopic changes
in the small or large intestine. Disseminated infection has been reported with
microsporidia involving the liver (hepatitis), the peritoneum and also the cornea
producing keratopathy. In some of these disseminated cases, infection has been
attributed to the organism Encephalitozoon cuniculi, a genus closely related to
enterocytozoon.
Diagnosis. Spores can be detected in faeces by light microscopy using a modified
trichrome stain (Orenstein et al., 1990). In view of their small size, spores are difficult
to detect if the observer is inexperienced. The various forms of microsporidia (sporonts,
plasmodia, schizonts) can also be detected by light microscopy in paraffin-embedded
sections of small intestinal biopsies stained with haematoxylin eosin. Resolution of the
organisms may be improved by using semi-thin plastic sections stained with methylene
blue azure II followed by basic fuchsin or toluidine blue (Bryan et al., 1991). However,
identification of microsporidian species relies on resolution of their ultrastructural
features by electron microscopy. E. bieneusi can only be reliably distinguished from S.
intestinalis by transmission electron microscopy of small intestine (Kelly et al., 1994).
Serologic diagnosis is available for E. cuniculi but not for other microsporidial species
as yet. Although partial responses have been described with co-trimoxazole there is
increasing evidence that albendazole is effective in microsporidiosis at a dose of
400-800 mg twice daily for 14-28 days (Blanshard et al., 1992). Infection with E.
bieneusi can be suppressed but usually not eradicated whereas in infection with S.
intestinalis, the symptoms of disease resolve rapidly and the infection is usually
eradicated.
Microbial enteropathy and HIV infection
67
Cyclospora
In 1983, some Haitian's with AIDS and chronic diarrhoea were found to have acid-fast
cryptosporidium-like organisms in faecal specimens. The organism was intermediate in
size between C. parvum and /. belli but thought to be morphologically similar to
cryptosporidium. This observation was closely followed by an investigation of travellers
and residents in Nepal with persistent diarrhoea who were found to have
cyanobacterium-like bodies (CLB) in their stools (Taylor et al., 1988; Shlim et al., 1991).
In 1993, this organism was eventually shown to be an organism of the genus Cyclospora
and has been tentatively named, Cyclospora cayetanensis (Ortega et al., 1993).
The organism. The genus Cyclospora was formally established in 1881 and infection
has been described in a variety of animal species including reptiles, insectivores,
myriapods and rodents. Human infection has only been described recently and probably
involves a new species, since the human Cyclospora is morphologically distinct from
those seen in animals. The infective form of the parasite is the oocyst which on light
microscopy appear as non-refractile spheres 8-10 /im in diameter containing a cluster
of refractile, membrane-bound globules enclosed within a membrane to produce a
morula (Ortega et al., 1993). Oocysts sporulate in vitro when cultured for 5 days at 25
or 32°C. Complete sporulation with the appearance of sporozoites within sporocysts
was evident by day 7-13. Potassium dichromate promotes sporulation even at 4°C.
Electron microscopy of the immature oocysts reveals an outer fibrillar coat beneath
which there is a cell wall. Within a sporulated oocyst, two sporocysts can be identified
each of which yields two sporozoites. In the intestine, the parasite takes up an
intracellular location within enterocytes and are contained within a parasitophorous
vacuole. Parasites have a single nucleus with a prominent nucleoli. Intracellular
parasites measure 6-8 /xm long and 1-4 nm wide.
Epidemiology. Cyclospora was initially identified in humans in Haiti, Nepal and Peru
but is now recognised to occur widely in Europe, Asia, North Africa and throughout
the Americas (Hoge et al., 1993). Infection has been reported in immunocompetent
travellers, children and in HIV-infected adults. Overall, the world prevalence is not
known but transmission is assumed to occur by the faecal-oral route, mainly through
water. In Nepal, there is good evidence of seasonal variation with peak incidence during
the rainy season (Hoge et al., 1993). Well-documented waterborne outbreaks have been
described in Nepal and Chicago. Currently, major risk factors for acquiring cyclospora
infection appear to be HIV infection and travel in the developing world.
Pathogenesis. The mechanism by which Cyclospora produces diarrhoea is unknown.
However, the organism takes up an intracellular location and one might assume that
this results in enterocyte dysfunction and possibly enterocyte loss (Bendall et al., 1993).
This may account for the partial villous atrophy and crypt hyperplasia that are features
of this infection in the small intestine (Conner et al., 1993; Pape et al., 1994). In
addition, there is an increase in chronic inflammatory cells in the lamina propria and
also focal increases in intra-epithelial lymphocytes. Evidence to support a direct effect
on the enterocyte comes from microscopic examination of the epithelial cells in which
there is focal vacuolisation, disruption of the microvillus membrane and a tendency for
enterocyte shape to change from columnar to cuboidal. Whether the organism secretes
enterotoxins to account for the watery diarrhoea remains to be established.
Clinical features. Infection with Cyclospora cannot reliably be distinguished from
cryptosporidiosis, microsporidiosis or isosporiasis on clinical grounds. The major
68
M. J. G. Farthing et al.
feature is persistent watery diarrhoea which in immunocompetent individuals
lasts between 3-6 weeks but may continue for up to 15 weeks in patients infected with
HIV (Shlim et al., 1991; Hoge et al., 1993). Diarrhoea may be cyclic or relapsing.
Prolonged illness is associated with profound loss of weight and fatigue. In HIV-infected
patients, the diarrhoea is usually persistent and unremitting and not usually
self-limiting.
Diagnosis. Using an acid-fast stain, such as Ziehl-Neelsen, oocysts 7 /itn in
diameter can be detected in faecal specimens (Ortega et al., 1993). The organisms also
autofluoresce blue when examined with ultraviolet epifluorescence microscopy.
Inexperienced microscopists can confuse cyclospora with C. parvum although the
latter are smaller. If there is doubt, ultrastructural examination of the parasite in
small intestinal biopsies by electron microscopy can provide confirmatory evidence.
Preliminary evidence suggested that cyclospora infections were responsive to
co-trimoxazole. A double-blind placebo controlled trial has recently been reported from
Nepal indicating that 960 mg co-trimoxazole given twice daily for 7 days will eradicate
approximately 90% of infections in immunocompetent adults (Hoge et al., 1995). If
diarrhoea continues and the stools are still positive for cyclospora, then a more
prolonged course of therapy may be required.
Summary and conclusion
Persistent diarrhoea and small bowel enteropathy are important features of HIV
infection. Although HIV infection itself may in some instances produce enteropathy and
diarrhoea, at least 80% of cases of persistent diarrhoea in patients with HIV can be
attributed to a specific infective agent. The coccidian parasites C. parvum, I. belli,
microsporidia and cyclospora are emerging as important enteropathogens which can
now be reliably diagnosed on faecal examination and for some of these infections at
least there is curative therapy available. The mechanisms by which they produce
enteropathy and diarrhoeal disease remains unknown and largely unexplored.
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