H
u m a n m i c r o s p o r i d i o s e s a n dAIDS:
RECENT ADVANCES
I. DESPORTES-LIVAGE*
Summary :
Microsporidia, unicellular parasites frequent in animals, were rarely reported in humans before the advent of AIDS. This immunodeficiency syndrome induces the emergence or resurgence of opportunistic infections such as microsporidioses. Since 1 9 8 5 , five species of microsporidia have been found in HIV-infected patients. O ne of these species w as already known in animals whereas all others are new. An increasing number of cases of microsporidioses is detected due to the improvement of methods of diagnosis. According to a study conducted in USA, intestinal microsporidia appear to be the first cause of diarrhea in patients with AIDS. These parasites are intensively investigated as shown by the increasing number of studies published since 1 9 9 3 . Most data concern the diagnosis, pathology, therapy and epidemiology of human microsporidioses as well as the characterization of their agents. Experimental studies aiming to define the immune context of these infections are also reported.
KEY WORDS : microsporidia, microsporidiosis, diagnosis, pathology, therapy, SIDA.
R ésum é : Mi c r o s p o r i d i o s e sh u m a in e s e t SIDA
Les microsporidies, parasites unicellulaires fréquents ch ez les animaux, étaient peu connues ch ez l'homme jusqu'à l'apparition du SIDA. C e syndrome entraîne la résurgence ou l'émergence d'infections opportunistes parmi lesquelles figurent les microsporidioses. Cinq espèces d e microsporidies dont quatre nouvelles ont été trouvées chez les patients atteints de SIDA et, a vec l'amélioration des méthodes d e diagnostic, le nombre des ca s rapportés d e microsporidioses ne cesse d'augmenter. Les atteintes digestives sont les plus fréquentes et, selon une enquête récemment effectuée aux Etats-Unis, les microsporidies intestinales seraient la première cause d e diarrhées ch ez les patients atteints d e SIDA. C es parasites font l'objet d'un effort d'investigation intense comme le montre la quantité d'études publiées depuis
19 9 4 . Les informations ici rapportées sont relatives au diagnostic, aux aspects cliniques, thérapeutiques et épidémiologiques des microsporidioses ainsi qu'à la caractérisation d e leurs agents.
L'approche expérimentale visant à définir le contexte immunitaire d e ces infections est également évoquée.
MOTS CLÉS : microsporidies, diagnostic, pathologie, thérapie, SIDA.
INTRODUCTION
M
icrosporidia are unicellular parasites which infect a wide range o f invertebrates including protozoa such as ciliates (Foissner & Foissner, 1995). Also known in fish, birds and mammals (Canning& Lom, 1986), they were rarely reported in humans before the advent o f AIDS. Since 1985, four new species of microsporidia have been found in AIDS patients (Table I) and these parasites are gaining increasing attention as opportunistic pathogens responsible for significant mor
bidity. Most recent data, concerning human microspori
dioses and their agents, are herein reported.
GENERAL CHARACTERS OF MICROSPORIDIA
T
hese protists are considered to be ancient eukaryotes. They have the nuclear organization o f eukaryotes but they lack mitochondria, per-
* INSERM U313, CHU Pitié-Salpêtrière, 91, boulevard de l’Hôpital, 75013 Paris, France.
Tél. : 45.83.46.84 - Fax : 45.83.88.56.
Microsporidia Clinical symptoms
Enterocytozoon bieneusi Abdominal cramping, nausea, weight loss, malabsorption, zinc deficiency, chronic diar
rhoea, cholecystitis, cholan
gitis, rhinosinusitis E ncephalitozoon
(= Septata) intestinalis Diarrhoea, nausea, malabsorp
tion, weight loss, fever, chole
cystitis, rhinosinusitis, bron
chiolitis, nephritis
Encephalitozoon hellem Keratoconjunctivitis, sinsusitis, bronchiolitis, nephritis, urete
ritis, dysuria, prostatitis Encephalitozoon cu n iculi Dysuria, nephritis, sinusitis,
bronchitis, hepatitis, peritonitis Trachipleistophora hom inis Myositis
Nosema connori Disseminated infection
Nosema ocu lam m Corneal stroma infection Vittaforma c o m e a e
(= Nosema com eu m ) Corneal stroma infection
Table I. — Microsporidian species parasite of man and their clinical manifestations according to the literature cited in text. N. oculam m Cali et al., 1991 and V. co m e a e Silveira & Canning, 1995 have been only found in HIV-negative patients. The unique case of infection by N. connori Sprague 1974, has been reported in an athymic infant. E.
cuniculi is also parasitic in animals (carnivora, lagomorphs, rodents).
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1996032107
DESPORTES-LIVAGE I.
oxysomes, Golgi membranes, and their ribosomal RNA sequence is similar to that o f prokaryotes (Vossbrinck et al., 1987). Their developm ent occurs within cells o f their hosts. It consists o f a division phase follow ed by a spore producing phase. The only extracellular stages
are mature spores w hich are released in the extracel
lular medium after the host cell death. Microsporidian spores possess a typical polar tube o f w hich extrusion ensures the inoculation o f the infecting sporoplasm into a host cell (Fig. 1).
Fig. 1. - Transmission electron micrograph showing three spores of Enterocytozoon bieneusi at different stages of maturation in the cyto
plasm of an enterocyte. Sections o f the polar tube (arrowheads) are seen in the sporoplasm. These spores measure approximately 1.5 x 0.8 pm. Scale bar = 0.5 μm.
108 M ise au point Parasite, 1996, 3, 107-113
Humanmicrosporidiosesand AIDS
AIDS RELATED M ICRO SPO RIDIO SES
C
lin ical m an ifestatio n s o f m icro sp o rid io ses depend on the site on the infection. E n terocy- to z o o n bien eu si, (Desportes et al., 1985), develops in the enterocytes o f the small intestine. The spe
cies causes chronic diarrhea, abdominal cramping and nausea; it is also responsible for the malabsorption and the severe weight loss reported in AIDS patients, and appears to be involved in zinc deficiency as shown by the low levels measured in patients (Asmuth et al.,
1994). This microsporidian could thus contribute to the progression to AIDS since deficiency in zinc is known to b e related w ith pro g ressio n to this syndrom e (Graham et al., 1990 cited in Asmuth et al., 1994). The parasite has also been found in HIV infected patients w ithout ch ron ic diarrhea (R ab en eck et a l., 1995).
Although small intestine is the primay site o f infection, Ent. b ien eu si can also spread to the biliary ducts and nasopharyngeal epithelium, causing thereby some cases of cholangitis and rhinosinusitis (W eber et al., 1994).
E n c ep h a lito z o o n (= S ep tata) in testin alis is the second species know n to cause diarrhea in AIDS patients (Cali et al., 1993; Hartskeerl et al., 1995). E n c e p h a lito z o o n b e lle m has b ee n isolated from ocular biop sies o f patients with keratoconjonctivitis (Didier et al., 1991).
W hereas Ent. B ie n e u s i appears to be restricted to epi- th elia, all sp e cie s o f the g en u s E n c e p h a li t o z o o n develop in a variety o f cells including macrophages thus causing disseminated infections. E n c ep h a lito z o o n in testinalis, E n c. h ellem and E n c. cu n icu li, a species already know n to infect mammals, can cause rhinosi
nusitis, bronchiolitis, nephritis, ureteritis, hepatitis as well as peritonitis (Cali et a l , 1993; W eber et a l , 1994; Gunnarsson et al., 1995; Sobottka et al., 1995;
Didier et al., 1996). Three cases o f myositis w ere im puted to P leis to p h o ra sp. (Ledford et al., 1985;
Chupp et al., 1992; Hollister et al., 1995). Peculiarities observed in the sporogony o f this parasite have lead to the creation o f the genus T ra ch ip leisto p h o ra with the species T. h o m in is (Hollister et al., 1995). O f all these species, Ent. b ie n e u s i is the most frequent in AIDS patients. It is reported in 14 to 40 % o f HIV- infected patients with weight loss and chronic diarrhea (Asmuth et al., 1994; W eber et al., 1994). A recent eva
luation o f the prevalence o f enteric pathogens in HIV- infected patients has show n that microsporidia are the m ajor cau se o f diarrhea and other gastrointestinal manifestations (Kotler, 1995).
The interest o f biologists and clinicians is mainly focused on the diagnosis and treatment o f human m icrosporidioses. Most inform ation concerning the characterization o f these pathogens and their identifi
cation is provided by the study o f in vitro models o f microsporidial infections.
T A X O N O M IC A L DATA _ _
^ he com parison o f isolates o f E n c ep h a lito z o o n (= sep ta ta ) in testin alis obtained from cultures on RK13 cell line (Van G ool et al., 1994) with those o f E n c e p h a lito z o o n c u n ic u li and E nc. h ellem was performed by SDS-PAGE, w estern blotting and DNA analysis (Hartskeerl et al., 1995). Due to the hom ology (90 % ) found betw een gene sequences encoding for small subunits o f ribosomal RNA, the spe
cies previously described under the generic name o f S ep tata has been reclassified in the genus E n c e p h a litozoon .
G enom ic analyses have also shown that the micro
sporidian isolated from the urine and sputum o f an AIDS patient corresponded to E n c e p h a lito z o o n c u n i
cu li, a species already know n to infect carnivora, rodents or lagomorphs (De Groote et al., 1995). Similar results w ere obtained by Hollister. Interestingly the human isolate differed from canine and murine isolates (Hollister et al., 1995). A rabbit isolate has b een also recently characterized (Didier et a l , 1995).
DETECTION
T
he identification o f intracellular stages o f microsporidia requires histological studies o f samples obtained by invasive procedures. Therefore, the
detection o f the spores released in excreta such as faeces, urine, bile, duodenal, bronchial or nasal fluids appears to be the most practical method o f diagnosis.
However it is hampered by the small-size o f micro
sporidian spores. Actually, the uneasy diagnosis of microsporidia has been a cause o f underestimation of the num ber o f infected patients. Electron m icroscopy remains the best method for the specific identification o f developmental stages and also for confirming the diagnosis performed with other techniques such as the staining by the fluorochrom e Uvitex 2B (Van G ool et al., 1993) and the trichrome o f W eber et a l . (1992) modified by Kokoskin et al. (1994) which are exten
sively used for the identification o f microsporidia in stools, urine and any other sample (D e Girolami et al., 1995). New specific diagnostic tools have been pro
posed using im m unological characterization or the PCR amplification o f genes coding for conserved ribo
somal RNA subunits.
Im m u n o d ia g n o sis
The production o f specific antibodies directed against microporidian species as well as the detection o f anti
bodies in patients’ sera require large quantities o f puri
fied antigens. In vitro cultures o f E n c e p h a lito z o o n
DESPORTES-LIVAGE I
h ellem and E nc. c u n ic u li provide millions o f spores w hich are released in the supernatants. This material has been used for immunological identification and characterization (Aldras e t al., 1994; Didier et al., 1991, 1995; Hollister et al., 1995; Schwartz et al., 1993 ; Vis- vesvara et al., 1995). Fluorescent antibody staining enabled the distinction betw een the two m orphologi
cally similar species E nc. h ellem and E nc. c u n ic u li (Schwartz et al., 1993). In the absence o f experimental m odels o f Ent. b ien eu si, other m icrosporidia have b een tested for their serologic cross-reactivity with this species. Both Ent. b ien eu si and E nc. in testin alis w ere thus aspecifically detected by indirect immuno- fluorescent antibody tests using murine polyclonal antisera raised in BALB/c mice immunized with spores of Enc. h ellem or E nc. c u n ic u li (Aldras et al., 1994;
Zierdt et al., 1993). A w estern blot technique using G lu gea a th erin a e, a microsporidian parasitic in fish, has show n the cross-reactivity o f sera from patients with microsporidiosis to G lu g ea antigens (Om brouck et al., 1995). Since 1994, in vitro cultures o f E nc. in testin alis permits the developm ent o f diagnostic procedures (D oultree et al., 1995; Hartskeerl et al., 1995). Poly
clonal antibodies selectively directed against this spe
cies have been raised in rabbits (Aldras et al., 1994;
Visvesvara et al., 1995) and m ice (Hartskeerl et al., 1995). O m brouck has recently proposed a method com bining the non specific labeling o f microsporidian spores by the fluorochrom e Uvitex 2B and an indirect im m unofluorescent assay using a polyclonal antibody specifically directed against Enc. intestinalis (Ombrouck et al., 1996). Additionally this method enables the dis
tinction betw een spores o f this species and those o f E n tero cy to z o o n b ie n e u s i w hich are only stained by fluorochom e.
P C R DETECTION
The genom ic amplification by the polymerase chain reaction for the detection and characterization o f micro- sporidial infections in humans has b een first investi
gated in the Netherlands and in the USA (Shuitema et al., 1993; W eiss e t al., 1993; Zhu et al., 1994). Primers are selected in the conserved DNA sequence coding for the small subunit ribosomal RNA which contains species-specific seq u ences and has a characteristic small size. Using these primers, (SSU)rRNA gene frag
ments o f Ent. b ien eu si w ere detected in biopsies after amplication by the PCR method. Fedorko e t al. (1995) have proposed a PCR assay for the detection o f micro
sporidia in stool specim ens. Presently, the species identification o f Ent. b ie n e u s i and Enc. in testin alis in stool samples can be performed within some hours due to a recent improvement o f the PCR m ethod (O m brouck et al., 1996).
1 1 0
HOST IM M U N IT Y
A N D MICROSPORIDIAL IN FE C TIO N S
C
linical manifestations o f microsporidioses as those o f other opportunistic infections correlate with the intensity o f infection and the d ecrease in the num ber o f CD4+ cells (CD4+ cell counts < 50 per mm3) (Kotler, 1995). Animals models are helpful for understanding human infections and stu
dying the relationships betw een microsporidia and the host immune system. A model o f human encephalito- zoonosis (E n c. c u n ic u li) has b een obtained using SCID m ice (Herm anek et al., 1993; Koudela et al., 1993). Experimental infections by Enc. cu n icu li, Enc.
h ellem and V ittaform a c o m e a e (= N o sem a c o rn eu m ) w ere also obtained by Didier et al. (1994) in different murine strains and m onkeys. All these microsporidia cause asymptomatic and chronic infection in immu- nologically com petent animals w hereas im m unocom promised ones die more or less rapidly according to the infecting species. Lethality was observed in g en e
ralized infections caused by the dissemination o f the parasites by the m acrophages. Experimental infections of BALB/c m ice have show n that the destruction o f E nc. c u n ic u li by m acrophages occurred in the pre
sence o f factors released by T-lym phocytes (Schmidt
& Shadduck, 1984 cited in Didier et al., 1994, 1995).
In vitro studies have demonstrated that murine m acro
phages could kill Ent. cu n ic u li w hen they w ere treated with IFN-gamma in com bination with lipopolysaccha- ride; a m icrobiostatic acitivity w as obtained w hen m acrop h ages w ere treated w ith IFN -gam m a only (Didier & Shadduck, 1994). According to Didier (1995), microbicidal or microbiostatic activities o f activated macrophages occur through the nitric oxyde-dependent mechanism also involved in the killing o f L e is h m a n ia m a jo r and other pathogens (Vouldoukis et al., manus
cript in press). These in vitro data suggest that the lack o f activating factors such as IFN-gamma due to TH1- cell depletion (Del Prete & Romagini, 1994) is involved in the spreading o f Encephalitozoonosis in athymic mice.
TREATMENT
C
linical assays confirmed the selective eradication o f all E n c e p h a lito z o o n species by alben
dazole. In vitro assays have show n the altera
tio n o f th e d e v e lo p m e n t o f E n t. c u n i c u l i. T h e diassem bly o f m icrotubules caused by albendazole inhibits the formation o f mitotic spindles and prevents the nuclear divisions thus producing enlarged and disorganised stages (Colbourn et al., 1994). The deve
Parasite, 1996, 3, 107-113 M ise au point
Human micbosporjdioses and AIDS
lopm ent o f this species is also inhibited by fumagillin, 5-fluorouracil and sparfloxacin (Beauvais et a l , 1994).
Patients infected with Ent. in testin alis are successfully treated with albendazole as shown by the improvement in symptoms within days o f start o f therapy and the clearance o f spores o f the parasite in stools (Sobottka et al., 1995). It is noteworthy that spores o f the para
site are still detected in the urine o f som e treated patients after the improvement o f clinical symptoms.
The persistence o f infected sites could explain the para
sitic relapses frequently observed in patients with severe im m unodeficiency despite low -dose m ainte
nance therapy with albendazole (Molina et al., 1995).
Fumagillin has been used with success in the treatment o f keratoconjunctivitis caused by Ent. h ellem (Diesen- house et a l , 1993). Up to this date no therapy has proven to be effective against E. b ien eu si.
C O N C L U S IO N
T he increasing num ber o f studies on human m icrosporidioses have contributed to the better characterization o f their agents. They have also improved the diagnosis and the treatment o f ence- phalitozoonoses. However specific treatments o f E nte- ro cy to z o o n b ien eu si are not yet available. In addition the in vitro model o f Ent. b ie n e u s i is still lacking. No doubt that the obtention o f long term culture o f this sp ecies will facilitate the developm ent o f specific immunological tests and facilitate pharmacological stu
dies. The method o f isolation o f microsporidian spores by flow cytometry developed by Challier et al. in 1994 can provide samples o f spores o f Ent. bien eu si. This material is presently selected in our laboratory for the production o f antibodies and the infection o f cell lines.
The epidem iology o f human microsporidioses is under investigation. Spores o f Ent. b ie n e u s i have been found in stools o f an im m unocom petent traveller returning from tropical areas and without HIV infection (Sand- fort et al., 1993). Similarly a case o f infection by this microsporidia has been reported in a turkish child living in Germany on her return from a vacation trip in Turkey (Sobottka et al., 1995). These patients pre
sented diarrhoea w hich resolved without treatment.
Theses observations suggest that Ent. b ie n e u s i cause intestinal infections which are spontaneously resolutive in immunocompetent people. The presence o f Ent. b ie n eu si in an HIV-negative patient w ho was treated with cyclosporin after transplant therapy confirms the dra
matic involvement o f the im m unodeficiency in the developm ent and pathogenesis o f m icrosporidioses (M onneret et a l , 1995). It must be pointed out that som e cases o f microsporiodiosis observed in immu
nocom petent patients are limited to immunoprivileged
sites as shown by corneal stroma infections caused byN osem a o c u la ru m (Cali et al., 1991) and V ittaform a c o rne a e (Silveira & Canning, 1995). These data confirm the opportunistic character o f microsporidial infec
tions.
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