SPECIFIC AND RAPID DETECTION OF M I C R O S P O R I A IN STOOL SPECIMENS FROM aids PATIENTS BY PCR

SPECIFIC AND RAPID DETECTION OF M I C R O S P O R I A IN STOOL SPECIMENS FROM aids PATIENTS BY PCR

O M B R O U C K C.*, C I C E R O N L.* & D E S P O R T E S - L I V A G E I.*

Summary:

Two microsporidian species, Enterocytozoon bieneusi and

wasting syndrome in AIDS patients. A new PCR assay is proposed for the rapid and specific detection of these parasites in stools.

KEY WORDS : AIDS, microsporidia, PCR, stools.

Résumé : DÉTECTION SPÉCIFIQUE ET RAPIDE DES MICROSPORIDIES PAR LA PCR DANS LES SELLES DE PATIENTS ATTEINTS DE SIDA

Deux microsporidies, Enterocytozoon bieneusi et Encephalitozoon intestinalis, causent la diarrhée et l'amaigrissement de patients atteints de SIDA. Un nouveau test par la PCR permet la détection rapide et spécifique de ces parasites dans les selles.

MOTS CLÉS : SIDA, miaosporidie, PCR, selles.

During the last d e c a d e , intestinal microsporidia h a v e b e c o m e r e c o g n i z e d as an important c a u s e o f o p p o r - tunistic infections in i m m u n o c o m p r o m i s e d patients, especially t h o s e with AIDS. T w o s p e c i e s are the c a u s e o f diarrhoea a n d o t h e r gastrointestinal diseases in HIV infected patients: ENTEROCYTOZOON BIENEUSI ( D e s p o n e s ET AL., 1 9 8 5 ) and ENCEPHALITOZOON INTESTINALIS (Hartskeerl ET AL., 1 9 9 5 ) . Diagnosis o f gastrointestinal microspori- diosis c a n b e m a d e b y detecting s p o r e s o f the para- sites in stool s p e c i m e n s with W e b e r ' s modified tri- c h r o m e stain o r optical brightening agents such a s Uvitex 2 B ( D e Girolami ET AL., 1 9 9 5 ) . H o w e v e r the identification o f microsporidia to t h e s p e c i e s level cur- rently d e p e n d s o n the o b s e r v a t i o n o f cytological c h a - racteristics o n l y v i e w e d with the e l e c t r o n m i c r o s c o p e . T h e specific identification o f microsporidia is o f impor- t a n c e b e c a u s e E . INTESTINALIS r e s p o n d s to a l b e n d a z o l e therapy. Up to this date, n o convincing therapy is avai- lable for E . BIENEUSI. Molecular studies have shown that the small-subunit ( S S U ) rRNA g e n e s o f microsporidia s h a r e d o n l y limited h o m o l o g y with t h e S S U rRNA g e n e s o f o t h e r eukaryotic organisms, pointing out that t h e s e s e q u e n c e s m a y b e useful as g e n e p r o b e s in hybridization a n d p o l y m e r a s e c h a i n reaction ( P C R ) assays ( W e i s s ET AL., 1 9 9 4 ) . W e therefore evaluated the diagnostic value o f amplification o f microsporidian DNA b y PCR with template DNA extracted from stool s p e c i m e n s from HIV-infected patients with and without intestinal microsporidiosis, as confirmed b y light-micro- s c o p y . Positive controls for PCR w e r e o b t a i n e d from cultures for E . INTESTINALIS a n d from s p o r e s isolated by flow cytometry for E . BIENEUSI (Challier ET AL., 1 9 9 4 ) .

* Unité INSERM 313. CHU Pitié-Salpêtrière, 91, boulevard de l'Hôpital, 75013 Paris.

Parasite, 1996, 3, 85-86

DNA w a s r e l e a s e d from microsporidia b y boiling the s a m p l e s . T h e PCR r e a c t i o n w a s p e r f o r m e d u n d e r conditions d e s c r i b e d b y Weiss ( 1 9 9 4 ) . T h e primer pair V1 a n d E B 4 5 0 w a s u s e d to amplify DNA from E . BIE-

NEUSI a n d the internal 3 0 m e r o l i g o n u c l e o t i d e E B 1 5 0 w a s u s e d to confirm b y southern blotting that the amplified fragment w a s from E . BIENEUSI. T h e primer pair V I and SI500 w a s u s e d to amplify DNA from E. INTESTINALIS.

In s a m p l e s containing E . BIENEUSI, amplification o f the predicted 3 5 0 b p fragment w a s indisputable o n ethi- dium stained gels (Fig. 1A). Using an internal oli- g o m e r , hybridization w a s a p p a r e n t in all o f t h e s e s a m p l e s (Fig. I B ) . In negative stools n o amplification w a s s e e n b y ethidium staining o r detected by hybri- dization. In addition, primer pair V I a n d SI500 ampli- fied a 3 8 0 b p fragment from s t o o l s infected with E . INTESTINALIS and from cultured parasite but not from u n i n f e c t e d stools. No amplification w a s s e e n with E . BIENEUSI infected stool s a m p l e s (Fig. 1A). No hybri- dization w a s o b s e r v e d w h e n using E B 1 5 0 the specific p r o b e o f E. BIENEUSI (Fig. I B ) .

PCR has already b e e n used by F e d o r k o ( 1 9 9 5 ) t o identify intestinal microsporidia in stools. However, the PCR assay p r o p o s e d b y this author required harsh conditions e m p l o y i n g b o t h m e c h a n i c a l and c h e m i c a l disruption and a laborious 4-day p r o c e d u r e . Compa- ratively, boiling t h e s a m p l e s to release DNA from microsporidia a p p e a r s to b e a simple a n d time-saving m e t h o d . I n d e e d o u r PCR assay c a n b e p e r f o r m e d within o n e day and its specificity is high s i n c e w e u s e t w o pairs o f primers selective o f e a c h intestinal s p e - cies. T h e s e qualities indicate that the PCR p r o c e d u r e p r o p o s e d herein for the species-specific detection o f

Note de recherche

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1996031085

OMBROUCK С , CICERON L. & DESPORTES-LIVAGE I.

Fig. 1. — Analysis o f PCR products by agarose gel electrophoresis. A) Analysis by agarose gel electrophoresis. B ) Southern blot and hybri­

dization with EB150 probe to confirm that primer set V1/EB450 amplifies SSU-rRNA gene from E. kieneusi.

Lanes 1-6: FCR was based on the amplification o f a 350 bp fragment o f the SSU-rRNA of E. bieneusi; 1-2: stools with no known micro- sporidia; 3-4: E. bieneusi from stool; 5: E. bieneusi isolated by cytometry; 6: E. intestinalis from culture.

Lane 7: DNA fragments o f the Hae Ill-digested OX174. Lanes 8-14: PCR was based on the amplification of a 380 bp fragment o f the SSU- rRNA of E. intestinalis-, 8: E. intestinalis from culture; 9: E. intestinalis from stool; 10: E. bieneusi from stool; 11: E. bieneusi isolated by cytometry; 12-13: stools with no known microsporidia; 14: control.

intestinal microsporidia would b e very valuable for cli­

nical a n d epidemiological investigations.

Study supported b y a grant o f t h e A g e n c e Nationale d e R e c h e r c h e s sur le SIDA (ANRS).

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Reçu le 29 décembre 1995 Accepté le 3 janvier 1996

86 Note d e recherche Parasite, 1996, 3, 85-86