papers diagnosis patients hiv by new - from bmj and acp · uvitex 2b method. faecal samples, which...

J Clin Pathol 1993;46:694-699

PapersDiagnosis of intestinal and disseminatedmicrosporidial infections in patients with HIV bya new rapid fluorescence technique

T van Gool, F Snijders, P Reiss, J KM Eeftnck Schattenkerk, MA van den Bergh Weerman,J FWM Bartelsman, J JM Bruins, E U Canning, J Dankert

AbstractAims-To assess the value of a new rapidfluorescence method for the diagnosis ofmicrosporidiosis in HIV seropositivepatients.Methods-Microsporidian spores instools were demonstrated by using thefluorochrome stain Uvitex 2B. The newtechnique was evaluated in three groupsof HIV seropositive patients with diar-rhoea. Group 1: 19 patients with biopsyconfirmed E bieneusi infection (186 stoolsamples); group 2: 143 consecutivepatients from whom faeces were submit-ted for routine investigation of diarrhoea(318 samples); group 3: 16 patients withsmall intestinal biopsy specimens nega-tive for microsporidia (55 samples). Thenew method was used to monitor sporeshedding during experimental treatmentwith paromomycin and albendazole infour patients.Results-Brightly fluorescent sporeswere detected in all stool samples ofpatients in group 1. In group 2 16 (11%)patients had spores in their stool sam-ples. E bieneusi was found in 11 patients;in the other five another genus ofmicrosporidia, Encephalitozoon, wasrecognised. Encephalitozoon spores werealso found in the urine of three of thesepatients and in the maxillary sinusaspirate of two of them, suggesting dis-seminated infection. The results wereconfirmed by electron microscopic exaniin-ation. In group 3 negative biopsy speci-mens were confirmed by negative stoolsamples in all cases. Treatment withalbendazole and paromomycin did notaffect the spore shedding in threepatients with E bieneusi infection. Bycontrast, in a patient with Encephalito-zoon sp infection albendazole treat-ment resulted in clinical improvementtogether with complete cessation of sporeexcretion in the stool.Conclusion-The Uvitex 2B fluorescencemethod combines speed, sensitivity, andspecificity for the diagnosis and treat-ment evaluation of intestinal and dissem-inated microsporidiosis.

(i Clin Pathol 1993;46:694-699)

Microsporidia are obligate intracellular proto-zoan parasites that infect most animal phyla.'In HIV seropositive people infections with

different types of microsporidia are beingdiagnosed increasingly often. The dominantspecies infecting the enterocytes of the smallintestine is Enterocytozoon bieneusi, thought tobe responsible for as many as 30% of cases ofchronic diarrhoea for which no other micro-bial cause can be found.'4 More recently, Ebieneusi has also been detected in the bile andbiliary epithelium from patients with a clinicalsyndrome of AIDS related sclerosing cholan-gitis5 (Abstracts presented at the 3rdEuropean Conference on Clinical Aspectsand Treatment of HIV Infection, Paris, 1992;187, 189). Occasional infection with anothermicrosporidian genus, Encephalitozoon, hasbeen associated with hepatitis,6 peritonitis,7and keratoconjunctivitis.8 Recently, a specieswith some of the characteristics ofEncephalitozoon has been identified in theintestine of a patient with AIDS.9Traditionally, diagnosis of E bieneusi intesti-nal microsporidiosis has depended on inva-sive endoscopic procedures to provide biopsyspecimens from the distal duodenum or thejejunum, for examination by light microscopyor electron microscopy. Recently, the detec-tion of E bieneusi spores in faeces has beendescribed: this could replace endoscopicbiopsy as the favoured procedure for diagnos-ing this infection.'1"2We initially reported a Giemsa staining

procedure for faecal smears.'0 Although use-ful, this procedure is time-consuming becauseit requires lengthy microscopic examinationto recognise the very small spores, especiallyif present in low numbers. Most recently, achromotrope-based staining technique for thedetection of E bieneusi in faeces has improvedthe discrimination between spores and otherfaecal elements, but the method remainslaborious and lengthy to perform and has alimited sensitivity.'2To allow diagnosis of microsporidiosis to

be part of routine faecal examination, wedeveloped a new, fast, and more generallyapplicable fluorescence method based onstaining with the fluorochrome Uvitex 2B.Uvitex 2B binds to chitin, a component of themicrosporidian spore wall. During intracellu-lar maturation of spores, the chitin compo-nent of the spore wall increases.'The method was evaluated in HIV

seropositive patients with known biopsy con-firmed E bieneusi infection and in unselectedHIV positive patients from whom faeces wereroutinely submitted for parasitological exami-nation.We were also able to use the method for

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Department ofMedical MicrobiologyT van GoolF SnijdersJ DankertJ J M BruinsDepartment ofInternal Medicine(AIDS Unit)F SnijdersP ReissJ KM Eeftinck

SchattenkerkDepartment ofPathologyM A van den BerghWeerman

Department ofGastroenterologyJ FWM BartelsmanAcademic MedicalCenter, University ofAmsterdam,Meibergdreef 9, 1105AZ, Amsterdam,The NetherlandsDepartment ofBiology, ImperialCollege LondonE U CanningCorrespondence to:Dr T van GoolAccepted for publication17 February 1993

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Diagnosis of intestinal and disseminated microsporidial infections in patients with HIV

diagnosis of intestinal and disseminatedEncephalitozoon-type infections. Finally, anattempt was made to assess whether themethod could be used to monitormicrosporidian spore shedding during drugtreatment.

MethodsThe Uvitex 2B method was evaluated in threegroups of HIV seropositive patients withdiarrhoea.

GROUP 1This group comprised 19 patients with biopsyconfirmed E bieneusi infection. In 14 theinfection had been diagnosed by light micro-scopic examination of Giemsa stained duo-denal biopsy smears. In the remaining fivediagnosis was established by examiningGiemsa stained faecal smears, with subse-quent confirmation by biopsy. In total 186faecal samples were collected (two to 80 sam-ples per patient) and examined with theUvitex 2B method. Faecal samples, whichhad been obtained from one patient on 40consecutive days, were examined to studydaily variation in spore shedding. All 19patients had persistent diarrhoea andrepeated tests for Salmonella sp, Shigella sp,Yersinia sp, and Campylobacter sp, as well asfor parasitic pathogens, including Giardia,Cryptosporidium and Isospora spp were nega-tive. Faecal samples were not routinely exam-ined for mycobacteria. In 14 of 19 patientsthe duodenal biopsy specimens were exam-ined for the presence of mycobacteria byZiehl-Neelsen stain and culture; all speci-mens were negative.

GROUP 2Faecal samples from 143 HIV seropositivepatients with diarrhoea, consecutively submit-ted for routine parasitological examinationbetween April 1991 and February 1992, wereexamined with Uvitex 2B. If microsporidianspores were detected with both Uvitex 2Band Giemsa, patients were asked to undergoduodenoscopy with biopsy for confirmationof infection. In cases where a biopsy speci-men was not available, electron microscopicexamination of faeces was performed. Urinespecimens, sinus aspirate, nasal discharge andsputum from four patients were examined.All patients had their stools routinely culturedfor pathogens. In five of the 10 patients biop-sied, the biopsy specimen was also examinedfor the presence of mycobacteria.

GROUP 3Sixteen of the 143 patients from group 2, inwhom stool examination for microsporidianspores and other pathogens was negative,were examined more extensively. Small bowelbiopsies were performed either shortly after(eight patients), or before (eight patients) fae-cal examination with Uvitex 2B. These speci-mens were examined "blind" by lightmicroscopy for the presence of microsporidia.The results from this group were used to

assess the specificity of the new method.

EVALUATION OF DRUG TREATMENTParasitological and clinical response to treat-ment with paromomycin and albendazolewere evaluated in two patients with AIDS foreach drug. The patients experienced persis-tent diarrhoea; biopsy confirmed microspori-diosis was the only recognised microbialcause. Faecal samples were obtained dailyand examined with the Uvitex 2B method.

PREPARATION OF SPECIMENS FROM PATIENTSFresh faeces (0-5-1 g) were homogenised in 8ml distilled water and filtered through a 300,um pore mesh sieve. After diluting the filtratein distilled water to an appropriate density(MacFarland No 9) smears were preparedeither by spinning an aliquot of 100 ,ul in acytospin centrifuge at 1500 rpm for 2 min-utes, or by air-drying 50 pl on a circular(5 mm) area on a slide. The effects of priordisinfection of the faecal homogenates, with4-10% formalin, ethanol 70%, or heating to56°C for 30 minutes on the staining proper-ties of spores was examined. Urine samplesand maxillary sinus aspirates were centrifugedat 1250 x g for 10 minutes. The pellet waswashed once with phosphate buffered saline(PBS). Smears were then prepared. Smallintestinal biopsy specimens were obtained byupper gastrointestinal fibreoptic endoscopy.Biopsy specimens taken from the distal partof the duodenum were processed as describedbefore.4

Material for electron microscopy (faeces,duodenal biopsy specimens, urine, maxillarysinus aspirate, nasal discharge and sputum)was fixed in Karnovsky's fixative for 10 min-utes, post-fixed with osmium tetroxide for 10minutes, and embedded in Epon resin.Polymerisation was at 80°C for 12 hours.Sections were stained with uranyl acetate andlead citrate and examined with a Philips CM10 electron microscope.

STAININGThe slides were fixed with methanol for 2minutes for fluorescence staining, stainedwith 1% Uvitex 2B (Ciba-Geigy Basel; PH3-9/CH 6 25) in PBS (pH 7-2) for 10 min-utes, rinsed with PBS (2-5 seconds), counter-stained with 0.5% Evan's blue in PBS (30seconds), rinsed with PBS (2-5 seconds) andleft to dry at room temperature. Smears wereexamined at x 1250 magnification under aLeitz fluorescence microscope equipped witha 50 Watt mercury high pressure lamp and anexcitation filter with a transmission rangefrom 355 to 425 nm and a suppression filterof 460 nm (Ploemopak filter block D). Theactive substance of Uvitex 2B has a X lightchain of 355 nm. Uvitex 2B stained faecalsmears were stored at room temperature inthe dark and re-examined after six and 10months. The average number of maturespores in five fields/slide (x 1250) was esti-mated.To examine the staining properties of

Uvitex 2B, smears ofE cuniculi obtained from

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Van Gool, Snijders, Reiss, Eeftinck Schattenkerk, van den Bergh Weermnan, Bartelsman, Bruins, Canning, Dankert

Figure 1 Enterocytozoonbieneusi in faeces stainedwith Uvitex 2B x 3000.Mature spores (thickarrow) and immaturedevelopmental stages(presumably sporoblasts;thin arrow). Bar = 2,um.

in vitro culture were prepared. E cuniculi wasmaintained in RK13 cells with Dulbecco'sminimal essential medium supplemented with10% heat-inactivated fetal calf serum andgentamicin (20 ,ug/ml) and amphotericin B(20 ,g/ml). The cultures were incubated at37°C in an atmosphere of 5% CO, in air.Tissue culture supernatant fluid, containingimmature stages and mature spores, was col-lected, washed once with PBS, pelleted, andresuspended in PBS. For smear preparation,100 ,ul of suspension was spun in a cytospincentrifuge at 1500 rpm for 2 minutes.

Figure 2 Encephalitozoonsp in small intestinalenterocytes. Parasitophorasvacuoles with spores (s) inthe centre, sporoblasts (sb)at the periphery and somegranular material (gm)separating the stages.Bar = 1 plm.

ResultsSTAINING PROPERTIES OF MICROSPORIDIA WITHUVITEX 2BUvitex 2B stained spores of E cuniculi,obtained from culture supematant fluid, fluo-resced bright white. Immature developmentalstages (presumably sporoblasts), appearedreddish brown. In culture supernatant fluid,mature spores considerably outnumberedimmature developmental stages.

In faecal samples two different micro-sporidia, confirmed by electron microscopy asE bieneusi and a species of Encephalitozoon,were recognised after staining with Uvitex2B. The same staining properties-namelybright white fluorescence or reddish brown-as observed with the cultured E cuniculi, wereexhibited respectively by mature and imma-ture stages of both species. In the faecalsmears, however, the immature stages greatlyoutnumbered the mature spores.The shape and size of mature spores of E

bieneusi (broadly oval, about 1 6 x 1 0 ,im)showed little variation (fig 1), but the largerspores of Encephalitozoon varied considerably,both in shape and size, being broad and rod-like or kidney shaped, and measuring 2-5-3-3x 1 3-2 1 ,um. Mature spores and immaturedevelopmental stages of Encephalitozoon spwere also seen in urine samples, nasal dis-charge, maxillary sinus aspirate, and sputum.A morphological characteristic, always pre-sent in a proportion of the mature spores ofEbieneusi, and (less frequently) in those ofEncephalitozoon sp, was a concave inner stripeand shadow. This characteristic was usedthroughout the study as an important crite-rion for identification of microsporidianspores. The inner stripe and shadow were notobserved in mature spores of E cuniculiderived from culture supematant fluids.The background was dark brown or black

and discrimination between microsporidianspores and fluorescing structures such asyeasts and fungi was possible on the basis ofclearly visible differences in size and mor-phology. Only small fungal spores showedsome resemblance to E bieneusi andEncephalitozoon sp spores, but they were morerod-like and the concave inner stripe andshadow were always absent.The time needed to read the slides varied

from 30 seconds to 2 minutes; to excludemicrosporidia infection a 3 minute search wassufficient.

Fixation with ethanol 70% or heating offaecal suspensions to 56°C for 30 minutes didnot alter the staining properties of maturespores, but fixation with formalin reducedconsiderably the intensity of fluorescence ofmature spores.

Storage of Uvitex 2B stained smears atroom temperature in the dark for six monthsdid not influence fluorescence of maturespores but a slight decrease of intensity wasobserved after 10 months.

Electron microscopic examination ofEncephalitozoon infections in small intestinalbiopsy specimens showed all developmentalstages other than sporoplasms (fig 2).

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FaecesUvitex 2B positive

0

Figure 3 Results offaeces and duodenal biopsy examination in patients with biopsyconfirmed E bieneusi infection (group 1), with diarrhoea ofunknown origin routinelysubmittedfor parasitological examination (group 2), and with negative biopsy specimensfor microsporidia (group 3).

Meronts were found lying adjacent to themembrane of a parasitophoras vacuole;sporogon phases were found lying free withinthe vacuole. Around the parasites in the vac-uole a variable amount of loose granularmaterial was frequently observed. Sporogonywas disporous, not in a sporophorous vesicle,and the nuclear arrangement was monokary-otic. These characteristics are diagnostic ofthe genus Encephalitozoon.

DIAGNOSIS OF MICROSPORIDIOSIS WITH UVITEX2B IN THE THREE GROUPSGroup 1Mature spores and immature stages wereseen in all 186 Uvitex 2B stained faecalsmears from the 19 patients with known Ebieneusi small intestinal infection (fig 3).Faecal samples obtained from one patient on40 consecutive days were all positive. A varia-

Examination of various specimens from five patients with AIDS by Uvitex 2B, electronmicroscopy or light microscopy for Encephalitozoon sp, infection

Specimen examined:

Duodenal Maxillary sinusFaeces biopsy Urine aspiratelnasal discharge

Case Uvitex 2B Uvitex 2BNo Uvitex 2B EM LM and EM andEM andEM

1 + + + + ND2 + ND + + +3 + + + - ND4 + + _ + +5t + ND ND ND ND

LM light microscopy; EM electron microscopy; + positive; - negative; ND not done; *No EMavailable; tPatient died several days after diagnosis of intestinal microsporidiosis.

tion in spore numbers of 1-30 maturespores/field (x 1250) was observed; no regu-lar daily or weekly pattern was discernible.

Group 2Intestinal infection-Using Uvitex 2B, intesti-nal microsporidiosis was diagnosed in 16 of143 (11-2%) patients with diarrhoea. Ebieneusi was found in 11 of 16 (69%), andEncephalitozoon infection in five of 16 (31%)patients (fig 3). Concomitant (potential)pathogenic bacteria and parasites weredetected in the stools in six of 16 patients;Entamoeba histolytica (twice), Entamoebahistolytica, and Campylobacter jejuni (once),Blastocystis hominis (once), Shigella flexneri(once) and Mycobacterium avium intracellulare(once). All duodenal biopsy specimens exam-ined for mycobacteria were negative.

In patients with E bieneusi infection confir-mation was obtained by light microscopicexamination of duodenal biopsy specimens insix cases and by electron microscopic exami-nation of faecal samples from the remainingfive cases. In one patient with low numbers ofE bieneusi spores in faeces, confirmed by elec-tron microscopy, microsporidia were notdetected in biopsy specimens either by lightmicroscopic or electron microscopy. In twoother patients, with numerous spores in thestool, extensive light microscopic examinationof duodenal biopsy smears was requiredbefore any infected cell was detected.

Biopsy specimens for electron microscopyexamination were available from four of thefive patients in whom Encephalitozoon hadbeen found in the faeces with Uvitex 2B;Encephalitozoon infection was confirmed inthree (table). In the fourth patient nomicrosporidia were detected in biopsy speci-mens even after prolonged examination byboth light microscopic and electronmicroscopy (case 4). The CD4 + counts inthe five patients with Encephalitozoon sp infec-tion ranged between 0 01 and 0a 14 x 109/1.

Extraintestinal infection-Urine samples wereexamined from four of the five patients inwhom Encephalitozoon sp had been found asan intestinal infection. In three of themspores and immature stages characteristics ofEncephalitozoon sp were detected with Uvitex2B; electron microscopic confirmation wasobtained in two (table). Parasites werelocated both in extracellular as well as inintracellular positions. The morphology ofinfected epithelial cells by light microscopysuggested that they had originated from thelower urinary tract. There was no clinical orlaboratory evidence for the existence of uri-nary tract disease. Bacterial cultures fromurme were negative.

In two patients with an Encephalitozooninfection (cases 2 and 4) spores were alsodetected in maxillary sinus aspirate, nasal dis-charge, and sputum. Ultrastructurally, themature spores and immature developmentalstages observed seemed to be identical withthose present in duodenal biopsy specimens.

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Van Gool, Snijders, Reiss, Eeftinck Schattenkerk, van den Bergh Weerman, Bartelsman, Bruins, Canning, Dankert

Group 3In this group of 16 patients with diarrhoealight microscopy of small intestinal biopsyspecimens did not provide evidence ofmicrosporidiosis in any of the patients. Atotal of 55 stool samples (two to 10 per

patient) were examined with negative results(fig 3).

EVALUATION OF DRUG TREATMENTTwo patients with intestinal E bieneusi infec-tion as the sole known cause of their chronicdiarrhoea were treated with oral paro-

momycin 500 mg three times a day for 12days. Both patients experienced a decrease indiarrhoeal symptoms, but spores were

detected in daily stool samples throughoutthe treatment period without obvious alter-ation in the shedding pattern of the spores.

One patient with E bieneusi-associatedchronic diarrhoea was treated with oral alben-dazole 400 mg twice a day for 15 days. Thisdid not result in any changes, either in diar-rhoeal signs and symptoms, or in sheddingof spores in faeces. Another patient withchronic diarrhoea and biopsy confirmedEncephalitozoon infection of the small intes-tine with massive spore excretion in the stoolreceived oral albendazole 400 mg twice a day.His diarrhoea lessened in a few days and fullydisappeared after three weeks. Spore excre-

tion stopped completely after six days oftreatment and the stool remained negative fortwo months. A duodenal biopsy specimentaken after two months treatment was free ofparasites. After 10 weeks, in spite of contin-ued albendazole treatment, diarrhoearecurred and stool examination at 16 weekscontained Encephalitozoon spores again, con-

firming the clinical relapse.

DiscussionThe diagnosis of E bieneusi intestinalmicrosporidiosis has, until recently, dependedon invasive endoscopic procedures.2A13 Non-invasive means for diagnosis became feasibleafter spores had been recognised in faeces.'2-14Unfortunately, Giemsa stained faecal prepa-

rations require experienced interpretation.Although a recently described chromotropebased staining technique'4 may overcome

these restrictions to some extent, the lengthy(2 hours) and laborious staining proceduremakes it unsuitable for routine use. The

Uvitex 2B method is a considerable advanceon previous methods for the diagnosis ofmicrosporidial infections because of its sim-plicity and the brilliant white fluorescencewhich facilitates rapid recognition of spores.The staining procedure takes less than 15minutes to perform, and the time to read theslides reliably is only of the order of minutes.The staining properties are not reduced bythe use of ethanol 70% or heating to 56°C for30 minutes. Therefore, these methods for dis-infection of the specimens can be used beforethey are processed further in the laboratory.

As Uvitex 2B binds to chitin, it is not

strictly specific for microsporidia. 14 The

microsporidian spores, however, can easily bedifferentiated by morphological character-istics from other chitin-containing micro-organisms.A valuable feature of the Uvitex 2B

method is that it distinguishes betweenmature chitin-containing spores and non-infective immature spores of both E bieneusiand Encephalitozoon sp. Mature spores of Ecuniculi were abundantly present in super-natant fluid from in vitro cultures. In con-trast, immature spores of E bieneusi andEncephalitozoon greatly outnumbered maturespores in faeces. This suggests that the life-span of small intestinal epithelial cells inpatients with microsporidiosis is too short forcomplete development of most of themicrosporidia. The concave inner stripe andshadow observed in a large part of the maturespores probably represents a partly collapsedinner spore wall resulting from discharge ofthe spore contents into the environment.

In all patients with intestinal micro-sporidiosis, spores were detected with theUvitex 2B method in all faecal specimens,irrespective of day to day variation in sporeshedding and faeces consistency. This is incontrast with observations by Weber et al,who, using the chromotrope-based stainingmethod in two patients with intestinalmicrosporidiosis, found only five of 25 faecalsamples to be positive.'2

Furthermore, some studies suggest ahigher sensitivity of Uvitex 2B compared withsmall intestinal biopsy for diagnosis of intes-tinal microsporidiosis. In two patients stoolexamination with the Uvitex 2B methodrevealed microsporidia (confirmed as Ebieneusi and Encephalitozoon sp by electronmicroscopy of stool and urine sediment,respectively), while duodenal biopsy speci-mens obtained subsequently were negative byboth light microscopy and electronmicroscopy. In two other patients withnumerous spores of E bieneusi in their stoolsparasites could be found in biopsy specimensonly after a prolonged search at lightmicroscopy.

In previous studies with HIV seropositivepatients with chronic diarrhoea not explainedby other intestinal pathogens, microsporidio-sis has ranged from 20 to 30%.2A The preva-lence of 11% (E bieneusi and Encephalitozoonsp combined) found in our study reflects theprevalence of intestinal microsporidiosis inunselected HIV positive patients presentingwith diarrhoea.

Until now small intestinal infection with Ebieneusi has been the predominant type ofmicrosporidiosis in HIV seropositive individ-uals. Our staining technique also led to therecognition of small intestinal infection withanother genus of microsporidia, which couldbe distinguished from E bieneusi on the basisof differences in size and shape, and whichwas ultrastructurally similar to Encephalito-zoon.

Serological surveys suggest that antibodiesto E cuniculi are relatively common amongcertain human populations, with rates up to

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20% in HIV seropositive individuals.'5 Theidentification of Encephalitozoon sp in five ofthe 16 (31%) patients with intestinal micro-sporidiosis indicates the potential importanceof this genus as a cause of chronic diarrhoeain HIV positive subjects. As has beenreported for E bieneusi in this study and oth-ers,4 12 infections with Encephalitozoon sp werefound only in patients with greatly reducedCD4 + lymphocyte numbers.Whether the tendency of the Encepha-

litozoon sp to disseminate beyond the gut willresult in increased recognition of new clinicalsyndromes remains to be elucidated. A recentstudy suggests that Encephalitozoon-likemicro-organisms can cause renal pathology inpatients with AIDS.16 The presence ofEncephalitozoon spores in sputum as observedin two patients in our study does not provepulmonary infection by microsporidia aspostnasal drip may be mixed with the spu-tum.

Currently, no proved effective treatmentfor human microsporidial infections exists. InHIV seropositive patients with E bieneusiintestinal infection clinical improvement ofdiarrhoeal signs and symptoms has beenreported in uncontrolled studies with a smallnumber of patients treated with metronida-zole4 or albendazole."7 Neither drug eradi-cated the parasites from the small intestine,although ultrastructural changes in E bieneusiin biopsy specimens following albendazoletreatment have been reported.'9 Paromo-mycin, an aminoglycoside which shows mini-mal absorption after oral administration, hasrecently shown promise for the treatment ofcryptosporidial diarrhoea in HIV seropositivepatients. 8

In the patients with microsporidiosis due toE bieneusi who were treated with paro-momycin or albendazole, no changes in theshedding pattern of spores in stools wereseen; although some clinical improvementwas observed with paromomycin. This effectcould be the result of changes in the intestinalflora by the drug, leading to reduction ofintraluminal digestion of malabsorbed nutri-ents and thus stool volume. An effect of paro-momycin on another unrecognised pathogenalso cannot be excluded.

In contrast, in the patient withEncephalitozoon infection treatment withalbendazole led to clinical improvement aswell as cessation of spore shedding in thestool. Recently, we made a similar observa-tion in another patient with Encephalitozooninfection (case 4), with complete cessation of

spore shedding in faeces and also in urine andsinus discharge. These findings suggest thatalbendazole may well exert a different effecton Encephalitozoon and E bieneusi in vivo.Our new fast and easy staining technique

with Uvitex 2B will bring the diagnosis ofmicrosporidial infections within the range ofroutine practice. Furthermore, it may facili-tate research regarding epidemiology, patho-genesis, and treatment of microsporidiosis.We thank Ms JM de Bruin and Mr H Gilis for technical assis-tance, Dr JCM Vetter and Dr J Veenstra for advice and clini-cal support, and Dr LM Kortbeek (National Institute ofPublic Health and Environmental Protection, Bilthoven) forproviding E cuniculi for culture.

1 Canning EU, Lom J. The microsporidia of vertebrates. NewYork: Academic Press, 1986.

2 Peacock CS, Blanshard C, Tovey DG, Ellis DS, GazzardBG. Histological diagnosis of intestinal microsporidiosisin patients with AIDS. Jf Clin Pathol 1991;44:558-63.

3 Orenstein JM, Chiang J, Steinberg W, Smith PD,Rotterdam H, Kotler DP. Intestinal microsporidiosis asa cause of diarrhoea in human immunodeficiency virus-infected patients: a report of 20 cases. Hum Pathol1990;21:475-81.

4 Eeftinck Schattenkerk JKM, van Gool T, van Ketel RJ, etal. Clinical significance of small-intestinal micro-sporidiosis in HIV-1-infected individuals. Lancet 1991;337:895-8.

5 McWhinney PHM, Nathwani D, Green JF, et al.Microsporidiosis detected in association with AIDS-related sclerosing cholangitis. AIDS 1991;5:1394-5.

6 Terada S, Reddy RK, Jeffers U, Cali A, Schiff ER.Microsporidian hepatitis in the acquired immunodefi-ciency syndrome. Ann Intern Med 1987;107:61-2.

7 Zender HO, Arrigoni E, Eckert J, Kapanci Y. A case ofEncephalitozoon cuniculi peritonitis in a patient withAIDS. AmJClin Pathol 1989;92:352-6.

8 Didier ES, Didier PJ, Friedberg DN, et al. Isolation of anew human microsporidian, Encephalitozoon hellem(n.sp.) from three AIDS patients with keratoconjunc-tivitis. J Infect Dis 1991;163:617-21.

9 Cali A, Orenstein JM, Kotler DP, Owen R. A comparisonof two microsporidian parasites in enterocytes of AIDSpatients with chronic diarrhoea. Jf Protozool 1991;38:96-8.

10 Van Gool T, Hollister WS, Eeftinck Schattenkerk J, et al.Diagnosis of Enterocytozoon bieneusi microsporidiosis inAIDS patients by recovery of spores from faeces. Lancet1990;336:697-8.

11 Orenstein JM, Zierdt W, Zierdt C, Kotler DP.Identification of spores of Enterocytozoon in stool andduodenal fluid from AIDS patients. Lancet 1990;336:1127-8.

12 Weber R, Bryan RT, Owen RL, et al. Improved light-microscopical detection of microsporidia spores in stooland duodenal aspirates. N Engl J Med 1992;326: 161-6.

13 Desportes I, le Charpentier Y, Galian A, et al. Occurrenceof a new microsporidian: Enterocytozoon bieneusi n.g.,n. sp., in the enterocytes of a human patient with AIDS.J Protozool 1985;32:250-4.

14 Koch HH, Pimsler M. Evaluation of Uvitex 2B: a nonspe-cific fluorescent stain for the detection and identifying offungi and algae in tissue. Lab Med 1987;18:603-6.

15 Hollister WS, Canning EU, Willcox A. Evidence for wide-spread occurrence of antibodies to Encephalitozooncuniculi (microspora) in man provided by ELISA andother serological tests. Parasitology 1989;102:33-43.

16 Orenstein JM, Dieterich T, Kotler DP. Systemic dissemi-nation by a newly recognized intestinal microsporidiaspecies in AIDS. AIDS 1992;6:1143-50.

17 Blanshard C, Ellis DS, Tovey DG, et al. Treatment ofintestinal microsporidiosis with albendazole in patientswith AIDS. AIDS 1992;6:311-3.

18 Clezy K, Gold J, Blaze J, Jones P. Paromomycin for thetreatment of cryptosporidial diarrhoea in AIDS patients.AIDS 1991;5:9.

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