J Clin Pathol 1991;44:558-563

Histological diagnosis of intestinalmicrosporidiosis in patients with AIDS

C S Peacock, C Blanshard, D-G Tovey, D S Ellis, B G Gazzard

AbstractFifty nine patients seropositive forhuman immunodeficiency virus (HIV)and diarrhoea and 20 with weight losswere investigated for microsporidiosisusing light and electron microscopicalexamination of duodenal and jejunalbiopsy specimens. Eight cases of micro-sporidiosis were found, in five ofwhom itwas the sole pathogen. In all eight casesthe organism was identified at lightmicroscopy without prior knowledge ofthe electron microscopical findings. Allstages of the life cycle are best seen inresin sections cut at 1 pm and stainedwith Giemsa, but spores could easily beidentified in paraffin sections cut at 5 pmand stained with haematoxylin andeosin. In all cases the parasite was iden-tified both in duodenal pinch and jejunal"Crosby" capsule biopsy specimens. Allcases of microsporidiosis occurred inpatients with diarrhoea. Both electronand light microscopical examinationsuggested that the pathogenic mechan-ism involves the shedding of infectedenterocytes containing large numbers ofspores.

It is suggested that the optimal way todiagnose microsporidiosis is by lightmicroscopical examination of duodenalpinch biopsy specimens.

AIDS Unit,Westminster Hospital,Horseferry Road,London SWlP 2APC BlanshardB G GazzardThe London School ofHygiene and TropicalMedicineC S PeacockD G ToveyD S EllisCorrespondence to:Dr B G Gazzard

Accepted for publication7 February 1991

Microsporidial infection may cause diarrhoeain up to 30% of patients positive for humanimmunodeficiency virus (HIV) in whom noother pathogen is found.' The organism wasfirst discovered in the small intestine of aHaitian patient in 1980,2 and infection hasbeen reported ever since with increasingprevalence in patients from Europe,3 NorthAmerica' and Africa.4 Four genera of micro-sporidia have been identified by electronmicroscopy in man,5 three as rare isolatedinfections but occurring in many animal hosts.The fourth genus, Enterocytozoon, has onlyone recognised species, E bieneusi, and hasonly been found in HIV positive patients withmalabsorption and diarrhoea.6At present the diagnosis of microsporidial

infection depends on the examination of sec-tions, imprints, or smears of small bowelbiopsy specimens as oocysts have only re-cently been shown in human faeces7 and noserological tests have yet been developed.Although the organisms can be seen with thelight microscope using fresh impression

smears of jejunal biopsy specimens stainedwith Giemsa,8 this technique is not routinelyperformed and the recommended methodof detection has been by electron micro-scopy.'"" This is time-consuming, expensive,restricted in its availability and inappropriatefor large numbers of samples.The aim of this investigation was to deter-

mine the prevalence of microsporidial infec-tion in groups of HIV antibody positivepatients and to assess whether such infectioncan be diagnosed reliably at light microscopy.Both jejunal biopsy specimens obtained by aWatson capsule and duodenal pinch biopsyspecimens were examined as the latter wouldbe more convenient to use for routine diag-nosis.

MethodsGroup A comprised 59 HIV positive patientswith diarrhoea (defined as three or more liquidmotions a day for more than a month). Fortyfive had pathogens other than microsporidia(19 Cryptosporidium, eight Cytomegalovirus(CMV), five Salmonella species, four Campy-lobacter species, three Mycobacterium aviumintracellulare, six Giardia lamblia, two Herpessimplex virus). In 14 patients no other causeof diarrhoea was found despite micro-biological analysis of multiple stool samples,duodenal aspiration, and rigid sigmoidoscopyand rectal biopsy. One patient had cyto-megalovirus duodenitis, but a rectal biopsyspecimen could not be obtained because of apainful anal fissure.Group B comprised 20 HIV positive

patients with weight loss (greater than 10%ideal body weight lost) but no diarrhoea, whowere being investigated for causes of mal-absorption.

In all the patients the HIV antibody testwas confirmed by two methods. All patientshad duodenal and jejunal biopsy specimensobtained during the course of fibre opticendoscopy. Spiked, fenestrated biopsy forcepswere used to take six randomly sited pinchbiopsy specimens from the second part of theduodenum and a Watson capsule waspositioned in the distal duodenum endo-scopically and then advanced into the jejunumto obtain jejunal biopsy specimens.

Rectal biopsy specimens were obtained inall patients during rigid sigmoidoscopy.Each jejunal biopsy specimen was bisected.

One piece was processed for light and theother for electron microscopical examination.Specimens for light microscopy were fixed for

558 on July 13, 2020 by guest. P

rotected by copyright.http://jcp.bm

j.com/

J Clin P

athol: first published as 10.1136/jcp.44.7.558 on 1 July 1991. Dow

nloaded from

Histological diagnosis of intestinal microsporidiosis in patients with AIDS

1

6.IFigure I An early meront with a single cleft (small arrow) but no discernible nucleiyet, and another at a later stage containing several clefts (largerarrow) and several nuclei,found in enterocytes of the small intestine (a I gm resin section stained with haematoxylin and eosin).

Figure 2 A multinucleate sporont, or plasmodium, produced by nuclear divisions in the meront is arrowed (a 3 pm parqffin wax section stained withhaematoxylin and eosin).

Figure 3 An enterocyte containing two sporonts (arrowed), the lower one indenting the host cell nucleus; section prepared as in fig 1.

Figure 4 A sporont deforming the host cell nucleus (arrowed). Note afurthersporont at the upper right region of thisfigure: section preparedas in fig 1.

Figure 5 Developing spores, which at this stage are freely dispersed throughout the host cell cytoplasm (a I gm resin section stained with Giemsa).

a minimum time of three hours in 10% formolsaline. Specimens were then processed follow-ing a routine schedule by dehydrating throughgraded alcohols, clearing in Citroclear, andembedding in paraffin wax. Sections 2-5 gmthick were cut using a rotary microtome,placed on albumin coated slides, and heatedon a hotplate for 15 minutes. Slides from eachspecimen were then stained with haematoxy-lin and eosin, Giemsa, Gram's stain, periodicacid Schiff and Ziehl Neelsen stain.

In the eight patients in whom micro-sporidial infection was found, further biopsymaterial was processed through graded alco-hol and embedded in glycolmethacrylateresin. Sections of 1 gm were cut and stainedwith haematoxylin and eosin and Giemsastains.

Specimens for electron microscopicalexamination were fixed in 3% gluteraldehydein 0-1 molar cacodylate buffer, pH 7-3, post-fixed in 1% osmium tetroxide, dehydratedthrough graded methanols, impregnated viapropylene oxide and embedded in Taab resin.

Sections 70 nm thick were stained with uranylacetate and lead citrate and examined using aJEOL 100 CX microscope.

ResultsEight patients with microsporidiosis wereidentified; in five it was the sole pathogen(one had Mycobacterium avium intracellulare;one had Mycobacterium avium intracellulareand Campylobacter; one had cytomegalovirus(CMV). Microsporidiosis was not diagnosed inany patient who did not have diarrhoea. In alleight cases the organism was identified by lightmicroscopy and electron microscopy. In all thecases the microsporidia were seen indepen-dently at light microscopy using standard cutsections stained with haematoxylin and eosinwithout knowledge of the electron micro-scopical findings. None of the rectal biopsyspecimens examined either by light or electronmicroscopy had evidence of establishedmicrosporidiosis, although in some specimensdead cysts suggestive of attempted infestationwere found.

559

on July 13, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.44.7.558 on 1 July 1991. D

ownloaded from

Peacock, Blanshard, Tovey, Ellis, Gazzard

6 * ... !.i,5: c .. ,..._

*. .0 '. '.:t

.>. '"f'S;. e>t X* . ' :S . . . . : . .., .:: ,',:: ' . ., ,.,.' ;:, v .. - -*: ' ;: . ::.''. :."::"';: . '',,;5:,.' :... .. ':: ,. .... ct

.

.: .: ..... :.:,: : . .... ::* . ' ' °: '., . ' ' ;

.: . §J, :*. . ,. . . ' !: 8.: .; :. :F #'.. ;., , .. . i . ^. ,lS,.:. . . ..... ';; ,:. !:: ... ; . : .:: ' .

*: ,'i''.... :*' 2.'St'''..,.^....,'S's.wt -'..' ,,'.:;

,,: .: ': ....'+ Ms::_'; .' ........ 'z''. . :'j 'S, ,.' " ........... .. ,: .. :, . ' : ..

9 :X,

*

Figure 6 Spores at various stages of maturation. An infected enterocyte (arrowed) has already broken away into the gut lumen: section prepared asinfig 2.

Figure 7 A host cell containing infective spores being extruded into the gut lumen (arrowed). The enterocyte microvilli have been lost and thecytoplasm become vacuolated: section prepared as in fig 1.

Figure 8 Three infected host cells (arrowed) containing mature spores lying free in the lumen of the gut. All these cells have lost their microvilli andhave severely vacuolated cytoplasm: section prepared as infig 1.

Figure 9 A 1 gm resin section stained with Giemsa showing a very heavy microsporidiosial infection of enterocytes. (This type ofpreparationprobably gave the clearest results, but proved unnecessary for normal diagnostic purposes.)

LIFE CYCLE AT LIGHT MICROSCOPYThe various stages in the life cycle of themicrosporidium were identified in the smallintestinal biopsy specimens of all eightpatients, except the initial infective sporoplasmwhich is propelled into the cell from the spore.The next stage, the meront, proved the most

difficult to identify with certainty. In theearliest recognised forms it appeared as aweakly basophilic body, about 3-5 pm indiameter, often with a clear line or slashthrough it (fig 1). In later stages more individualnuclei were contained within the structure andmore clear lines became visible (fig 1).A multinucleate structure, the sporont, or

plasmodium was identified, which waspresumed to occur as a result of nuclear divi-

sion (merogony) of the meront (figs 2 and 3).This structure was seen to be in direct contactwith the host cytoplasm at electron micro-scopical examination. Individual units werenot refractile but were strongly basophilic. Theplasmodia varied in size between 3-8 gm indiameter, and more than one parasite could befound in infected host cells, giving rise to up to40 nuclei. Sporont and meront stages werefound apposed to the host cell nucleus in manydifferent orientations, identing the latter insome instances (figs 3 and 4).The plasmodia were presumed to enlarge by

cell division (sporogony) and to differentiate toproduce spores 2-3 pm in diameter (fig 5) whichwere distributed between the nucleus and theapex of the luminal surface of the cell. Unlike

560

on July 13, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.44.7.558 on 1 July 1991. D

ownloaded from

Histological diagnosis of intestinal microsporidiosis in patients with AIDS

0

4'

K .&

Figure 10 Heavily infected enterocytes in a patient with AIDS. Four parasites are arrowed in the central cell, the adjacent ones containing threeandfive meronts, respectively. Note the clefts in meronts and indented host cell nuclei.

Figure 11 Spores containing sixfilament coils, typical of Enterocytozoon bieneusi.

Figure 12 Enlarged view of thefilaments, aggregated as infig 11, showing their internal structure.

the plasmodia, the individual units were oftendissociated from one another and not present ina tight group (fig 6).

Enterocytes containing mature sporesappeared to be degenerate in all eight patients(fig 7), with vacuolated cytoplasm and thedisappearance of the microvilli. The cells con-taining mature spores appeared to be activelyextruded from the mucosa (fig 8). This processdid not resemble normal cell replacement andadjacent uninfected cells were normal inappearance.

COMPARISON OF DIFFERENT STAINING METHODSAND DEPTH OF SECTIONSSpores were the most readily identified forms ofmicrosporidia. When the condenser on themicroscope was lowered or removed, theirrefractile nature became more apparent makingidentification much easier. The spores couldreadily be identified on paraffin wax sectionsstained with haematoxylin and eosin, even insections cut at 5 pIm. The sporont and merontstages could be identified in sections cut at 3 gumor less, although all stages of the life cycle werebest seen in resin sections cut at 1 pm. Theoptimal stain for light microscopy washaematoxylin and eosin for wax embedded (fig2) and Giemsa (fig 9) for resin embeddedmaterial. These stains yielded a clear contrastbetween parasite and background, allowingdiagnosis at lower magnification, which wasparticularly striking using Giemsa. In all casesthe parasite was identified on both duodenalpinch biopsy specimens and "Crosby" capsulesamples.

ELECTRON MICROSCOPYAll stages other than the sporoplast werereadily identified on electron microscopy.Infection in each case was due to E bieneusibecause there were six coils ofthe polar filamentof the spores present (figs 1 1 and 12) and therewas no parasitopherous vacuole. The earlymeront stages (fig 10) were easily identifiedbecause of their pale appearance within thedarker staining host cytoplasm and thepresence within them, even when mono-nucleate, of transparent clefts which seemedto become more numerous as developmentproceeded. The polar filaments were first seen asdark osmiophilic aggregations from which oneindividual filament became allocated later as itbecame formed, still lying within the intactparasite plasmalemma-that is, formation byendodyogeny (fig 13). The outer walls of thesespores stained intensely (fig 14) and were foundto be the easiest features to identify at electronmicroscopy when scanning the infected biopsyspecimens at low magnification.There was evidence ofprogressive damage to

the microvilli of the infected cells (fig 15) andthe mature spores seemed to be liberated intothe gut lumen following the breakdown andextrusion of the latter (figs 14-17).

DiscussionThis study seems to show that microsporidiosisis more common in selected groups of patientsin the United Kingdom than in the previouslypublished unselected series where infestationwas only found in one of more than 100 HIVantibody positive patients with diarrhoea.'2

561

on July 13, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.44.7.558 on 1 July 1991. D

ownloaded from

Peacock, Blanshard, Tovey, Ellis, Gazzard

.E

0

Figure 13 A meront containing at least 16 nuclei (three marked "n") and extensive central endoplasmic reticulum. The arrow marks a group of, asyet, unallocatedfilaments awaiting their incorporation individually into the developing spores.

Figure 14 Profiles of some 30 spores no longer surrounded by any sporont plasmalemma. Once these latter have disintegrated it is impossible to tell, ashere, whether one or more sporont gave rise to these spores. This host cell still appears viable with an intact brush border.

Figure 15 Parasite spores lying within a vacuolated and dying enterocyte before the process of its extrusion.

Figure 16 Profiles of two spores (arrowed) within a dead enterocyte which is being extruded into the gut lumen, but whose plasmalemma is stilladherent to those of the adjacent cells. Note disappearance ofany proper brush border.

Figure 17 Thefinal stage in the extrusion process of an infected enterocyte (two spores arrowed). Note the healthy condition of the adjacentgut cellswith their normal microvilli.

The incidence in the present study, where fiveof 15 patients in whom no other pathogen hadbeen found as a cause of diarrhoea had evidenceof microsporidiosis, is very similar to a largeNew York experience reported recently.'Our study suggestes that light microscopy is

a satisfactory technique for showing thepresence of microsporidia. The organism wasseen in all patients in whom it could also beidentified by electron microscopical examina-tion. This contrasts with the study from NewYork' where only five of 20 cases were initiallysuspected by light microscopy, and it wassuggested that this might have been because of

low numbers of infecting particles or poortissue preparation. The lower definition andmagnification of light microscopy comparedwith that of electron microscopy, however,should be compensated for by the ability toscan larger areas of tissue, and poor tissuepreservation should only be a problem withnecrotic tissue or necropsy material. The use of1 gm glycolmethacrylate resin sections stainedwith Giemsa and examined at light microscopygave the clearest identification of the variousstages of microsporidiosis, but thicker cutparaffin wax sections stained with haematoxylinand eosin clearly showed microsporidial infec-

562

f-?..11.

I

z. .k

on July 13, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.44.7.558 on 1 July 1991. D

ownloaded from

Histological diagnosis of intestinal microsporidiosis in patients with AIDS

tion, particularly the spore stage which wasbasophilic and highly refractile.

It is still only possible to classify theindividual species of microsporidia using elec-tron microscopy although the characteristicsize, site, and appearance at light microscopyshould suggest the diagnosis of E bieneusi.Other intracellular parasites of enterocytesincluding Cryptosporidium, Isospora, andSarcocystis species are easily distinguishedfrom microsporidial infection at the lightmicroscopic level.The light and electron microscopic

appearances of the life cycle of this parasitestudied in this paper confirm many of thepreviously reported features. Electron micro-scopy also clearly showed division of thesporont by endodyogony, which has not beenreported before. Both light and electronmicroscopy showed extrusion of necrotic cellswith large numbers of spores contained withinthem, which is a method of shedding andtransmitting this parasite that has not beenpreviously recorded.

Proofthat microsporidiosis is responsible fordiarrhoea in immunodeficient patients is un-likely to be obtained until a suitable animal hostis identified or there is an effective treatmentcapable of eradicating the protozoan. Micro-sporidia were only found in our patients withdiarrhoea, however, which might be caused bythe observed degeneration ofinfected epithelialcells.Much remains to be learnt about the clinical

characteristics and prognosis of patients with

microsporidiosis. This should become mucheasier ifthe organism can readily be identified inlight microscopic samples. Duodenal pinchbiopsy specimens are particularly easy to per-form, and clinically it seems that this procedureis as likely to yield a positive histologicaldiagnosis as larger "Crosby" capsule samples.

1 Orenstein J, Steinberg W, Chieng J, Smith P, Rotterdam H,Kotler D. Intestinal microsporidiosis as a cause of diar-rhoea in HIV infected patients. A report of 20 cases.Human Pathol 1990;21:475-81.

2 Desportes I, Le Charpentier Y, Gallan A, et al. Occurrenceof a new microsporidian: Enterocytozoon bieneusi n.g.n.sp. J Protozool 1985;32:250-4.

3 Curry A, McWilliam LJ, Habousi NY, Mandal BK. Micro-sporidiosis in a British patient with AIDS. J Clin Pathol1988;41:477-8.

4 Lucas SB, Papadaki L, Conlon C, Sewankambo N, Good-game R, Serwadda D. Diagnosis of intestinal Micro-sporidiosis in patients with AIDS. J Clin Pathol 1989;42:885-90.

5 Shadduck JA. Human microsporidiosis and AIDS. RevInfect Dis 1989;11:203-7.

6 Canning EU, Hollister WS. Enterocytozoon Bieneusi(Microspora): Prevalence and pathogenicity in AIDSpatients. Trans Roy Soc Trop Med Hyg 1990;84:181-6.

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

8 Rijpstra AC, Canning EU, Van Ketel RJ, Eeftinck Schatten-kerk JKM, Laarman JJ. Use of light microscopy todiagnose small intestinal microspordiosis in patients withAIDS. J Infect Dis 1988;157:827-31.

9 McWilliam LJ, Curry A. Intestinal microsporidiosis inAIDS. J Clin Pathol 1989;42:173-4.

10 Dobbins WO, Weinstein WN. Electron microscopy of theintestine and rectum in the acquired immunodeficiencysyndrome. Gastroenterology 1985;88:738-49.

11 Cali A, Owen RL. Microsporidiosis. In: Balows A, HauslerWRJ Jr, Lenette EH, eds. The laboratory diagnosis ofinfectious diseases: Principles and practice. Vol 1. NewYork: Springer-Verlag, 1988:929-50.

12 Canning EU, Hollister WS, Cann K, et al. Microsporidianinfection due to Enterocytozoon: Relationship withimnmunosuppression and HIV. Trans Roy Soc Trop MedHyg 1988;82:651.

563

on July 13, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.44.7.558 on 1 July 1991. D

ownloaded from