Intracellular Protozoan Infection in Small Intestinal Biopsies of Patients with AIDS

Path. Res. Pract. 192,249-259 (1996)

Intracellular Protozoan Infection in Small Intestinal Biopsies of Patients with AIDS Light and Electron Microscopic Evaluation R. Boldorini, A. Tosoni 1 , G. Mazzucco2 , M. Cernuschi 3 , P. Caramello 4 , E. Maran2 , G. Costanzi 1 and G. Monga Dipartimento di Scienze Mediche, Facolta di Medicina e Chirurgia di Novara; 1 V Cattedra di Anatomia Patologica, Istituto di Scienze Biomediche tiL. Sacco," Universita di Milano; 2Dipartimento di Scienze Biomediche e Oncologia Umana, Facolta di Medicina e Chirurgia, Universita di Torino; 3Clinica delle Malattie Infettive, Ospedale S. Raffaele, Milano; 41stituto di Malattie Infettive, Universita di Torino, Torino, Italy

SUMMARY Small intestinal biopsies of 21 patients with acquired immunodeficiency syndrome (AIDS) with light microscopic findings diagnostic or suspicious for parasite infection were investigated" by transmission electron microscopy (TEM). TEM allowed us to identify and specify the genus and species of involved parasites in 16 out of the 21 cases: 7 Cryptosporidium parvum, 5 Enterocytozoon bieneusi and 4 Isospora belli. Cryptosporidium was easily identified on light microscopy (LM), and only slightly influenced by parasite burden in all the 7 cases; TEM confirmed LM diagnosis and made it possible to characterize the parasites as C. parvum. The identification of Microsporidium on LM in our cases was related to the burden of parasite; its presence was certainty identified in 2 cases and suspected in 3. TEM allowed to identify these parasites as E. bieneusi. Intracytoplasmic coccidia could be detected with certainty in semithin sections in all 4 cases, but TEM was always needed to specify the infectious agent as I. belli. In 5 cases the suspicious of protozoan infection on LM (3 microsporidia, 1 intracytoplasmic coccidia and 1 Cryptosporidium) was not confirmed by TEM. Our data suggest that TEM is an appropriate diagnostic tool in this field of pathology and necessary in most of the cases.

Introduction Parasitic infections of the gastrointestinal (GI) tract are increasingly found to be responsible for severe diarrhea and weight loss in patients with acquired immunodeficiency syndrome (AIDS)2, 11, 27, 36. Since they share similar symptoms, the identification of the causative agent solely on the ground of the clinical setting is difficult, if not impossible. Extensive search for the © 1996 by Gustav Fischer Verlag, Stuttgart

parasites in either stool samRles or in small intestinal biopsies is therefore needed 4,27. Biopsies are considered a useful diagnostic tool because one can identify infectious agents in them and also evaluate the severity of the mucosal inj ury 3, 14, 15, 17. Intracellular parasites may be identified by light microscopy (LM) using conventional or special stains when they are numerous or when the severe mucosal damage and inflammation suggest their presence and 0344-0338/96/0192-0249$3.50/0

250 . R. Boldorini et al.

prompt a close search 35 . However, AIDS patients may have severe GI symptoms with only a few parasites and normal mucosal architecture lO • Also, the identification of different agents or the various stages of their life cycle may be difficult with LM alone. Transmission electron microscopy (TEM) has been pro~osed as an appropriate diagnostic tool in such cases 0. Recent reports have investigated the ability of LM to diagnose Microsporidium in paraffin sections, using standard or special stains 16, 19,21,30,34, 39,40. In some of these studies the LM results were compared with those of TEM16, 21, 30. To our knowledge no investigators have compared the abilities of LM and TEM to differentiate microsporidia from other intracellular parasites, and, especially, to distinguish the different species of coccidia. It must be stressed that the correct diagnosis is of clinical importance, because of different prognostic and therapeutic implications27 . Indeed, there are no specific therapies for some of these (i.e., Cryptosporidium and Sarcocystis 12 ), but others (such as Cyclospora, Microsporidium and Isospora belli) can be successfully treated 12, 13,24. In order to evaluate the usefulness of TEM in this field of pathology, we studied a group of small intestinal biopsies of patients with AIDS and severe GI symptoms where parasitic infection was diagnosed or suspected by LM, and compared those results with the results of the TEM examination. Special attention was paid to differentiate various species of infecting protozoa on the grounds of LM and TEM.

Results The major clinicopathological findings for the selected patients are shown in Table 1, and the results of the LM and TEM examinations are shown in Tables 2 and 3. Cryptosporidium could be easily identified by LM, either in paraffin or semi thin sections, in cases number 1,2,3 and 4, due to high parasitic burden. HE disclosed roundish particles of 2-5 /lm in diameter, with small intracellular vacuoles at high magnification, free in the lumen or resting on the brush border of the small intestinal mucosa. The parasites were intensely PAS positive, dark-blue with toluidine blue and brownish with the PTAH stain (Fig. 1). TEM investigation confirmed the presence of Cryptosporidium and disclosed the various phases of its life cycle, including both asexual (trophozoites, Fig. 2, and meronts, Fig. 3 A), ranging from 1.5 to 4 /lm, and sexual forms (micro and macrogametocytes, Fig. 3 B), ranging from 3 to 4 /lm. All the phases were found within parasitophorous vacuole, covered by the cell membrane of the absorptive cells. Cases number 5 and 6 showed only rare forms of Cryptosporidium by LM, which were confirmed by TEM through the identification of both asexual and sexual forms in case 6, and only asexual forms in case 5. In case number 7, infection by Cryptosporidium was suspected in only a few cells in the paraffin sections, confirmed in semi thin sections stained with tolui-

Table 1. Clinicopathological findings of the studied population Age/Sex

Risk factor

CD4* count

Other pathologies

1 2 3

29M 27M 37M

IVDUo Unknown Homosexual

3 10 2

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

25 M 35 M 32M 26 F 52M 31 M 32 F 59 M 30M 23 F 39M 40M 36 M 38 M 44M

Homosexual IVDU IVDU IVDU Homosexual IVDU Unknown Homosexual IVDU Unknown Homosexual Homosexual IVDU IVDU Homosexual

28 17 15 15 30 11 24 5 5 35 8 8 45 21 57

C. albicans esophagitis Bacterial pneumonia. C. albicans esophagitis Kaposi's sarcoma disseminated. T. gondii encephalitis. CMV chorioretinitis Kaposi's sarcoma disseminated

19 20 21

60M 36 M 31 M

Homosexual Homosexual Homosexual

24 20 25

Case

CD4* count: cells/ill; IVDUo: Intravenous drug user.

C. neoformans disseminated Non-Hodgkin's lymphoma M. avium lymphadenitis CMV chorioretinitis CMV, disseminated. C. albicans esophagitis C. albicans esophagitis P. carinii pneumonia. C. albicans esophagitis M. avium esophagitis Salmonellosis Kaposi's sarcoma. cutaneous; T. gondii encephalitis CMV esophagitis T. gondii encephalitis; C. albicans esophagitis HSV retinitis; P. carinii pneumonia

Intestinal Protozoa Infections in AIDS· 251 Table 2. Light and electron microscopic findings of biopsy samples Case

Histology

Semi thin sections

TEM

Inflammation

Atrophy

Parasite

Burden of parasite

Parasite

Parasite

1 2 3

Chronic, moderate Absent Chronic, mild

Absent Absent Mild

Cryptosporidium, DO Cryptosporidium, D Cryptosporidium, D

+++ +++ +++

C. parvum C. parvum C. parvum

4 5 6 7 8

Chronic, mild Absent Chronic, mild Chronic, mild Absent

Mild Absent Absent Absent Absent

Cryptosporidium, Cryptosporidium, Cryptosporidium, Cryptosporidium, Microsporidia, D

+++ + + 0/+ +++

Cryptosporidium, D Cryptosporidium, D Cryptosporidium, D Microsporidia, S" Cryptosporidium, D Cryptosporidium, D Cryptosporidium, D Cryptosporidium, D Microsporidia, D

9

Chronic, mild

Absent

Microsporidia, S

+

Microsporidia, S

10

Chronic, mild

Absent

Microsporidia, S

0/+

Microsporidia, S

11

Chronic, mild

Mild

0/+

Microsporidia, S

12

Chronic, mild

Absent

0/+

Microsporidia, D

13

Chronic, mild

Mild

++

14

Chronic, mild

Moderate

15

Chronic, mild

Moderate

16

Chronic, mild

Absent

Microsporidia, S Coccidia, intracytoplasmic, Coccidia, intracytoplasmic, Coccidia, intracytoplasmic, Coccidia, intracytoplasmic, Coccidia, intracytoplasmic,

NA

17

Chronic, mild

Absent

0/+

18 19 20 21

Chronic, mild Absent Chronic, mild Chronic, mild

Absent Absent Absent Absent

Coccidia, intracytoplasmic, S

Coccidia, intracytoplasmic, Coccidia, intracytoplasmic, Coccidia, intracytoplasmic, Coccidia, intracytoplasmic,

Microsporidia, S Cryptosporidium, S

0/+ 0/+

Findings for parasite presence: DO

N N

D D D S

S S D S S

+ 0/+

N

Microsporidia, S Microsporidia, S

N N

D D D D

C. parvum C. parvum C. parvum C. parvum Enterocytozoon bieneusi Enterocytozoon bieneusi Enterocytozoon bieneusi Enterocytozoon bieneusi

Enterocytozoon bieneusi Isospora belli Isospora belli Isospora belli Isospora belli

N N N N N

= diagnostic, S" = suspicious, NA = negative

Table 3. TEM evaluation of the phases of life cycle of the infecting parasites Case

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16

Parasite

Phase of life cycle

C. parvum C. parvum C. parvum C. parvum C. parvum C. parvum C. parvum E. bieneusi E. bieneusi E. bieneusi E. bieneusi E. bieneusi I. belli I. belli I. belli I. belli

Meronts I and II; trophozoites; micro and macrogametocytes Meronts I and II trophozoites, micro and macrogametocytes Meronts I; microgametocytes Meronts I and II; trophozoites; micro and macrogametocytes Meronts II; trophozoites Trophozoites; macrogametocytes Meronts II; trophozoites Meronts, early and proliferative phases, sporonts, spores Meronts, proliferative stages, spores Meronts, proliferative stages; spores Meronts, proliferative stages; spores Meronts, proliferative phase; sporonts; spores Schizonts; merozoites (from 6 to 7 each P.Vo.); trophozoites Schizonts; merozoites (from 1 to 6 each P.Y.); trophozoites Schizonts; merozoites (from 5 to 13 each P.Y.); trophozoites Merozoites (from 2 to 4 each P.Y.)

P.VO. = parasitophorous vacuole.

252 . R. Boldorini et al.

Fig. 1. Two cryptosporidia (arrows) are evident on the epithelial surface of intestinal mucosa. Semithin section, PTAH, 960 x.

dine blue, and with TEM, through the finding of a few trophozoites and meronts in the parasitophorous vacuoles of rare enterocytes. Semi thin sections of case 3 also disclosed features suspicious for the presence of microsporidia which was not confirmed by TEM. In all? cases the infection by Cryptosporidium caused several types of injuries in the infected cells: the microvilli were rare, blunted or joined at their bases, with loss of the glycocalyx (Fig. 2); the cytoplasm of the enterocytes contained autolysosomes, and there was dilatation of smooth endoplasmic reticulum.

In case 8, Microsporidium was diagnosed on paraffin sections stained with Giemsa and semi thin sections stained with toluidine blue and PTAH (Fig. 4). In spite of a close search, the parasites were not identified in the HE and PAS-stained sections. Clusters of egg-shaped spores, from 1 to 1.5 /lm, were seen in the cytoplasm of enterocytes, often located in a supranuclear site. More rarely, the spores were identified in a subnuclear region of the enterocytes and in degenerating cells within the intestinal lumen. On occasion, intermediate (plasmodial) forms were identified in the semithin sections, as elliptical, intracytoplasmic bodies, about 2.5 -5.5 /lm, staining lighter than the surrounding cytoplasm and with clefts and small nuclei in intimate contact with the cytoplasm of the enterocytes. Electron microscopy revealed spores and intermediate forms typical of Enterocytozoon bieneusi in the cytoplasm of the enterocytes. In detail, we could find the early proliferating plasmodium, which was a roundoval, anucleated intracytoplasmatic body, about 1 /lm in diameter, surrounded by a ring of mitochondria, and containing only free ribosomes. This form was identified in only a few cells. Later-stages meronts (about 2-3.5 /lm by 1.5-2 /lm in size) and sporonts (about 2.5-5.5/lm by 1.5-3/lm in size) were frequently found. They were characterized by the presence of nuclei, clefts and precursors of the polar tube complex. Finally, several mature spores, about 1 /lm in diameter, some of these with six turns of the polar tube (about 60 nm) and anchoring plate, typical of E. bieneusi, were also identified in some cells (Fig. 5 A-D).

Fig. 2. Trophozoites of Cryptosporidium parvum in their typical extracytoplasmic, but intracellular site. Microvilli are severely damaged. 21 000 x.

Intestinal Protozoa Infections in AIDS . 253

The infection by Microsporidium was suspected in cases 9, 10 and 11 by LM due to the presence of sparse apical bodies in a few enterocytes. Nevertheless, these bodies were not diagnostic for Microsporidium in Giemsa stained sections nor in semi thin sections. Moreover, the presence in case 11 of rare egg-shaped forms, 2.5 -4 /lm in diameter, "cupping off" the nuclei of apical enterocytes and sometimes surrounded by a clear halo, similar to a parasitophorous vacuole, created suspicion of coinfection with intracytoplasmic coccidia. TEM examination of these cases showed spores and plasmodial forms diagnostic for E. bieneusi. After careful examination, the presence of other parasites was excluded. Histologic findings were suspicious for coccidia infection in case 12, whereas semithin sections and TEM revealed a few spores of Microsporidium. All the phases of the life cycle of E. bieneusi were identified in this case by TEM. Enterocytes infected with E. bieneusi showed only mild injuries, such as the presence of many lysosomes and autophagosomes associated with spores in the cytoplasm of the enterocytes. No damage of the microvilli was observed. Severely damaged enterocytes in the intestinallumen containing a lot of mature spores, were rarely found. Intracytoplasmic coccidia infection was identified in paraffin sections in only one case (number 13), due to its high parasitic burden. In HE, they appeared as poorly stained elongated structures within the cytoplasm of the apical pole of the enterocytes, often surrounded by a clear space (Fig. 6). The parasites were not seen with PAS and Giemsa stains. Parasitic forms were too rare in cases 14 and 15 to make a sure diagnosis. No parasites were found in paraffin sections of case number 16. In semithin sections, intracytoplasmic coccidia were identified with certainty in cases 13, 14, 15 and 16. They were recognized as elongated, bananashaped forms, with small poorly distinct nuclei, darkerblue than the cytoplasm of the infected cells and separated from that by a clear halo. Single, or, more frequently, three to six forms could be seen in a clear vacuole clearly distinct from the cytoplasm. Parasitic forms (ranging from 2 to 5/lm in length and about 1 /lm in width) were found in the enterocytes alone, in all but one case (number 16), where they were also observed in the macrophages of the lamina propria and embolizing capillary vessel. Isospora belli was identified by TEM in all the 4 cases. Only asexual forms were found. Case number 16 showed a few merozoites, whereas trophozoites and schizonts prevailed in cases 13, 14 and 15. All the developing stages were found within the parasitophorous vacuoles inside the cytoplasm of enterocytes and also in macrophages of the lamina propria (case 16). In the latter case, a few extracellular merozoites were also seen. Depending on the sectioning, merozoites appeared as elongated or roundish structures (about 1 by 1.5 -5 /lm in size), with prominent nuclei and large nucleoli. Structures typical of apicomplexa, such as conoid, rhoptries, mitochondria, and

two-layer pellicle interrupted at the polar rings, were better identified in the elongated forms. Trophozoites were identified as roundish structures about 1.53 /lm, schizonts were egg-shaped, contained 2-5 nuclei and measured about 2-3 by 4-5.5 /lm (Fig. 7 A, B). Infected enterocytes showed a wide spectrum of injuries, ranging from a few lysosome-like intracytoplasmic structures to severe damage of microvilli. The damage to the enterocytes was not related to the presence of parasites: some normal enterocytes contained parasites, whereas some others which were severely injured, appeared to be free of microorganisms. Five false positive cases, according to LM data (17, 18, 19,20 and 21), were found. Infection by intracytoplasmic coccidia was suspected from paraffin sections in case 17. Cases 18, 19 and 20 revealed some round-oval 1-1.5 /lm bodies in the apical cytoplasm of the enterocytes, suggestive of micro sporidia infection. TEM examination disclosed electron dense apoptotic bodies, probably representing fragments of lymphocytes or enterocytes, in the apical pole of a few enterocytes (Fig. 8). Case 21 showed rare spherical PAS-stained bodies on the luminal surface of the enterocytes, suggestive of cryptosporidia infection, which were disregarded with TEM examination. No HIV particles or other infectious agents were found in our cases; TRI were frequently found (15/ 21) in endothelial cells of the lamina propria.

Discussion Among 21 biopsy samples for which the diagnosis of parasitic infections was made with certainty or suspected from paraffin or semithin sections, TEM made the identification and typization of the microorganisms possible in 16 cases. The identification of Cryptosporidium in paraffin sections was easy and only slightly influenced by the parasitic burden. Indeed, it was also identified with certainty in two cases in which the number of parasites was low. Mucin droplets entrapped by the microvilli can be confused with the parasitophorous vacuole of the Cryptosporidium2o • This could be the case for patient 21, in which the presence of Cryptosporidium was suspected in paraffin sections but not validated in semithin sections nor by TEM. Ultrastructural investigations did not add further data about the diagnosis of Cryptosporidium spp, but made it possible to diagnose C. parvum, due to the dimensions of the asexual forms 1• In addition, the ultrastructural examination of the biopsy specimens disclosed several injuries of the absorptive enterocytes, not identifiable by LM; these lesions did not depend on the parasitic burden, and were constant findings for our cases. The diagnostic sensitivity of LM to recognize microsporidia in paraffin sections in our cases was low. Kotler et al. 16, in a similar study performed in a larger series of small intestinal biopsies, reported a high spe-

254 . R. Boldorini et al.

Fig. 3. A, B. Various phases of life cycle of C. parvum: I type meront, with 8 merozoites (A, 25000 x), and macrogametocytes (B, 11 000 x) in their parasitophorous vacuole.

cificity but low sensitivity of LM to identify microsporidia, either in HE or in specially stained sections. A large number of their cases were defined only "suspected for," or "consistent with" the presence of microsporidia because of the low parasite burden rather than the technical limits of the method. In our cases also, diagnostic accuracy was related to the burden of parasite, but was connected more closely to the presence and the numbers of spores than of the plasmodial forms. Indeed, the former were difficult to distinguish from intraepithelial lymphocytes and from giant lysosomes or pha£osomes, also because of their unpredictable staining 2; identification of the spores allows a

sure diagnosis of microsporidia 32 only when they are numerous. On the contrary, the plasmodial forms are not recognized in HE and Giemsa stains, because they stain lightly and poorly, making it difficult to identify the parasitic clefts, and, especially, to distinguish them from the dilated endoplasmic reticulum frequently found in the absorptive epithelium 22 . In two cases (11 and 12), the presence of intracytoplasmic coccidia in paraffin sections was erroneously suspected. These microorganisms are separated from the cytoplasm by a parasitophorous vacuole in all phases of the life cycle 13, while all the stages of E. bieneusi are in intimate contact with the cytoplasm of the

Fig. 4. Spores of Microsporidium in supranuclear site of one enterocyte are evident in semithin sections (arrow). PTAH,

Fig. 6. Particular of inflammed mucosa. Several coccidia are evident into the enterocyte cytoplasms of inflammed mucosa (arrowheads). HE, 800 x.

960 x.

Intestinal Protozoa Infections in AIDS· 255

Fig. 5. Various phases of life cycle of Enterocytozoon bieneusi. - A: early proliferating plasmodium, surrounded by a ring of mitochondria (23000 x). - B: spores (arrows), and meront (arrowhead), "hollowing out" the nucleus of the enterocyte (7000 x). - C: sporont with polar tube complex (arrows) and sporoblast (arrowhead) (24000 x). - D: sloughing, degenerating enterocyte containing spores (arrowheads) (7000 x).

256 . R. Boldorini et al.

Fig. 7. Merozoites (A, 28000 x) and trophozoites (B, 15000 x) of Isospora belli in enterocytes. Note conoid (arrow) and intracellular mitochondria (arrowheads), in A, and parasitophorous vacuole (arrows), in B.

host ce1l 26 • Moreover, intracytoplasmic coccidia, when cut perpendicularly to their major axis, may look like egg-shaped forms 8 , similar to the spores of Microsporidium. This misleading interpretation may be an artifact of fixation, which causes retraction of parasitic

forms of microsporidia. More reliable results were obtained with the semi thin sections, but the diagnosis still remained doubtful for 3 out of 5 cases. At present, only two species (Enterocytozoon bieneusi and Septata intestinalis) of microsporidia are

Intestinal Protozoa Infections in AIDS . 257

known to infect the GI tract of humans. Both have been characterized by TEM and their major differences reported in the literature 6,7. S. intestinalis does not form plasmodial stages and also infects macrophages and fibroblasts of the lamina propria, and, most importantly, the developmental phases occur within a parasitophorous vacuole surrounded by a fibrous lamina. Differentiation between two microsporidia is possible only on the grounds of ultrastructural findings, and this is important in view of the different therapeutic approaches 9, 23. Our ultrastructural findings are specific for E. bieneusi: plasmodial forms were identified in all the positive cases, and spores within a true parasitophorous vacuole were never found. The infection by intracytoplasmic coccidia could be recognized confidently for all the cases from semithin sections stained with toluidine blue, even in cases with low parasitic burden. Conversely, it was not easy to identify it in paraffin section. Two false-positives and one false-negative case, interpreted on the ground of LM examination, were found. In two cases the presence of microorganisms was suspected and in only one certainly identified. The difficulty might be due to several factors, primarily the low burden of parasites which was a constant finding in all undiagnosed cases. In addition, the poor staining characteristics of the parasite prevent a sure differentiation from intraepitheliallymphocytes and mucin droplets 28 . Nevertheless, it is worth emphasizing that LM alone (even with semithin examination), cannot differentiate the various genera of the intracytoplasmic coccidia 12. Sarcocystis spp, Eimeria spp and Cyclospora spp, other than Isospora spp, are recognized causative agents of severe GI symptoms also in AIDS patients, and can be identified by finding the oocysts in fecal smears S,24,27,31,38. Since 00cysts may be shed intermittently or not at all, the identification of the parasite may be impossible for some cases 8, 13, 37. Therefore, ultrastructural examination of biopsy samples becomes necessary for correct diagnosis. I. belli infection was the only coccidia infection in our 4 cases. Only asexual stages of developing parasites were found. In all the cases, they were within the cytoplasm of the enterocytes, and, in one case, also within macrophages and free in the lamina propria. These features are clearly different with those reported for intestinal infection with Sarcocystis hominis, S. suihominis and S. lindemanni in which the morphologic key of the diagnosis is the finding of sexual forms in the enterocytes, and the absence of asexual forms in the same cells4, 5, 12,26. Our results rule out the possibility of sarcocystosis, since gametocytes were never found within enterocytes and no parasites were identified within endothelial cells. In case 16, asexual forms were found free in the lamina propria and within the macrophages, and merozoites were found into the lumen of small capillary vessels on semithin sections. These finding are similar to those of Restrepo et a1. 28 , and may explain the possibility of dissemination of I. belli, also reported in AIDS patients.

Fig. 8. Electron dense apoptotic body, resembling a parasite in the cytoplasm of one enterocyte (21000 x).

To our knowledge, there are no detailed reports of the ultrastructural aspects of human intestinal mucosa infected by Eimeria spp and Cyclospora spp, making it impossible to diagnose differentially from I. belli. Licois et al. 18 described light and ultrastructural aspects of Eimeria intestinalis in experimentally infected rabbits. The data reported show many differences from I. belli; the most important ones are the presence of Eimeria within intraepitheliallymphocytes and the large number (up to 30) of merozoites within each vacuole, never reported in I. belli infection nor found in our cases. In conclusion, our results further point out that electron microscopy supplies some not otherwise available data which make it possible to identify the species and genus of the parasite involved and, therefore, to undertake appropriate therapy in most instances. Material and Methods Between January 1992 and August 1994, 21 small intestinal biopsies of AIDS subjects (defined according to the Centers for Disease Control criteria 29 ) with chronic diarrhea and weight loss, were investigated by LM and TEM in our Units. The biopsies were taken at duodenal bulb. One to three tissue samples were fixed in 10 % neutral buffered formalin and embedded in paraffin. Five 11m thick sections were routinely stained with Haematoxylin and Eosin (HE), Periodic Acid-Schiff (PAS) and Giemsa. Another sample was fixed in 2.5 % buffered glutaraldehyde, post-fixed in 1 % osmium tetroxide and embedded in the epoxy resin. Semithin sections were stained with toluidine blue and Phosphotungstic-Acid Haematoxylin (PTAH). The ultrathin sections were

258 . R. Boldorini et al. stained with uranyl acetate and lead citrate and examined with TEM.

Light Microscopy Evaluation Paraffin sections were evaluated considering the following parameters: a) grading of atrophy25; b) intraepithelial lymphocytic infiltration, graded as mild, moderate and severe; c) identification of intracellular parasites, evaluated as negative, suspicious or positive; d) burden of parasites graded semiquantitatively from negative (0) to high (+++); e) classification of parasite.

Semithin sections were evaluated as negative, suspicious or positive for the presence of parasites; classification of parasites and, whenever possible, the phase of life cycle of the protozoa, was also determined.

Transmission Electron Microscopy Evaluation All the cases were evaluated to identify the genus and species of microorganisms, phases of life cycle and type of cells infected. Presence and severity of cell damage, Human Immunodeficiency Virus (HIV) particles, or equivalents of viral infection, such as tubuloreticular inclusions (TRI) or cilindrical confronting cysternae (CCq33, and the presence of other viruses or microorganisms were also searched for.

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Received July 24, 1995 . Accepted in revised form November 10, 1995

Key words: AIDS - Small intestinal biopsy - Protozoa - Diagnostic electron microscopy Dr. Renzo Boldorini, Servizio di Anatomia Patologica, Ospedale Maggiore della Carita, Corso Mazzini 18,1-28100 Novara, Italy, Phone: ""39-321-373374, Fax: 39-321-373485