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J Clin Pathol 1991;44:182-193

Opportunistic protozoan infections in humanimmunodeficiency virus disease: Reviewhighlighting diagnostic and therapeutic aspects

A Curry, A J Turner, S Lucas

IntroductionOpportunistic protozoan infections are amongthe most serious infections in patients withAIDS."A They cause severe morbidity andmortality; because many are treatable, it isimportant that early and accurate diagnosesare made. This is normally accomplished bydirect microscopic visualisation of the parasitein infected tissues or body secretions. Rigidadherence to normal diagnostic proceduresmay not be appropriate in patients with AIDSbecause the site and manifestation of some ofthese infections may be unusual. Experienceof these conditions among histopathologistsand microbiologists is extremely variable.Furthermore, treatment of the protozoaninfections in patients with AIDS is often com-plicated by severe side effects and a high rateof recurrence.We review the known protozoan infections

in human immunodeficiency virus (HIV)seropositive people, the appropriate tissues tosample, the light and electron microscopicappearances, and briefly outline appropriateantibiotic treatment. The protozoa identifiedto date in these patients are listed in the table.

AIDS. The AIDS epidemic has considerablyincreased our awareness of this organism,which is now known to be a common child-hood infection among the immunocompetentin whom the infection is self-limiting.8 Inpatients with AIDS and in other immuno-compromised groups infection can be bothprotracted and life threatening. It has a par-ticularly high incidence in HIV positivepatients with diarrhoea in Africa" and it isfound in up to 10% of most series of HIVpositive patients with diarrhoea in the UnitedKingdom and the United States of America.The gastrointestinal tract from oesophagus torectum, the biliary tract including intra-hepatic ducts, and the bronchial tree can beinfected in patients with AIDS.'2"3 Infectionis caused by the ingestion of oocysts in food orwater.

Cryotosporidium under both the lightmicroscope and the electron microscopeoccupies an extracytoplasmic location withinan intracellular parasitophagous vacuole (figs1, 2, and 3). Laboratory diagnosis of Crypto-sporidium infection requires the staining ofoocysts in faecal smears with modified Ziehl-Neelsen or auramine stains.'4 Monoclonal

Predominantly intestinal infectionsVarious infectious agents have been found inthe gastrointestinal tract of patients withAID 5,-7 some of which may be associatedwith symptoms. For potential protozoan in-fections, several stool samples should beexamined for cysts or oocysts before invasiveprocedures are considered. If microscopicalexamination of wet preparations or fixed andstained stool samples fails to show thepresence of an infectious agent, then biopsymay be necessary. A rectal biopsy specimen,which is easily taken, may indicate thepresence of a protozoan infection, but it is notalways the most appropriate site of the intes-tine to sample. For some protozoan infections,such as microsporidiosis, diagnosis is impossi-ble without biopsy of the small bowel.Coccidioses Coccidian protozoa are allintracellular parasites with complex life cyclescomprising asexual (schizogony), sexual(gametogony), and sporogenous phases. Atsome stage in their respective life cycles, allpossess characteristic structures, which can beseen under the electron microscope.Cryptosporidium Now the most well recog-nised of intestinal coccidia, °0 humanCryptosporidium infection was considered tobe both rare and zoonotic before the advent of

Protozoa found in HIV seropositive patients

Phylum sporozoa (alt: Apicomplexa)Class CoccideaOrder Eimeriida

CryptosporidiumIsosporaSarcocystisToxoplasma

Class PiroplasmeaOrder Piroplasmida

BabesiaClass HaemosporideaOrder HaemosporidaPlasmodium

Phylum Microsporidia (alt: Microspora)Class MicrosporeaOrder Microsporidia

EncephalitozoonEnterocytozoon

Phylum RhizopodaClass Lobosea

EntamoebaAcanthamoeba

Phylum MetamonadaClass AnaxostyleaOrder Diplomonadida

Giardia

Phylum KinetoplastaOrder Trypanosomatida

TrypanosomaLeishmania

Possible protozoon: Blastocystis

Protozoon or fungus: Pneumocystis

Public HealthLaboratory,Withington Hospital,Manchester, M20 8LR.A CurryA J TurnerDepartment ofHistopathology,University College andMiddlesex School ofMedicine, UniversityStreet, LondonS LucasCorrespondence to:Dr A CurryAccepted for publication26 July 1990

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Opportunistic protozoan infections in HIV disease

Figure 1Cryptosporidium in arectal biopsy specimen.Note the superficiallocation of this parasite(haematoxylin and eosin).

antibodies have been used on faecal smearsand paraffin wax sections, but they stain onlyoocysts and not the smaller trophozoites.15 Arectal biopsy specimen shows the parasite inmany cases, but a duodenal biopsy specimenis probably more sensitive; both tissue biopsyspecimens and faecal smears can identify asmall proportion of organisms that the othermodality has missed.'6Attempts to treat Cryptosoporidium infec-

tion have met with limited success. Mostreports concern the use of spiramycin17;relapses are common, occurring in up to 50%of patients at three months. Although a largenumber of other drugs have been used, thereis no established effective treatment.Isospora Before the advent of AIDS therewere few recorded human intestinal infectionscaused by Isospora belli.5 In some countries or

in ethnic groups within countries, however,isosporiasis is relatively common in patientswith AIDS.'8 In the United States of Americathe incidence among Hispanics with AIDS is8%; the incidence among others with AIDS is1%.19 A similar high incidence of I belli hasbeen noted in Haitian patients with AIDS(12%) and in Africa." 1618 20 This organism hasalso been recorded in patients with AIDS inthe United Kingdom (S Lucas, personal ob-servations). The zoonotic reservoir-if thereis one-is unknown.

Chronic watery diarrhoea is the majorsymptom of isosporiasis. Infection is pre-sumably through ingestion of oocysts in foodor water. The infected enterocytes of the smallintestine (the main organ infected) show com-pletely intracellular and intracytoplasmicparasites (figs 4-7). There is some evidence for

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Figure 2 Electron micrograph of a human rectal biopsyspecimen showing intracellular but extracytoplasmiclocation of a late trophozoite stage of Cryptosporidium.

Figure 3 Electron micrograph ofa schizont ofCryptosporidium.

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Curry, Turner, Lucas

Figure 4 Four zoites of Isospora seen in cross section(arrowhead) within a duodenal enterocyte(haematoxylin and eosin).

latency, as isosporiasis may develop severalmonths or years after presumed exposure.'9Mild to severe mucosal inflammation, oftenwith eosinophilia, and crypt hyperplasticatrophy occur; the parasites vary in abun-dance. Extraintestinal infection by Isosporahas been described in a patient with AIDS inwhom both the small and large bowel wereinfected; the organism was also found inmesenteric lymph nodes.2'

Figure S Six sickle-shaped zoites of Jsospora seen inlongitudinal section in a duodenal biopsy specimen(arrowhead) (haematoxylin and eosin).

Figure 6 Early schizonts of Isospora-large granularmononuclear cells arrowed (haematoxylin and eosin).

Laboratory diagnosis of Isospora dependson faecal examination for oocysts (fig 8) or abiopsy of the small intestine where the entericstages may be found in the gut wall. Com-petent histological diagnosis is complicated bythe similarity of this organism to both Sarco-cystis and Toxoplasma; it is larger than micro-sporidia. Like Cryptosporidium, tissue biopsyspecimens and faecal smears identify smallnumbers of cases the other modality has

Figure 7 Multinucleatedgametocyte of Isospora in anenterocyte (haematoxylin and eosin).

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Figure 8 Faecal smearwith four ovoid oocysts ofIsospora belli (modifiedZiehl-Neelsen).

missed, but faecal smears are more sensitive.'6In some reports patients with isosporiasis

have responded well to co-trimoxazole, al-though recurrence is common (about 50%).Prophylaxis with co-trimoxazole or pyrimeth-amine-sulphadoxine prevents recurrence forup to 16 months, with a low rate of adversereactions." In a Zambian study, however,patients with diarrhoea associated with iso-sporiasis did not improve on co-trimoxazole.'6Sarcocystis Sarcocystis hominis andS suihominis intestinal infections are rare. Arecent report in an HIV infected person23 andthe case illustrated here were attributed toSarcocystis (fig 9). Infection is probablyexclusively zoonotic in nature. Consumptionof poorly cooked, infected muscle releaseszoites which infect the enterocytes; the sexualphase of the life cycle then takes place, caus-ing diarrhoea. Oocysts are excreted in faeces(fig 10) and these are similar to those of Isosp-ora, although they have more delicate wallsand are more spherical.

Other enteric protozoaMICROSPORIDIOSISMicrosporidia (or microsporans) are a wide-spread group of obligate intracellular parasiticprotozoans, characterised by the possession ofacoiled filament seen by electron microscopywithin the small resistant spores.24 Once thespores have been ingested, this filament uncoilsand injects the infective sporoplasm directlyinto the host enterocytes, where proliferationoccurs. Recorded cases of human infectionremain uncommon, but this group of organ-isms is now assuming some prominence in thespectrum of opportunistic infections found in

patients with AIDS. Enterocytozoon bieneusi,which was first described in such a patient,25 26seems to be the commonest human micro-sporidian infection. This organism has onlybeen found in symptomatic patients (up to6 5%)27 in whom the major features are mal-absorption and diarrhoea. Its role in thepathogenesis of diarrhoea is still being debated.It seems to replicate and produce sporesexclusively within the small intestinal wallwithout evoking any major tissue response. Aduodenal or jejunal (not rectal) biopsyspecimen is therefore necessary for diagnosis.Demonstration of the organisms within smallintestinal enterocytes is possible using simplehistological strains27 (figs 11 and 12), but elec-tron microscopical examination is required fordefinitive diagnosis, and is more sensitive (figs13 and 14).28 Faecal smear examination is notuseful because the small size of the spores (1 x1-7 gum) and the ability of the walls to retainGram stain make them indistinguishable fromGram positive coccal bacteria. Tissue dabsfrom small bowel biopsy specimens, however,can be stained with Giemsa to demonstratemicrosporans.29

Encephalitozoon cuniculi has been reported ina patient with AIDS with peritonitis30 and inanother with hepatitis.3' Several patients in theUnited States of America have developed con-junctival lesions (keratitis) caused by thisorganism. Perforation may occur. There islittle inflammation; swabs or biopsy specimensofconjunctiva show Giemsa and Gram positive1-2 im ovoid organisms, and electron micro-scopical examination confirms the diagnosis.3233

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Figure 9 Duodenal biopsy specimen: oocysts ofSarcocystis adjacent to enterocytes (arrowhead)(haematoxylin and eosin).

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Figure 10 Faecal smearof Sarcocystis, showingoocysts as both colourlessrefractile spheres withinternal granular zooites,and as acid-fast sphere(arrowhead) (modifiedZiehl-Neelsen). 4

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There is no established treatment for micro-sporidian infection in any site.26 In a recentreport of five cases of ocular microsporidiosisvarious topical antibiotics were used with noobvious benefit.33

GIARDIASISGiardia lamblia is a flagellate protozoon spreadby the faecal-oral route, with a high incidencein homosexual men.3 Enteric infection is oftenasymptomatic. The infection is found globallyin HIV seropositive people; so far, there is noevidence that it is more or less common in suchpatients with diarrhoea compared with thosewho are HIV negative. Conventional thera-peutic regimens are adequate for these infec-tions in patients with AIDS.34

AMOEBIASISIntestinal infection with Entamoeba histolyticamay be found in 20% of homosexual men,irrespective of gastrointestinal tract symptoms,and similar prevalences are seen in HIVpositive homosexual men with diarrhoea.35 Sofar, all these infections have taken the form ofnon-invasive luminal carriage of amoebae, andnot invasive amoebiasis proper.36 Isoenzymecharacterisation of pathogenic compared withnon-pathogenic strains ofE histolytica that aremorphologically identical may be done inspecial centres, and confirms the lack ofpotentially invasive strains in HIV positivepatients in developed countries.36 In fact, giventhe lack of cases of invasive amoebiasis inAfrica," 20 there may even be a significantnegative association between HIV infectionand the carriage of potentially invasive strainsofE histolytica.37When non-invasive infections are found in

faeces, parasitological examination showstypical cysts and trophozoites, but the latterrarely contain phagocytosed red cells.Similarly, in biopsy material trophozoites maybe abundant, but they are not seen to invademucosa or have red cells within the cytoplasm(fig 15). Amoebic serology, which is sensitiveand specific for invasive amoebiasis, is negativein these patients. Conventional therapeuticregimens are adequate for Entamoeba infec-tions in patients with AIDS.34Blastocystis hominis Blastocystis is anamoeboid organism of uncertain taxonomywhich seems to have protozoan features (fig16).38 Its role in enteric disease is just ascontroversial, with some workers convinced ofits pathogenic role.39 It does not invade tissue,and has not been confidently noted in rectalbiopsy specimens.

Beneficial effects have been claimed fortreated Blastocystis infection; drugs usedinclude metronidazole, 2 g daily for twoweeks,40 and co-trimoxazole,39 but the evidenceso far is limited.

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Figure 11 Duodenal biopsy specimen: two enterocytes(arrowheads) contain spores of Enterocytozoon, seen assmall refractile haematoxophylic dots (haematoxylin andeosin).

Figure 12 Enterocytozoon spores in duodenal enterocyte(arrowhead); Semithin (Giemsa).

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Figure 13 Electronmicrograph of a spore ofEnterocytozoon bieneusi inhuman small intestinalbiopsy specimen. Noteprofiles of internal coiledfilament, characteristic ofthis group of intracellularparasites.

Predominantly extra-intestinalinfectionsTOXOPLASMOSISThe coccidian Toxoplasma gondii is a commoninfection which is not usually pathogenicin immunocompetent hosts. In developedcountries 50-90% of adults are seropositive forToxoplasma antibodies. The life cycle begins incats in which the sexual reproductive phaseoccurs, and continues in man (or othermammals). Ingestion of the infective oocystscontaining sporozoites by humans initiates theasexual cycle. The sporozoites are taken up by

macrophages in which they rapidly multiplyand are carried to various organs. Rupture ofthe macrophages releases the tachyzoites whichdisseminate the infection. Ultimately theorganisms become enclosed in dormant (latent)tissue cysts in brain and muscle. Clinicallyimportant disease in man is largely restricted tocongenital infection, generalised lymph-adenopathy in primary infection, and reac-tivated infections in the immunocompromisedincluding HIV positive patients.4' The mostimportant manifestation in HIV infectedpatients is encephalitis, often necrotising and

Figure 14 Electronmicrograph of an earlysporont of Enterocytozoonbieneusi indentingenterocyte nucleus. Noterandom profiles of sporefilaments.

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Figure 15 Rectal biopsy specimen: abundanttrophozoites of non-pathogenic Entamoeba histolyticaadjacent to mucosa. The amoebae have not phagocytosederythrocytes (haematoxylin and eosin).

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often fatal.42 Toxoplasma myocarditis, De 11npneumonitis, enteritis and peritonitis are also patierseen.43" The incidence of active Toxoplasma Acaminfection in patients with AIDS can be as high amoebas 70% in some areas. ted

Laboratory diagnosis of toxoplasmosis is ted irnormally accomplished by serological testing, cerebbut such procedures are unreliable'4 when

necro,

dealing with patients with AIDS because of necrovarying antibody titres. Histological analysis of numbtissue (figs 17 and 18) or smears can provide the theremost dependable diagnosis and this mayrequire immunohistochemical techniques (fig19) to identify positively the parasite. The use atof such invasive procedures is controversial,and if Toxoplasma encephalitis is suspected on

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Figure 16 Three Blastocystis organisms in afaecalsmear, with surrounding bacteria (Giemsa).

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il grounds anti-toxoplasma treatment isstarted without a definitive diagnosis.- treatment of toxoplasmosis in patientsAIDS has been reviewed recently.4546Lethamine plus a sulphonamide is thelent of choice. A high rate of relapse hasrecommendations that prolonged treat-is used, but the role of prophylaxis for allits with AIDS and serological evidence oflasma infection is not yet established.455esia and Plasmodium species are sporo-,arasites which develop in erythrocytes inmammalian hosts and in the tick anduito vectors, respectively. In man, babe-is uncommon, but has now been reported:ients with AIDS in the United States ofica.47 The disease is associated with fever,aia, and splenomegaly. In aspleniclts the parasitaemia may reach 40% ofredand the haemolysis can result in

oglobinuria.'4 Diagnosis is made by iden-g organisms in blood films. Treatment ofia infections in patients with AIDS withlinations of pentamidine, co-trimoxazole,ne and clindamycin has been tried but theLs have been variable and relapses occur.smodium falciparum malaria occurs ininfected people in endemic zones, but theence and clinical manifestations seem to) different from those in HIV negativeits.37 49 50

thamoeba Like Naegleria, Acanth-ba is a genus of free-living amoebae. Twoof acanthamoebiasis have been documen-n HIV positive patients. One had focaloral signs and numerous foci of haemor-c necrosis in cerebrum and cerebellum at)psy." The trophozoites are seen in largebers around necrotic vessels (fig 20), andmay be granulomatous inflammation. The

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e 17 Cerebral toxoplasmic necrosis: theteritis and perivascular onion-skinningwhead) is typical of toxoplasmosis (haematoxylinsin).


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Figure 18 Brain toxoplasmosis: two cysts and a clusterof loose tachyzoites (arrowhead) (haematoxylin andeosin) .

other case had Acanthamoeba infection of theparanasal sinus and the dermis of the leg.52 Thetrophozoites are 20-40 gm across and possess aprominent nucleolus. Although morphologicaldistinction from Naegleria (which has not yetbeen reported in HIV positive patients) andEntamoeba is possible, confirmation via specificimmunostaining or culture in referencelaboratories is recommended. Usually Acan-thamoeba encephalitis is diagnosed at necropsy;but diagnosis through examination ofcerebros-pinal fluid and biopsy specimens is possible.Amphotericin and flucytosine have beenrecommended for treatment.5'The route of infection for Acanthamoeba is

uncertain, but it probably requires a skininoculation or nasal lesion before haemato-genous dissemination to brain occurs.

Leishmania is a kinetoplastid flagellateprotozoan, which characteristically possesses aDNA-rich organelle associated with theflagellar base called a kinetoplast. Most leish-manial infections in man are zoonotic, the lifecycle involving sandflies (the vector) andcanines or rodents (the reservoir), dependingon the Leishmania species. In man the parasiteis seen as the amastigote form (Leishman-Donovan or LD body). Infection is oftenasymptomatic in endemic areas. Clinico-pathologically, there are four main forms ofleishmaniasis: visceral (kala azar); cutaneous;muco-cutaneous; and diffuse cutaneous. Todate, only the first two patterns have beenreported in HIV positive patients, and all thesepatients have been infected with Leishmaniainfantum, the common cause of both cutaneousand visceral leishmaniasis in Europe.2

Cutaneous leishmaniasis generally healswith activation of macrophages, necrosis, andparasite elimination. A patient with AIDS anda single heavily parasitised leishmanial skinulcer was reported from Italy; the lesion healedwith standard antimonial chemotherapy, and atno time was there evidence of visceralisation."In regions of Italy where only the dermato-tropic strain of L infantum is prevalent,however, HIV infection can be associated withvisceral disease alone.'Mucosal leishmaniasis affecting the palate

and pharynx has been seen in immunocom-promised patients, including those who areHIV positive (S Lucas, personal observations).The pathology is granulomatous with manyLD bodies. These lesions are probably exten-sions from facial infections.Over 100 cases of visceral leishmaniasis have

now been reported in HIV positive patients, allof whom have travelled to, or lived in, theMediterranean countries of France, Spain,Portugal, Italy and Malta." Patients maypresent with typical hepatosplenomegaly,anaemia, and fever. Parasites can usually beshown by aspiration of spleen, marrow, lymphnodes or skin. Histologically, they are usuallyevident in a tissue biopsy specimen such as liverand marrow; Kupffer cells and macrophagescontain abundant LD bodies, and there isassociated plasmacytosis (fig 21).Unusual lesions are also found. A skin biopsy

specimen for diagnosing Kaposi's sarcoma mayshow LD bodies in dermal macrophages amidthe tumour lesion before there is clinicalsuspicion of visceral leishmaniasis (fig 22).56

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Figure 19 Immunostaining with Bioquote monoclonalanti-toxoplasma antibody showing numerous zoites, withmany in the vessel wall (immunoperoxidase).

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Figure 20 Brain biopsyspecimenfrom patient withAIDS withAcanthamoebaencephalitis. Largetrophozoites with centralnucleolus seen clusteredtogether and around ablood vessel (haematoxylinand eosin).

Intestinal tumour-like lesions composed ofabundant leishmanial bodies ih macrophages57and incidental observation of parasitised rectallamina propria are also features (fig 23). Invisceral leishmaniasis without HIV infectionparasitism of alveolar macrophages hasoccasionally been seen, but in a case of pleuraleffusion in an HIV positive man, LD bodieswere identified in macrophages from a pleuraltap.58

Visceral leishmaniasis may be diagnosed byserology, but it is not always seropositive inHIV positive patients.59 Chemotherapy clearsthe parasite from most patients, but the relapserate is high. Treatment for visceral leishman-iasis has been reviewed in detail elsewhere.55Although a number of drugs have been used,6'the antimonial sodium stibogluconate (Pento-stam) remains the treatment of choice.Trypanosoma Like Leishmania, trypano-somes are kinetoplastid flagellates. Theyinhabit the bloodstream and other tissues ofvertebrates, including man; the insect vectorsare Tsetse flies (African) and Reduviid bugs(South American). African trypanosomiasis(sleeping sickness) is caused by Trypanosomabrucei. This infection does not seem to be moreprevalent in HIV positive people or moresevere compared with the normal population inAfrica.6'

In South America T cruzi infection causesChagas' disease. Seroepidemiological studiesin Brasil have found evidence of trypanosomalinfection among HIV positive people but with-out a significant association.6' A cerebralabscess caused by T cruzi in an HIV positivepatient has been reported (Kronfeld A, SprintzE, Cavacanti A, Carneiro A. Abstractpresented at Fifth International Conference onAIDS, Montreal, June 1990). Histologically,the amastigotes of T cruzi resemble toxo-plasmas; they possess a kinetoplast (likeLeishmania); immunostaining for toxo-plasmosis on sections is specific.Treatment for trypanosomiasis has been

reviewed in detail recently elsewhere.55 Thetwo drugs of confirmed efficacy for SouthAmerican trypanosomiasis are nifurtimox andbenznidazole.

Pneumocystis carinii Pneumocystis is an enig-matic micro-organism of uncertain taxonomy.It has been shunted backwards and forwardsbetween the fungi and the protozoa. Recentlyribosomal RNA homology studies62 haveshown identical nucleic acid sequences withsome fungi, but some mycologists remain to beconvinced that this is where this organismbelongs.63 The organism seems to beubiquitous, infecting man and other mammalsin many parts of the world. It leads anextracellular existence within the alveolar lungspaces.' Two distinct forms are apparentwithin the alveoli, a pleomorphic form (tro-phozoite) with a full complement of eukaryoticorganelles (including an enveloped nucleus andmitochondria), and a spherical thick-walledcystic stage, often enclosing several intracysticbodies with a similar appearance to the tro-phozoite. The wall of the trophozoite compr-

Figure 21 Bone marrow with visceral leishmaniasis.Arrowhead indicates LD bodies (Methacrylateembedded; haematozylin and eosin).

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Figure 22 Skin biopsyspecimen from a Kaposi'ssarcoma plaque: numerousLD bodies in macrophagesbetween the vascular slits(haematoxylin and eosin).Slide by courtesy ofDr RLindley, St Stephen'sHospital.

ises a single unit membrane covered by anexternal thin electron dense covering.Asymptomatic infection in childhood is

common, with up to two thirds of normalchildren of four years of age having antibody toPneumocystis.65 The route of infection ispresumably airborne. The most critical factorin the pathogenesis of Pneumocystis cariniipneumonia (PCP) in HIV positive patients isthe circulating CD4 lymphocyte count, anindicator of the degree of immunocompetence.It has been shown that until the CD4 countdrops below 200/mm3, PCP is rare.'The occurrence of a cluster of cases of PCP

was one of the first manifestations of the AIDSepidemic and PCP has remained the initialpresentation of AIDS in 60% of patients inNorth America, ultimately occurring in at least85% of patients.' It is fatal in 5-10% of initialepisodes; recurrences almost always occur. Forsecond or subsequent episodes, the averagesurvival is about 60%.67Although most Pneumocystis infection in

patients with AIDS remains pulmonary, thisorganism is occasionally found in non-pulmon-ary siteSI69 such as the ear,707' liver (fig 24),spleen, small intestine,72 lymph nodes, skin,7374pancreas, kidney, urethra, adrenals, thyroid,mesentery, heart,75 choroid,6976 bone marrow77and the Virchow-Robin spaces of the brain.78Some, but not all, of this extrapulmonaryinvasion is associated with the use ofnebulised pentamidine.79 The organismperhaps invades tissues which contain a muchlower concentration of pentamidine. Extra-pulmonary Pneumocystis, however, has alsobeen found in the absence of any clinicalpulmonary disease.72The macroscopic appearances of PCP are

well known-a pale dry pneumonic consolida-tion. Less common is cavitating PCP, where agranulomatous response is found aroundnecrotic areas of lung, and cysts are often more

scanty. PCP is in the differential diagnosis of acavitating pneumonia and may mimic tuber-culosis. The cysts also infiltrate under theendothelium of arteries and veins and causeocclusion.' This probably contributes to thepathogenesis of lung cavities; a similar processmay have led to necrosis of digits in widelydisseminated P carinii infection.74 In largedeposits of extrapulmonary pneumocystosis,such as in lymph nodes and liver, the grossappearance is of yellowish necrotic deposits.The cysts lie extracellularly, are often degen-erate, and only a small proportion take up themethanamine silver stain.

Early in the AIDS epidemic Pneumocystiscarinii pneumonia was diagnosed by directvisualisation of the parasite in open lung biopsyor needle biopsy specimens. The complicationsof such procedures, particularly pneumo-thorax, meant they were replaced by bron-chiolar lavage. Recently, induced sputum hasbeen advocated as a suitable diagnosticspecimen. This procedure is certainly lesstraumatic than either of the original methodsbut examination of sputum may be a lesssensitive technique in patients receiving pro-phylactic pentamidine.8" However specimensare taken, stains such as Giemsa and methan-amine silver are used to identify the organism,particularly the cystic stage. More recently,immunocytochemical techniques have beenintroduced82 which are easier to examine andinterpret. Sputum and lavage specimens,however, are classified as hazardous, requiringspecial handling facilities because infectiousHIV virus or Mycobacterium tuberculosis maybe present in samples before inactivation.Formalin fixation of the sputum or lavageovercomes this problem, but not all commer-

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Figure 23 Rectal biopsy specimen from HIV positiveseropositive haemophiliac. LD bodies (arrowhead) inlamina propria macrophages (haematoxylin and eosin).(Slide by courtesy of Dr A Vincenti, Winchester).

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Figure 24 Liver biopsyspecimen taken after deathshowing clusters ofextracellular Pneumocystiscysts adjacent tohepatocytes (haematoxylinand eosin). (Slidecourtesy of Dr R Goldin,St Mary's Hospital).

cially available Pneumocystis antibodies areraised to fixed material. In addition, attentionhas been drawn to the problem of false positiveindirect immunofluorescence results, possiblycaused by Candida cells becoming opsonisedwith anti-Candida antibodies and cross-reacting with fluorescein isothiocyanate anti-mouse antibodies in the sputum specimen.83

Extensive clinical experience has shown thatco-trimoxazole and parenteral pentamidine areeffective drugs for treating PCP in patients withAIDS, although side effects are common andoccur more frequently than in patients withother types of immune deficiency.67Treatment with aerosolised pentamidine

should permit delivery of high drug doses tothe lungs without achieving sufficient systemicconcentrations to cause toxicity.84 It may beeffective when the pneumonia is mild ormoderate, and is well tolerated. Pulmonary andsystemic toxicity, extrapulmonary disease, andrecurrence, however, all occur.' Aerosoltreatment is now used for primary and secon-dary prophylaxis of PCP.

ConclusionsHIV infection is here to stay and will spread,albeit with arguable rapidity. If they do notalready, histopathologists and microbiologistswill diagnose infections among HIV positivepatients and should be aware of the widespectrum of parasitic protozoa potentiallyinvolved, and the usual and unusual sites ofinfection. Undoubtedly, new infections,including protozoanones, are waiting to bedescribed.

We acknowledge the assistance of Drs E Dunbar, DM Jones,LJ McWilliam, and NY Haboubi.

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