cryptosporidium species new human pathogentrophozoite (fig. 5) then develops in a pseudo-external...

J Clin Pathol 1985;38:1321-1336

Review article

Cryptosporidium species a "new" human pathogenDP CASEMORE,* RL SANDS,I A CURRY$

From the *Public Health Laboratory, Glan Clwyd Hospital, Bodelwyddan, Clwyd, tthe School ofPharmacy,Liverpool Polytechnic, Liverpool, and tthe Public Health Laboratory and Department ofHistopathology,Withington Hospital, Manchester

SUMMARY Publications describing aspects of the coccidian protozoan parasite Cryptosporidium,increased greatly during 1983 and 1984 as a result of not only increasing veterinary interest but alsoin the role of the parasite in the newly recognised acquired immune deficiency syndrome (AIDS).The reports reflected widespread collaboration, not only between clinicians, microbiologists, andhistopathologists, but also between veterinary and human health care workers. Cryptosporidiumwas first described in mice in 1907 and subsequently in various other species; it was not described inman until 1976. Several likely putative species have been described, but there is probably little hostspecificity. Experimental and clinical studies have greatly increased the knowledge about theorganism's biology. The parasite undergoes its complete life cycle within the intestine, although itmay occasionally occur in other sites. The main symptom produced is a non-inflammatorydiarrhoea, which, in patients with AIDS and children in third world countries, may be lifethreatening: even in immunocompetent subjects this symptom is usually protracted. Attempts tofind effective chemotherapeutic agents have been unsuccessful. Epidemiologically the infection wasthought to be zoonotic in origin, but there is increasing evidence of person to person transmission.Diagnosis has depended upon histological examination, but simple methods of detection have nowbeen described: more invasive methods need no longer be used. The parasite, which is found morecommonly in children, occurs in about 2% of faecal specimens examined and seems to be closelyassociated with production of symptoms. A serological response has been shown. Much remains tobe learned about its epidemiology and pathogenic mechanisms, while the expected increase inincidence of AIDS makes an effective form of treatment essential.

The investigation of uncomplicated diarrhoeal dis-ease comprises a large proportion of the workload ofmicrobiological laboratories, and routine methodsoften fail to indicate a causative agent. The list ofcausative agents continues to increase. Recently theinvestigation of opportunistic infections affecting thegastrointestinal tract, particularly in immuno-compromised subjects, has increasingly entailed diag-nostic work by histopathologists and immunologistsas well as microbiologists.

Interest has focused on the coccidian protozoanorganism Cryptosporidium sp. This agent, already

Accepted for publication 21 May 1985

well known to veterinarians as a cause of diarrhoea inanimals, was first recognised in man in 1976. Theinfection has subsequently been recognised in bothimmunocompromised subjects (with severe and oftenlife threatening diarrhoea) and normal subjects (pre-dominantly children and young adults in whom itproduces a characteristic self limiting flu like gastro-enteritis). Cases occur both sporadically and in out-breaks. The illness is often more protracted than thatcaused by other agents, and invasive investigationsmay be carried out if Cryptosporidium is not diag-nosed.The infection probably has several reservoirs and

routes of transmission in both rural and urban com-munities. Early veterinary interest led to the assump-tion that infection was almost entirely zoonotic, but

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Casemore, Sands, Curry

Phylum

Class

Subclass

Order

Suborder

Family SarcocystkGenus Sarcocystii

Toxoplasmc

Apicompnxa

iSporozoeaI

ICoccidiaD_Eucoccidiida

Eimeriinac HemmospnaQmclarias)

IsrEirneria

isospora3

|jCryptosporidii`ae|Cryptosporidium(oocysts with 4naked sporozoites)

Fig. 1 Taxonomy of Cryptosporidium: simplified scheme, showing relations withother medically important species.

doubt has now been cast on this, and person to personspread is probably more common than was firstthought. The natural history of the organism andsome laboratory evidence suggests that environ-mental contamination is probably widespread andinfection may additionally be acquired through foodand water. Histologically, much is known about theinfection because many of the early cases weredetected in biopsy material, and infection has beenshown to extend beyond the gastrointestinal tract insome cases.

Little, however, is known about the pathogenicmechanisms, although Koch's postulates have largelybeen fulfilled. The parasite is notably resistant tochemotherapeutic agents, although a wide range havebeen tested in veterinary studies and administered inthe more serious cases in man. Recent work withexperimental models, including induced infection inlaboratory animals, cell culture, and fertile hens' eggs,has begun to elucidate the biological and pathogenicmechanisms of the organism. Microscopical and sero-logical methods for diagnosis have been developed. Itis now recognised that this organism is an importantcause of gastrointestinal infections in both normaland immunocompromised subjects throughout theworld. Routine laboratory investigation of such infec-tions should now include Cryptosporidium among thelist of suspected agents.

Historical aspects

The first description of Cryptosporidium is credited toTyzzer in 1907, who found the parasite in the pepticglands of laboratory mice and considered it to be anextracellular species related to the coccidian proto-zoa.' He subsequently suggested in 1910, probablyincorrectly, that it had already been described in1894-5 by J Jackson-Clark, who identified it as a coc-

cidian, Eimeria falciformii. The reports of Tyzzerwere remarkable for the quality of their descriptionand illustration.23 Before the first report of infectionin man cryptosporidia were described in a variety ofhost species and were each generally designated a spe-cies of their own. They were classified according tothe host in which they were found. The validity ofthese identifications was first questioned by Vetterlingin 1971.4 Subsequently, Tzipori et al suggested thaton the basis of wide host tolerance and lack of tissuespecificity there may be only a single species in thegenus Cryptosporidium.sThe report by Panciera et al in 1971 on the

importance of Cryptosporidium in diarrhoea incalves6 was a major stimulus to veterinary interestand subsequently to much experimental work: thiswas excellently reviewed by Tzipori,7 Angus,8 andCurrent et al.9 The importance of veterinary work inrelation to human medicine was amplified in a recentleading article.'0

Fig. 2 Light micrograph ofhuman rectal biopsy, showingCryptosporidium infection ofepithelial surface.(Haematoxylin and eosin.) x 650.

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Cryptosporidium species a "new" human pathogen

.-'.J,'.

Fig. 3 Electron micrograph ofmouse colonic crypt showingtwo individual organisms ofCryptosporidium derivedfrom ahuman case. One organism contains numerous polysaccharidegranules indicating that it is a sexual stage. Apparentlyempty organism is a schizont containing merozoites. Tissuewas initiallyfixed informalin and embedded in wax andsubsequently refixed and embeddedfor electron microscopicexamination. x 6250.

Taxonomy and life history

Fig. I summarises the taxonomy of Cryptosporidiumas a member of the coccidia. " 12 Sporozoan protozoaare usually specific to a particular host and tissue.

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Cryptosporidium is an unusual sporozoan for severalreasons. The endogenous stages seem superficially tobe extracellular parasites attached primarily to thegastric, intestinal, and rectal mucosa (Figs. 2 and 3) ofvarious animal species, including man, mammals,birds, and reptiles, and the epithelial surface of therespiratory tract can also be infected. 13 - 15 Electronmicroscopy, however, has shown that these parasitesare not intracellular but extracytoplasmic and thatthey cause the deformation of the luminal host cellplasma membrane, producing a false appearance ofsuperficial attachment. Several morphologicallydifferent developmental stages have been identified.

Reports on Cryptosporidium are confused becausemany species have been described."6 Conventionally,it would be assumed that each "species" was differentand would not infect other host animals. Studies oncross infection by several groups of workers bothsubstantiate416 and refute517 this concept. Mostrecent publications on Cryptosporidium have sug-gested that this parasite is not restricted to either aparticular host species or a specific epithelial surface.Current and Long infected chicken embryos withCryptosporidium isolated from humans and calves.'7These isolates were morphologically and devel-opmentally indistinguishable. The authors thereforestated that these observations supported the pro-posals that Cryptosporidium has little or no host

Life cycle of cryptosporidium

I Sexual

Fig. 4 Diagrammatic representation oflife cycle of Cryptosporidium. Infection starts with ingestion ofan oocyst containingfour sporozoites (SO). Digestive enzymes probably release sporozoites (S) from oocyst. Sporozoite enters an epithelial cell,takes up a pseudoexternal position, and matures into a trophozoite (T). Trophozoitefeeds and undergoes asexual multiplebudding process (schizogony) to produce a schizont (SC), which releases merozoites (M). Merozoites invade other epithelialcells and develop into trophozoites (T). Once again these undergo schizogony and release merozoites. Merozoites of this secondcycle ofasexual reproduction infectfurther epithelial cells but mature into either microgamonts (MI) or macrogamonts (MA)ofthe sexual cycle. Microgamont produces microgametes (MIG) which, on release, fertilise the macrogamont andproduce azygote (Z). Zygote mayfollow several sporogenous developmental routes: it may transform into an oocyst by secretion ofa-thick wall and development ofsporozoites, while attached to the host cell (A TOS), before becoming detached (DTOS), andpassing out ofthe gut; zygote may secrete a thick wall and oocyst produced may become detached (DTO) before developmentofsporozoites occurs (DTOS); or zygote may develop into a thin walled oocyst (TW) containing sporozoites (S). Thin walledoocysts (TW) may liberate sporozoites, thus spreading infection within the host. Microvilli have been omittedfrom infectedcellsfor clarity.

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Fig. 5 Young trophozoite ofCryptosporidium attached tomouse colonic mucosa. Note large nucleus with prominentnucleolus. x 25000.

specificity and that calves and other domestic animalsmay be a potential source of infection in man. Suchspeculation is supported by studies on the once con-fusing genus Toxoplasma. Toxoplasma gondii is nowthought to be a parasite with a wide host toleranceand, as a consequence, many previous names of spe-cies have been invalidated."8 If Cryptosporidium is asingle species genus then Cryptosporidium muriswould be the type species, as this was the first descrip-tion of this parasite by Tyzzer in 1907.' It is our view,

Fig. 6 Transverse section through attachment zone showingmembranousfolds present in this area. x 10000.

Fig. 7 Schizont containing merozoites.x 40000.

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Fig. 8 Merozoite inside schizont ofCryptosporidium. Note dense granulesat posterior end oforganism. x 62000.

however, and that of some veterinary workers (KWAngus, personal communication), that Cmuris maydiffer from Cparvum and that many of the morerecently identified organisms relate more closely toCparvum. At present there is insufficient evidence fora definitive statement. Workers should therefore con-tinue to use the name Cryptosporidium species untilsuch time as definitive evidence becomes available.The life cycle of Cryptosporidium (Fig. 4) has not

been completely elucidated, and, furthermore, docu-mented evidence is somewhat confusing because of

speculations about some facets of the cycle, notablythose outside the host and within the intestinal con-tents. The life cycle has been reconstructed, usingboth light microscopy and electron microscopy, byseveral authors.47 192023 These reconstructionsare, however, open to several different inter-pretations.The initial infective stage is the sporozoite ([-5 gm

x 0 75pm) liberated from an oocyst by partialdigestion after passage through the stomach.2 21 Thesporozoite must reach and penetrate a suitable host

Fig. 9 Thin section ofhumanfaecalspecimen positive for Cryptosporidium.Oocyst containsfour sporozoites andresidual cytoplasmic mass. x 40000.

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Fig. 10 Human rectal biopsy showingseveral cryptosporidial endogenousstages. Attachedform (right) is atrophozoite and detachedform a thinwalled oocyst which has yet to divideinternally toform sporozoites. x

40000.

cell, when a parasitophorous vacuole is formed. Thetrophozoite (Fig. 5) then develops in a pseudo-external location and derives nutrition from either thehost cell (Fig. 6) or the contents of the gut, or both.The mature trophozoite (1-5-6-0 pm in diameter) con-

tains the normal complement of eucaryotic cyto-plasmic organelles and undergoes schizogony (anasexual multiple budding process), producing eightmerozoites (2-5 pm x 0 4 gm) (Figs. 7 and 8), whichare liberated and invade the surrounding cells, thusspreading the infection within the host. A second gen-eration of trophozoites is derived from these mero-zoites. These again feed, mature, and undergo a fur-ther phase of schizogony. A smaller number ofmerozoites may be produced and liberated in this sec-ond phase of schizogony. Once again these penetratemore cells but now differentiate into either macro-

gametocytes or microgametocytes. This differ-entiation is the initial stage of the sexual cycle ofCryptosporidium and has been elegantly shown byGoebel and Braendler in the small intestine of mice.22The microgamont produces up to 16 microgametes

(2pm x 0-7 pm), which escape into the intestinallumen. They contain a large nucleus, a mito-chondrion, and a polar ring of microtubules. Theauthors speculated that this may be equivalent to aflagellum and, presumably, produces the motiveforce, enabling the microgamete to translocate to themacrogamete. No flagellar filaments, however, havebeen described that might account for their move-ment, and it is thought that locomotion is accom-

plished by a form of flexing or gliding.' 721 An alter-native suggestion is that the ring represents the"conoid" structure found in other coccidians, which

Table 1 Incidence ofCryptosporidiwn world wide 1983-4

Source Population No examined No positive %

Australia Hospital patients 884 36 (4.1)Children 697 33 (47)Adults 187 3 (1.6)

Bangladesh Animal attendants and families 320 14 (4 4)Boston All patients 1290 33 (2 6)Costa Rica Children 278 12 (43)

Rural 95 4 (4-2)Urban 183 8 (4.4)

Finland All patients (travellers) 1422 14 (0.98)Rwanda Children 193 20 (10.4)

Adults 100 3 (30)United Kingdom All 6580 140 (2 1)

Children 1363 59 (4.3)Adults 1739 35 (2 0)

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1327Cryptosporidium species a "new" human pathogenTable 2 Incidence ofCryptosporidium in 1983-4

Source Population No examined No positive %

Brighton All patients 800 10 (1-25)Blackburn All patients 2174 24 (1-1)

<15 NS 21>15 NS 3

Bristol All patients 867 43 (5-0)< 15 394 27 (6-8)>15 406 16(39)NS 67

Liverpool Children in hospital NS NS (1-3)North Yorkshire (rural) All patients 166 12 (7-2)North Wales (semirural) All patients 2573 51 (1-98)

<15 969 32 (3 3)16-40 524 17 (3 2)>40 809 2 (0-2)NS 211

Total 6580 140 (2 13)

NS = not stated.is thought to aid penetration. The initial stages ofattachment of the microgametes and macrogameteshave been described previously.172122The fusion of these sexual stages produces the

zygote. An oocyst (4-5-6-0pm in diameter) with athickened wall is derived from this zygote.23 Foursporozoites are formed inside the oocyst. The sporo-zoites may be produced while the oocyst is stillattached to the intestinal epithelial cells, but ourrecent observations suggest that the sporozoites mayform within the oocyst after detachment from theintestinal lining during passage in the faeces (Fig. 9).In addition, Current and Long suggested that thereare both thin walled and thick walled oocysts.'723 Asthe thin walled oocysts (Fig. 10) are infective theinfection is spread autogenously throughout the intes-tinal epithelium. Some confusion may arise becauseof the similarities between the oocysts of Cryptospori-dium and the sporocysts of Sarcocystis. Iseki regardedthe finding of endogenous stages-that is, thoseattached to the epithelial surface-as an essentialtenet for identifying Cryptosporidium."9 If biopsymaterial is available this may provide a definitivediagnosis. Non-invasive methods of diagnosis, how-ever, are preferable.

Pathology

The first recorded case of human cryptosporidiosiswas reported in 1976 in a 3 year old girl fromNashville who had symptoms of vomiting, waterydiarrhoea, and abdominal pains.24 She lived on a

farm, had been in excellent health and had developednormally. At rectal biopsy she was found to be infec-ted with Cryptosporidium, and she recovereduneventfully with supportive treatment. Follow upinvestigations on the domestic animals proved nega-tive, and thus the mode of transmission remainedunidentified. A review of three cases between 1976and 1979 focused attention on opportunistic crypto-sporidial infections in immunocompromisedpatients.25 -27 Each patient had presented with feverand chronic watery diarrhoea, ranging from 10 days'to 3 years' duration. In one patient the symptom ofdehydration was so severe that during 10 days 60litres of parenteral fluid were administered, in addi-tion to substantial oral intake.25 In every case histo-logical examination of jejunal biopsy specimensshowed the presence of cryptosporidia either as thesole suspected pathogen2527 or in association withGiardia lamblia.26 Moderate to severe abnormalities

Table 3 Cryptosporidium sp reported to Public Health Laboratory Service Communicable Disease Surveillance Centre June1983 to June 1984 (Table adaptedfrom figures provided by Communicable Disease Surveillance Centre, Colindale)

Age (years) Sex Diarrhoea Diarrhoea and Symptoms notvomiting stated

M F Unknown

< 1 9 10 1 12 5 31-4 50 38 4 56 24 125-9 15 20 2 20 10 710-14 8 2 1 10 1Child I 10 2 3 615-64 32 31 9 44 12 16>65 1 1 2Notknown 2 4 23 6 3 20

Total 117 107 50 150 59 65

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1328Table 4 Symptoms described in 23 published cases ofcryptosporidiosis in immunologically normal subjects

Symptom No of cases

Diarrhoea 19Cramps 12Fever 9Malaise 8Nausea 8Vomiting 4Anorexia 3Constipation 3Asymptomatic 2Headache 2

of villous architecture were reported with mildchronic inflammation of the lamina propria andslightly increased numbers of plasma cells, poly-morphonuclear leucocytes, and lymphocytes. Two ofthese patients had no known unusual exposure to ani-mals or house pets,2627 although the third patient25lived on a farm in a rural community. This patient,who had a history of ulcerative colitis and severe bul-lous pemphigoids, recovered uneventfully two weeksafter immunosuppressive treatment was stopped.

Between 1980 and 1983 more than 80 cases of cryp-tosporidial gastroenteritis in man were reportedeither as a self limiting gastroenteritis in normalpatients24 or as the severe symptoms produced inimmunocompromised patients, especially those withthe acquired immunodeficiency syndrome (AIDS).28

THE DISEASE IN IMMUNOCOMPETENTSUBJECTSA total of 159 cases have been reported as separatecase studies,2429 36 outbreaks 9 (unpublished obser-vations, DP Casemore et a!), and surveys.2937 42Selected reportS2429- 3740 identified an age rangefrom 2 months to 60 years with peak incidences dur-ing early childhood (less than 1 to 10 years) andbetween 21 and 30 years. Attention was first drawn byCasemore and Jackson to the incidence of sporadiccryptosporidiosis in children.37 Other observationsagree with this, suggesting that the incidence of cryp-tosporidiosis may peak in childhood (Tables 1, 2,and 3). Slightly more men than women have beeninfected.The outstanding clinical features of cryptosporidio-

sis are: a flu like illness with watery diarrhoea;cramps; fever; malaise; and nausea (Table 4). Diar-rhoea is often characterised by two to 10 waterystools a day, often beginning on the first or secondday of illness.Our experiences generally agree with the findings in

Table 4, although anorexia and vomiting werenoticed more often and, in a few cases, vomiting was

the predominant or initial symptom. The diarrhoea,which was usually foul smelling, was accompanied by


considerable weight loss (10% of body weight) andprostration in some cases. The abdominal pain ten-ded to occur in the upper right quadrant. The incu-bation period usually lasted between five days andtwo weeks after initial contact with theorganism,293032 36 inducing symptoms that couldlast for five to 14 days.9 24 31-34 To date, no figureshave been reported on the size of the infective dose,but it is thought to be low.3042 Verification of the sizeof the infective dose may indicate that the organism isefficient at causing the symptoms of disease and maycontinue to be excreted by patients for up to twoweeks after their diarrhoea has resolved.3042 Thiscontinued infectivity makes such patients a potentialhazard to contacts, including hospital staff.

THE DISEASE IN IMMUNODEFICIENTSUBJECTSIn contrast to the short term flu like gastrointestinalillness in immunocompetent patients, Cryptospori-dium may cause severe protracted diarrhoea inimmunodeficient patients. Most of the patients in the71 published records of immunocompromisedpatients had AIDS9 1343-55 but others hadhypogammaglobulinaemia9 26 ss 56 or were receivingimmunosuppressive treatment.25 27 57 58 The age dis-tribution in these reports was influenced by the largenumber of patients with AIDS, resulting in a peakincidence between 31 and 40 years. This was reflectedin a report on 21 patients with AIDS and Cryptospori-dium in whom the age range was 23-62 years (mean35 7 years).51 Interestingly, of seven patients who didnot have AIDS, five were aged under 20 years andthree under 10 years.

Transmission of cryptosporidium from animals toman was not confirmed in any of the 71 casesinvestigated, although two immunosuppressedpatients25 57 and one patient with AIDS45 had con-tact with farm animals. Contact with domestic petswas reported in three cases,4445 58 but follow upinvestigations were not made. In contrast to healthy

Table 5 Symptoms described in 43 immunocompromisedpatients

Symptoms No of cases among thosewith AIDS (n = 36)others (n = 7)

Diarrhoea 35/7Fever 25/3Weight loss 23/1Abdominal pain 12/3Vomiting 9/2Nausea 6/0Anorexia 5/1Headache 3/0Malaise 2/0Constipation 1/0

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Table 6 Organisms associated with Cryptosporidium in 57immunocompromised patients

Organism No of cases among thosewith AIDS (n 50)others (n = 7)

Candida 25/0Pneumocystis carinii 19/0Cytomegalovirus 15/0Mycoplasma avium-intracellulare 7/0Giardia 4/1Entamoeba histolytica 4/0Shigella 3/0Herpes simplex virus 3/0Neisseria gonorrhoeae 2/0Histoplasma 1/0Staphylococcus aureus 1/0Adenovirus 1/0Toxoplasma 1/0Salmomella 1/0Pseudomonas 1/0Isospora belli 1/0

subjects who had cryptosporidial infections,immunodeficient patients inhabited urban rather thanrural environments. Consequently, person to persontransmission may be an important factor in thespread of cryptosporidiosis, particularly in homo-sexuals.59 Among these subjects it may form part ofthe so called "gay bowel syndrome" of gastro-intestinal infection acquired through homosexualpractices.

Table 5 summarises the symptoms of 43 immu-nocompromised patients. All but one patient hadsevere protracted watery diarrhoea, either intermit-tently or continuously, for between seven days and sixyears. The mean duration of diarrhoea for patientswith AIDS was 20-6 weeks (range one to 78 weeks),with a fluid loss of between 1 and 12 litres a day. Fourof the seven patients who did not have AIDS com-

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plained of diarrhoea for between three and seven

weeks, and three patients suffered episodes of diar-rhoea lasting for three to six years. Fever, substantialweight loss (up to 50% of initial weight), and abdom-inal pain were often reported.

DIAGNOSIS IN IMMUNOCOMPROMISEDPATI ENTSCryptosporidium infection in immunocompromisedpatients was usually diagnosed by histological exam-

ination of biopsy material. Small bowel biopsy speci-mens were used most often, although in seven of 30cases biopsy together with faecal examination was

preferred. Necropsy studies showed that the proximaljejunum was the most heavily infected region of thegastrointestinal tract, but infection may extend fromthe pharynx to the rectum.27 5660 In addition, crypto-sporidia were also observed in lung biopsyspecimens131-5; they may have spread to the respira-tory tract either haematogenously52 or more proba-bly as a result of aspiration.13 Histologically, theorganisms may be easily overlooked (G Slavin, per-

sonal communication). In one case the organismswere found only after examination of six jejunal biop-sies.56 Histological changes are described as generallymild, consisting of slight blunting and distortion ofthe villi, lengthening of the crypts, and a modestmononuclear cell infiltration of the lamina pro-

25 27 52 56pria.Patients with AIDS have been coinfected with a

bewildering array of micro-organisms in stark con-

trast to those without AIDS and immunocompetentpatients (Table 6).1 13 284345-5461 Correlationbetween Cryptosporidium and AIDS has led to theproposal that it should be included as an importantfactor in the differential diagnosis.9 Of 57 patients

Table 7 Results oftreating 60 immunodeficient patients sufferingfrom cryptosporidiosis

Treatment (n) No improvement Improvement Recovery

Amphotericin (4) 4 0 0Amprolium (2) 1 1 0Bovine transmission factor (3) 3 0 0Chloroquine (1) 1 0 0Clindamycin (2) 2 0 0Co-trimoxazole (21) 21 0 0Diloxanide (5) 4 0 1Furazolidone (15) 13 1 1y Globulin (2) 2 0 0lodoquinol (2) 2 0 0Ketoconazole (5) 5 0 0Metronidazole (10) 9 1 0Paromomycin (2) 2 0 0Pentamidine (5) 4 1 0Primaquine (1) 1 0 0Pyrimethamine (8) 7 1 0Qumacrine (7) 7 0 0Salinamycin (1) 1 0 0Spiramycin (16) 8 4 4Tetracycline (6) 4 2 0

Total (n = 118) 101 11 6

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Table 8 Chemotherapeutic agents evaluatedprophylactically in calves, mice, or pigs listed alphabetically

Amprolium NicarbazinBleomycin OxytetracyclineDifluoromethylornithine PentamidineDiloxanide furoate PhenamidineDimetridazole QuinacrineEthopabate SalinomycinFuraltadone SulfadiazineHalofuginone SulfadimidineIpronidazole SulfamethazineLasalocid SulfaquinoxalineMetronidazole TrimethoprimMonensin

with AIDS and cryptosporidiosis, 42 died, and theparasite was rarely eradicated.

Although parenteral nutrition has been used to sus-tain patients with hypogammaglobulinaemia, thisprocedure has not been successful in managing otherimmunocompromised patients. The only successfulintervention occurred in subjects receiving immu-nosuppressive treatment, when the underlyingimmune deficiency could be reversed by stoppingtreatment.25 58

Immunosuppression occurs naturally as a directresult of measles infection. Cryptosporidiosis mayoccur in patients with measles,62 and local experiencewith more than one such case suggests that the sever-ity of symptoms is increased, more akin to the illnessseen in patients with AIDS, although with spontane-ous resolution. The first such patient, a 2 year oldchild, had as many as 19 bowel movements in one dayat the peak of the illness. The serological response waspoor. (DPC unpublished data).

Effective treatment for cryptosporidiosis, especiallyin cases of AIDS, has yet to be identified despite thelarge number of chemotherapeutic agents that havebeen tried (Table 7). Preliminary reports, however,suggest that a few patients may have responded totreatment with spiramycin given orally (Ig three orfour times a day).446364 Lack of a suitable laboratorymodel for assessing the efficiency of drugs in chronicinfection has been a major limitation. Drug evalu-ation studies have therefore been confined to the pre-vention of infection in calves, mice, and pigs.Investigators in Australia and in the United Stateshave concentrated on drugs known to be effectiveagainst coccidian parasites in animals or in treating

76.5-67 oprotozoan parasites in man. None of the drugshas been effective (Table 8).

Pathogenic mechanisms

Infection is initiated by the organism forming a stableattachment to the surface of the intestinal mucosa.Unlike many bacterial enteropathogens, this processis probably not mediated by extracellular organelles

Casemore, Sands, Curry(colonisation factors) but may proceed by cell to cellrecognition at the surface membrane. Electron micro-scopic studies on the ilea of infected calves showedthin, irregular, ruthenium red staining filamentsextending from the parasite glycocalyx to the host cellglycocalyx.68 Such a union of electronegativelycharged surfaces may be facilitated by the "bridging"action of divalent cations or, more probably, bysugar-sugar binding proteins (lectins).69 Adherenceof Entamoeba histolytica trophozoites has previouslybeen reported to be mediated by such a mechanism.70Possibly, similar proteins may be essential for crypto-sporidia to colonise the mucosal surface.The attachment phase is followed by penetration

into the epithelial cell. These initial host parasiterelations probably do much to change the appearanceof the villi. Heavy infection with Cryptosporidiumproduces depressions or craters within the mucosalsurface, strikingly illustrated by Snodgrass et al byscanning electron microscopy.7i Several investigatorsdescribed gross changes in the architecture of villiaffected by parasites. -27 52 56 72 73 Stunting andfusion of villi were most commonly reported, togetherwith damage to, and degeneration of, enterocytes.73This, coupled with the location of cryptosporidia pre-dominantly within the posterior small intestine,probably comprise the important enteropathogenicfactors.The relations between the main clinical symptoms

of disease and the pathophysiological eventsdescribed are likely to be multifactorial. A heavy pro-liferation of Cryptosporidium, particularly in theimmunocompromised host, may lead to impaireddigestion, malabsorption, and profuse watery diar-rhoea. The posterior small intestine is known to beparticularly efficient at net fluid absorption.74 Largenumbers of cryptosporidia adherent to villi are likelyto disturb normal villous function. Such a mechanismhas previously been suggested for the symptoms ofgiardiasis.75 Interestingly, however, lesions of similarseverity in the anterior small intestine caused by rota-virus result in only mild diarrhoea.76 Consequently,the distribution of cryptosporidia within the intestinemay be crucial in producing the symptoms of disease.Watery diarrhoea is the major symptomatic expres-

sion of cryptosporidiosis and indicates an appreciableincrease in lumenal water content. This may reflectTable 9 Incidence of Cryptosporidium by age 1983-4

Source No examined No positive (%)

World wide 11067 272 (2 46)Children 2531 124 (49)Adults 2026 41 (20)

United Kingdom only 6580 140 (2-13)Children 1363 59 (4-33)Adults 1739 35 (2-01)

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either a deficiency in the flow of water and nutrientsfrom the lumen to plasma compartments (absorption)or a net accumulation of fluid in which water andelectrolytes enter the lumen from the plasma (secre-tion), or both. Secretory diarrhoea is usually associ-ated with bacteria capable of elaborating an entero-toxin. Such a mechanism, however, has not beendiscovered for Cryptosporidium. In our experience theinoculation of faeces heavily infected with cryptospo-ridia into Vero and other cells sensitive to toxins doesnot produce a cytotoxic effect. Jervis et al suggestedthat coccidial enteritis produced in guinea pigs maybe caused by the response of the mucosa." The dis-charge of toxic metabolites directly from the parasiteto the infected enterocyte, coupled with the wide-spread reaction of the lamina propria, may provide apathogenic mechanism. Cryptosporidial infection inguinea pigs, however, seemed to have little outwardeffect. Most probably a reduction in the mucosal sur-face and a decrease ofmany mucosal enzymes may beresponsible for lowering the absorptive capacity ofthe small intestine, producing an osmotic diar-rhoea.78The intestinal mucosa acts as a semipermeable

membrane, and absorption of water occurs as a pas-sive response to physical forces, the most importantof which is that derived from osmotic pressure. Pres-sure gradients are established by absorption of sol-utes, and water follows across the membrane to main-tain osmotic equilibrium. Malabsorption of anywater soluble nutrient will result in the retention ofwater within the lumen, triggering an osmotic diar-rhoea. This is clearly the response in lactasedeficiency. The disaccharide remains in the lumenwith the attendant water until it reaches the colon.The lactose is then split by colonic bacteria,increasing the number of solutes, which in turn drawsmore water into the lumen. Many enteric infectionsare known to induce malabsorption of carbohydrates;

79 80 8 1including viral enteritis, cholera, shigellosis,Isospora belli,82 and giardiasis.8384 Tzipori reportedthat cryptosporidial infections had a pronouncedeffect on the activity of membrane bound lactase andsuggested that there was a strong correlation betweenthe degree of infection of the mucosa, the extent ofmucosal changes, and the severity of the clinical ill-ness.7 Similarly, malabsorption of fat and low serumcarotene concentrations were also detected in severalpatients with cryptosporidiosis.56 85

Malabsorption induced by infection may increasethe intestinal water load, causing fermentativeosmotic diarrhoea (LA Turnberg, personal commu-nication). It has been estimated that if5% of the aver-age carbohydrate content of the American dietreached the colon a load of 100 mmol(mOsm) capableof supporting 300 cm3 of water in the intestine would

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be produced. Bacterial fermentation of these sugarsto fatty acids would stimulate an additional sixfoldincrease to 18 litres of water.8687 Several workersreported that acid stools, containing large quantitiesof reducing sugars, characteristic in such condi-

88-9tions. Possibly, therefore, the offensive smellingstools which are often reported in cases of cryptospo-ridiosis may result from the bacterial fermentation ofunabsorbed nutrients. It is our experience that mostpatients when questioned complained of rumblingsensations in the bowel and extremely offensive fluidstools.Vomiting may exacerbate the loss of fluid produced

by diarrhoea. In some patients this may be the pre-dominant symptom with little or no diarrhoea. Themechanism for this emetic effect is unclear, but cryp-tosporidial endogenous forms have been detected inthe stomach.284492 We found cryptosporidia in thevomit of one of our patients.The severity of cryptosporidiosis in immu-

nocompromised patients may indicate that functionalcellular and humoral immunities are necessary toresolve infection. Although systemic antibodieswould not be thought to play a part in this because ofthe superficial position- of -the organisms on themucosa, circulating antibodies to Cryptosporidiumhave been detected,'2 93 94 and these were confirmedby our own findings. The presence of humoral anti-bodies against other enteric pathogens, occupying asimilar site, clearly shows that the response to Crypto-sporidium is not unique. It is known, for example, thatboth systemic and local antibodies are producedagainst Giardia lamblia,95 Escherichia coli,96 and Vib-rio cholerae.97 Campbell and Current concluded thatinfection with Cryptosporidium may not result in pro-tective immunity but may reduce the severity of sub-sequent infections.93 The actual role of circulatingantibodies to Cryptosporidium is uncertain, however,and the presence of locally produced antibodiesremains to be determined. Finally, the commonoccurrence of the parasite in newborn domestic ani-mals suggests that infection and perhaps disease mayoccur in infants before the development ofresistance98 when passive immunity might beexpected to be present. Studies on animals indicatedthat colostral antibodies fail to exert a protectiveeffect. 12

Epidemiological considerations

The ubiquitous nature of the parasite in man,wildlife, farm livestock, and pets and its apparentability to cross host species barriers implies that thereservoir of infection to man is large. The oocysts areinherently stable in excreta and resistant to chemicalagents99 100: oocysts have been found in the environ-

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1332ment.I0' These factors would imply that variousroutes of transmission are possible. Person to personspread has also been reported.7 30 3664101

There are a few reports of cryptosporidiosis in nor-mal subjects associated with other intestinal patho-gens, especially Campylobacter"'0 and Giardia.3839The findings of Jokipii et al suggested that their caseslargely represented a form of travellers' diarrhoea.39Our own findings included one subject who seemed tohave acquired mixed Cryptosporidium and Giardiainfection in Leningrad. In our experience, however,the most common organism, to occur with Cryptospo-ridium is Campylobacter. A few cases reported to theCommunicable Disease Surveillance Centre (CDSC)have been in people who have returned from abroad,and in some of these cases there seems to have been anassociation with the consumption of raw milk. Tworecent reports highlighted Cryptosporidium as a causeof travellers' diarrhoea. 102103

Water, raw milk, and foods have all been proposedas sources of infection, although this is difficult tosubstantiate directly in the absence of an enrichmentculture system. Concentration methods such asfiltration, Moore's swabs, and formol-ether centrifu-gation have been successfully used in environmentalstudies in one laboratory by (DPC)10' 120 but have sofar failed to yield positive results with milk, pipedwaters, and food. The need for such environmentalstudies has already been proposed.98A clear association between bovine and human

cryptosporidial infections was established in 26 sub-jects who had direct contact with the faeces of infec-ted calves9293234; one patient was a veterinary stu-dent in charge of calves used in transmissionexperiments with bovine cryptosporidium isolates.32Interestingly, the case reported by Nime et al mayalso have resulted from bovine transmission becausethe child was brought up on a cattle rearing farm.24Two other cases of possible transmission from ani-mals, though not calves, were also reported: onepatient was a 13 month old boy who was believed tohave become infected from a pet cat which was latershown to be positive for Cryptosporidium42; and theother was a professional athlete who had worked inhorse barns.35 Koch et al also reported an associationwith domestic cats.55 Possible sources of infectionwere not considered in 66 of an additional 130patients.93133-3841 Of the remaining 64 patients, 14had acquired their infection while abroad,39 26 mayhave been associated with rural rather than urbancommunities and 22 may'0l (DP Casemore,unpublished observation) have been associated withurban rather than rural communities.37 One case wasan accidental infection in a research worker,36 andone was reported as the first case of hospital crossinfection.30 More recent reports also highlighted

Casemore, Sands, Curryurban infections without evidence of zoonotic trans-mission.104-06 This evidence supports the proposalby Casemore and Jackson that cryptosporidiosis neednot be viewed as a zoonosis.107

Overall, Cryptosporidium seems to occur in about2% of specimens examined both abroad (Table 1) andin the United Kingdom (Table 2), although rates varygeographically and temporally. Some seasonal vari-ation has been suggested although further data arerequired to verify this. The observation that infectionamong immunologically competent subjects occursmore commonly in children has been confirmed37(Table 9). Outbreaks in children attending day nurse-ries have been reported'05 106 (DP Casemore,unpublished data). The incidence of positive findingsin relation to other recognised pathogens in man hasnot been widely reported, but studies in the UnitedKingdom106 together with our own experience'20suggest that Cryptosporidium may be found moreoften than many "common" pathogens.Most positive findings reported are from symptom-

atic subjects. The first report to the CDSC wasreceived in June 1983 from a case diagnosed at RhylPublic Health Laboratory. It was found as a result ofa prospective survey to determine the prevalence ofCryptosporidium in a semirural population. Resultsreported to the CDSC for 1983-4 (Table 3) include aproportion from subjects who were recorded asasymptomatic. This may reflect an absence of infor-mation rather than symptoms. It is our experiencethat such subjects, often contacts of cases, who areinitially reported as asymptomatic are found on per-sonal inquiry to have had mild symptoms. Occa-sionally, patients may have vomiting and abdominalpain without diarrhoea. Extended periods of excre-tion were also reported in those recovering frominfection.42 These mild cases and convalescentexcreters may provide an important reservoir of infec-tion in the community.

Laboratory methods

The methods now applied to detecting Cryptospori-dium are all essentially conventional, or aremodifications of conventional methods. Tyzzer testedvarious stains, including Romanowski stains, on his-tological sections and on smears of epithelial mucosafrom the guts of experimental animals. Subsequentreports including electron microscopy first reported in1966i08 established morphological details and anappreciation of the host parasite interaction. Oocystsin faeces were first detected and reported in 1978 byPohlenz et al, who worked on calf material stained byGiemsa and opened the way for diagnosis by non-invasive means,'09 despite the view of Iseki.'9 Thismethod was used by Tzipori on human faeces in

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Fig. 11 Immunofluorescence ofpurified cryptosporidialoocysts extractedftom faeces. x 2000.

1980," although a report in the same year suggestedthat oocysts were not present in stools.73 The appear-ance of oocysts in preparations stained by theRomanowski method varies according to the particu-lar stain and pH used. We found that consecutivestaining with Jenner and Giesma stains and washingat pH 6 8 gave results that were most useful as anadditional stain in equivocal cases.'20 The oocyststained blue to azure with a crescentic, more deeplystaining body and five or six eosinophilic granules:often a clear halo surrounded the cyst.The use of cold Ziehl-Neelsen stain was introduced

in 1981 by Henrikson and Pohlenz,1"0 who againworked on calf material, and this was rapidly adoptedby workers in human medicine.11' 112 116 With thismethod the internal structures, sporozoites, andresidual body are stained red: empty oocysts andzygote cases remain unstained but are readily recog-nised with practice. The use of strong carbol-fuchsineas a negative stain has been described"' but not gen-erally adopted. Auramine-phenol has been recom-mended as an alternative to the Ziehl-Neelsenstain"4 115 and probably stains the outer cases aswell as internal structures.120 The auramine-phenolmethod seems, therefore, to be more sensitive thanthe Ziehl-Neelsen stain. Carbol-fuchsine negativestaining combined with auramine-phenol was used ina simple rapid screening technique and has provedextremely useful in examining large numbers of speci-mens, including environmental material.10' 116 120Safranin staining followed by counter staining withmethylene blue is claimed to give more sensitiveresults."7 Our own findings and those of othersreported to us suggest that although the stain worksadmirably in some cases, failures occur owing to fac-tors that are as yet unclear. Interestingly, althoughincreased sensitivity is claimed, the incidence (1 3%)in a selected paediatric population was much lower

than that reported in children elsewhere (4%) (Table9).

In an effort to increase sensitivity concentrationmethods have been widely reported, especially by vet-erinary and American workers. The methods mostcommonly used are those of Sheather,118 using con-centrated sucrose and Ritchie's formol-ethermethod."9 In our experience concentration is rarelyrequired in acute cases, but it may have a role in theexamination of specimens from contacts and late orfollow up specimens. It is also of value for showingcryptosporidial contamination of environmental sam-ples.'0 Contrary to some American findings,"11112detailed analysis here has shown that the formol-ethermethod, when suitably modified, is considerably moresensitive than Sheather's method. Sucrose also inter-feres with various staining methods and with theattachment of the material to the slide.120Most light microscopy methods used for diagno-

sing cryptosporidiosis are not specific and can lead tomisidentification. The desirability of a specific test istherefore obvious. The fluorescence antibody test orother similar methods should provide the necessaryspecificity. Although commercial reagents are not yetavailable, experimental evidence from veterinaryworkers (KW Angus, personal communication) andfrom our laboratory shows that this method does pro-duce improved diagnostic specificity (Fig. 1 1).1 20

Availability of suitably sectioned biopsy materialfor examination in the electron microscope can pro-duce a definitive diagnosis.'9 Other workers haveused the negative staining method directly with faecalmaterial.'2' The sensitivity of this method is neces-sarily very low and in the absence of specific mor-phological features it is of limited diagnostic value.

Proof of an immune response is required both clin-ically and immunologically. Studies on antibodieshave been reported by Tzipori,94 Current, 12 andCampbell and Current.93 Similar studies in our labo-ratory showed that there was a response to infection,primarily rising titres of IgA and IgM. When serialspecimens were examined the expected rise in IgGtitres failed to occur the IgG titres remained low. Lowtitres of IgA and IgM were also present in some con-trol subjects. This contrasts with the findings ofCurrent' 2 that, almost without exception, theresponse is IgG.

ConclusionThe role of Cryptosporidium in human disease is nowestablished'22 Interest in human cryptosporidiosishas stemmed mainly from two sources: firstly, thecooperation and sharing of information between vet-erinary and human health care workers, and, sec-ondly, from the concern related to opportunisticinfections in immunocompromised patients,

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1334

especially those with AIDS. In view of the impendingincrease in the incidence of AIDS and the lack ofchemotherapeutic substances effective against Cryp-tosporidium it is essential that work on this parasiteshould continue. The incidence in immunologicallynormal subjects makes it imperative to pursue epi-demiological and environmental studies.

We thank the following: Dr F Bruce Jackson (direc-tor), Malcolm Armstrong, G Stringfellow, and othermembers of staff of the Public Health Laboratory anddepartment of microbiology, Rhyl; the Commu-nicable Disease Surveillance Centre, Colindale; SBloomfield (librarian, Central Public Health Labora-tory, Colindale); J Bladon (librarian, Ysbyty GlanClwyd); Professor L Turnberg, department of medi-cine, University of Manchester. We are particularlygrateful to Mr KW Angus and other staff of the Mor-den Institute, Edinburgh and clinical colleagueslocally.

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101Casemore DP, Jessop EG, Douce D, Jackson FB. Cryptospori-dium plus Campylobacter: an outbreak in a semi-rural popu-lation. JHyg (in press).

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107 Casemore DP, Jackson FB. Hypothesis: cryptosporidiosis inhuman beings is not primarily a zoonosis. JInfect 1984;9:153-6.

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120 Casemore DP, Armstrong M, Sands RL. The laboratory diagno-sis of cryptosporidiosis. J Clin Pathol 1985;38.

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122 Anonymous. Cryptosporidiosis [Editorial.] Lancet 1984;i:492-3.

Requests for reprints to: DP Casemore, Public Health Lab-oratory, Glan Clwyd Hospital, Bodelwyddan, Nr Rhyl,Wales LL18 5UJ.

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