Journal of Infection (I984) 9, 277-282

Preva lence o f Cryptospor id ium a m o n g pat ients with acute enteric infect ion

Winnie Holten-Andersen,* Jan Gerstoft,t Sv. Aa. Henriksen~ and Nils Strandberg Pedersen~

Statens Seruminstitut, *Department of Clinical Microbiology at Hvidovre Hospital and ~Rigshospitalet and tDepartment of Rubella, Copenhagen,

Denmark, and $State Veterinary Serum Laboratory, Bi~lowsvej 27, Copenhagen, Denmark

Accepted for publication 5 April I984

Summary Twelve hundred faecal samples from patients suspected of acute enteric infection were examined for Cryptosporidium oocysts by means of a modified Ziehl-Neelsen technique. Smears prepared from the formol-ether sediment of 4oo faecal samples were compared with those made directly from faeces. The numbers of positive patients were identical, but the diagnosis was easier to make with smears prepared from sediment. Crypto- sporidia were found in 2I samples and after correction for duplicates the net result was I6 cases (2%) of cryptosporidiosis diagnosed among 8oo patients. Except in one case Cryptosporidium was the only pathogen found. Fourteen patients had diarrhoea while excreting the parasite. All the patients recovered spontaneously within 3 weeks.

Nine of them had recently travelled abroad; for three others, infected calves may possibly have been the source of infection. Carriers were not found among I2o persons without gastro-intestinal symptoms.

Introduction

Cryptospor id ium is a coccidian parasite related to the coccidia species of domestic animals as well as to Sarcocystis species and to Toxoplasma gondii. It may infect the epithelial cells of the gastrointestinal tract in many species of mammals , birds and reptiles. T h e organism completes its life cycle in the microvillous border of the intestine and is excreted in an infective stage in the faeces of the host. 1

Cryptosporidia were discovered by Tyzzer in I9O72 but in the following 70 years only a few reports were publ ished on the association between this organism and disease. Recent investigations have revealed Cryptospor id ium as the causative agent in outbreaks of diarrhoea among young domestic animals, with an apparent lack of species specificity. 3

In I976, the first cases of human cryptosporidiosis were reported ~, 5 and up to I982 only a dozen cases had been described, half of which were in immunocompromised hosts. 6 Since then cryptosporidiosis has been shown to be an important complication in patients with acquired immunodeficiency syndrome (AIDS) among whom more than 2o cases have now been observed. 6

Correspondence to: Winnie Holten-Andersen, M.D., Statens Seruminstitut, Department of Clinical Microbiology at Hvidovre Hospital, Kettegaard AUe 30, DK-265o Hvidovre, Denmark.

oi63-4453/84/o6o27o+o6 $02.00/0 (~) I984 The British Society for the Study of Infection

278 W. H O L T E N - A N D E R S E N E T A L .

In immunologically normal patients, cryptosporidia 3, 7, 8 may cause diarrhoea, nausea, vomiting, abdominal pain, and often fever. The infection is self-limiting with spontaneous recovery within 3 weeks.

In immunocompromised patients, especially those with T-cell defects, as in patients with AIDS, infection with Cryptosporidium results in prolonged diarrhoea, malabsorption and loss of weight. 6

The purpose of the present study was to reveal the prevalence of Crypto- sporidium in patients suspected of suffering from acute enteric infection. Furthermore, the use of the formol-ether concentration technique for recover- ing the parasite from faeces has been studied.

M a t e r i a l s a n d m e t h o d s

Eight hundred consecutive faecal specimens submitted to the Department of Diagnostic Bacteriology, Statens Seruminstitut, Denmark for detection of enteropathogenic bacteria and 400 consecutive faecal specimens submitted to the Statens Seruminstitut, Department of Clinical Microbiology at Rigs- hospitalet for detection of parasites were examined. The specimens were submitted by general practitioners and from hospitals in Denmark during the spring of I983.

After correction for duplicates the material consisted of 655 specimens from patients for bacteriological examination and specimens from 265 patients for parasitological examination. The 655 samples included i2o samples from persons without gastro-intestinal symptoms who were employed in the food or drug industries. Subtraction of these healthy persons leaves 800 specimens originating from patients suspected of suffering from enteric infection or infestation.

The bacteriological examination routinely included culturing for Salmonella sp., Shigella sp., Yersinia enterocolitica, Campylobacter sp., and in selected cases for enteropathogenic Eschericia coli, Staphylococcus aureus and Vibrio cholerae. Examination for rotavirus and other viruses was not included in the present investigation. The parasitological examination included routine microscopy for helminths, eggs and cysts in formol-ether concentrated faecal samples), 10

The patients ranged in age from I month to 95 years with a median age of 28 years. Females and males were equally represented.

From I2oo original faecal samples and from 400 formol-ether sediments, smears were prepared and stained by a modified Ziehl-Neelsen technique. 11 The smears were examined microscopically at a magnification of x 400 for screening, and with an oil immersion objective at x IOOO for details.

Additional faecal samples and further information were obtained when cryptosporidia were found. When information about animal contacts was received, faecal samples from the animals in question were examined.

R e s u l t s

Cryptosporidia were detected in i6 of 80o patients suspected of suffering from enteric infection or infestation, giving a prevalence of 2 % (95 % confidence

Journal of Infection Plate I

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Plate i . Cryptospor id ium oocysts in faecal sediment obtained by means of the formol-e ther concentrat ion technique. In (a), (b) and (c) the modified Zieh l -Nee lsen staining technique was used; in (d) iodine staining of the wet sediment was used. T h e oocysts are indicated by arrows. (Magnification in (a) is x 27o, in (b) and (d) x 108o, and in (c) x 27oo.)

HOLTEN-ANDERSEN ET AL. (Facing p . 279 )

Prevalence of Cryptosporidium 279

limits I.I4-3.22 %). Examination of z2o samples from employees in the food or drug industries did not reveal any cryptosporidia or other enteric pathogens.

In smears stained by the modified Ziehl-Neelsen method, cryptosporidia presented themselves as small, round, red-stained organisms about 4 pm in diameter with a distinct cell wall and a few dark granules inside [Plate I (a), (b), (c)]. After iodine staining of the wet sediment, cryptosporidia appeared as bright, highly refractile, spherical bodies with dark granules [Plate r (d)]. Comparison was made between iodine staining of the sediments and modified Ziehl-Neelsen staining of, respectively, smears from the sediments and smears made directly from the samples. While positive cases found by the various methods were identical, a zo times greater number of cryptosporidia was found after concentration by the formol-ether technique than by direct examination. In iodine-stained sediments, cryptosporidia could be detected at x 40o magni- fication, but, to confirm the suspected diagnosis, Ziehl-Neelsen staining was required.

Clinical data from z6 patients with cryptosporidia are given in Table I. Ten cases were found among the 535 patients from whom samples of faeces were examined for enteropathogenic bacteria and six among the 265 patients from whom samples were examined for enteric parasites. Fourteen patients with Cryptosporidium had diarrhoea lasting for not more than 3 weeks. Examination of faecal samples taken after termination of the symptoms did not reveal cryptosporidia.

Among patients with cryptosporidia, two adopted children who had just arrived from South Korea did not have any signs of enteric disease at the time when cryptosporidia were detected, and examination I month after arrival did not reveal any parasites. For six children with cryptosporidiosis information about animal contacts was received and led to an examination of faecal samples from the animals in question. Cryptosporidium was found in three calves. Except for two brothers with cryptosporidiosis clustering of cases was not seen. Six of the adult patients had within the last month been travelling abroad. In one of these patients, admitted to hospital with severe gastro-intestinal symptoms both Cryptosporidium and Giardia lamblia were found. During treatment for giardiasis the patient recovered and examination of control faecal samples did not reveal any parasites.

The bacteriological findings in 535 samples were Campylobacter sp. (8), Yersinia enterocolitica (I5), Salmonella typhimurium (I3), various other Sal- monella sp. (8), and Shigella sp. (4), and enteropathogenic Escherichia coli (I).

The parasitological findings in 265 samples were Giardia lamblia (I8). Entamoeba coli (I3), Endolimax nana (7), Entamoeba histolytica (3), Entamoeba hartmanni (3), Chilomastix mesnili (2), Iodamoeba bi~tschlii (I), hookworm (I), Opisthorehis sp. (I), Trichuris trichiura (I) and Ascaris lumbricoides (I). Of these, Entamoeba coli, Endolimax nana, Entamoeba hartmanni, Chilomastix mesnili and Iodamoeba bi~tschlii are generally considered to be apathogenic.

Cryptosporidium therefore was among the most prevalent identified potential pathogens in the present groups of patients, surpassed only by Salmonella sp., Yersinia enterocolitica, and Giardia lamblia.

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Prevalence of Cryptosporidium 281

D i s c u s s i o n

Samples f rom patients with cryptosporidiosis were found to be positive whether the formol-e ther concentrat ion technique was used or not. The main difference was an increase in the number of organisms observed, thereby facilitating the diagnosis. 1° Since the formol-e ther concentrat ion technique is widely used for parasitological examination, it is suggested that i fcryptosporidia are suspected, such as by the presence of cryptosporidia-like cysts in the iodine-stained sediments examined at × 4o0 magnification, smears from the sediments should be stained by the modified Ziehl-Neelsen technique.

T h e prevalence of Cryptospor id ium found in this s tudy is compatible with results reported from Australia, 3 Finland, 7 and England. s In the present investigation, I4 of 16 patients with cryptosporidiosis had symptoms of an enteric infection during the I-3 week period in which cryptosporidia were excreted. In contrast, cryptosporidia were not found among the employees in food or drug industries, who may serve as a control group.

There was no definite proof that the presence of Cryptospor id ium was the causative agent of the enteric infection since the examination did not exclude all enteric infectious agents (rotavirus etc.). As the patients recovered, however, excretion of the organisms decreased and then stopped. Excretion of crypto- sporidia for more than 3 weeks should raise the suspicion that the patient has a cellular immunodefic iency e.g. AIDS. 6

In three cases cryptosporidia were found in animal contacts (calves) thereby suggesting these as a possible source of infection. I t has previously been shown that Cryptospor id ium may be contracted from animal contacts. 6 Otherwise the most obvious hazard of acquiring cryptosporidiosis seemed to be associated with travelling abroad, as is so with other enteric pathogens. 7,12

In conclusion, the present work indicates that Cryptospor id ium is among the most prevalent enteric pathogenic parasites, but that the infection is self-limiting in most patients. This is fortunate since efficient therapy does not so far exist. In patients suffering from A I D S , however, Cryptospor id ium may give rise to a severe l ife-threatening disease. Fur the r investigations concerning the occurrence, transmission, t rea tment and prevention of cryptosporidiosis in h u m a n beings are therefore needed.

(The authors wish to thank Mrs Inger Larsen for her secretarial assistance, and Mrs Grethe Gomme and Mrs Bente Michelsen, Statens Seruminstitut, for their skilful technical assistance.)

R e f e r e n c e s

I. Angus KW. Cryptosporidiosis in man, domestic animals and birds: a review. J R Soc Med 1983; 76: 62-7o.

2. Tyzzer EE. A sporozoan found in the peptic glands of the common mouse. Proc Soc Exp Biol Med I9O7; 5: I2-I3.

3. Tzipori S. Cryptosporidiosis in Animals and Humans. Microbiol Rev I983 ; 47: 84--96. 4. Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH. Acute enterocolitis in a human

being infected with the protozoan Cryptosporidium. Gastroenterology 1976; 7o: 592-598. 5. Meisel JL, Perera DR, Meligro C, Rubin CE. Overwhelming watery diarrhea associated

with a Cryptosporidium in an immunosuppressed patient. Gastroenterology 1976; 7o: II56--II60.

282 W. HOLTEN-ANDERSEN ET AL.

6. Current WL, Reese NC, Ernst JV, Bailey WS, Heyman MB, Weinstein WM. Human cryptosporidiosis in immunocompetent and immunodeficient persons. N Engl J Med 1983; 3o8: 1252--1257.

7. Jokipii L, Pohjola S, Jokipii A/VIM. Cryptosporidium: A frequent finding in patients with gastrointestinal symptoms. Lancet 1983; ii: 358-361.

8. Casemore DP, Jackson B. Sporadic Cryptosporidiosis in Children. Lancet 1983; ii: 679. 9. Allen AVH, Ridley DS. Further observations on the formol-ether concentration technique

for faecal parasites. J Clin Pathol 197o; 23: 545-546. IO. Garcia LS, Bruckner DA, Brewer TC, Shimizi RY. Techniques for the Recovery and

Identification of Cryptosporidium Oocysts from Stool Specimens. J Clin Microbiol 1983 ; I8: 185-19o.

I I. Henriksen SA, Pohlenz JFL. Staining of Cryptosporidia by a modified Ziehl-lXleelsen Technique. Acta Vet Scand 1981; 22: 594-596.

12. Dupont HL, Reves RR, Galindo E, Sullivan PS~ Wood LV, Mendiola JG. Treatment of travelers' diarrhea with trimethoprim/sulfamethoxazole and with trimethoprim alone. N EnglJ Med 1983; 307: 841-844.