- Email: [email protected]
Human cryptosporidiosis in China
639
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1992) 86, 639-640
Human
cryptosporidiosis
0.9%. These 3 children had high absorbence values at a serum dilution of 1:32 in an enzyme-linked immunosorbent assay (ELISA) for specific immunoglobulin (Ig) G and IgM antibodies to Cryptosporidium (the method described by UNCAR et al., 1986). However, in another community-based study in rural Fengyang, no positive stool was found from 239 children under 12 years of age. At the same time, blood samples were collected from the children in these 2 communities and examined by ELISA. About half of the children had IgG antibody against cryptosporidial oocysts (S.-X. Zu et al., to be published). Human cryptosporidiosis has been reported from all regions of China. Most surveys were carried out during the summer on patients presenting with diarrhoea. The cases reported were mainly children living in rural communities. The prevalences are shown in the Table. Sucking lingers seemed to be a risk factor for this infection in children (ZHU et al., 1990). Although no significant zoonotic transmission was found, both clinical and inapparent infections have been reported in 6 of 22 diarrhoeal calves and 1 of 4 kittens in Kunming, Yunnan (ZLJO et al., 1990) and in 28 of 49 healthy calves aged 2-5 months on one dairy farm, but in none of 19 calves aged 4-7 months on another dairy farm in the suburbs of Beijing (HUANG et al., 1990). We examined 28 normal dairy calves about 6 months of age in Hefei in the autumn of 1988, and found none with oocysts in their stool specimens. Garlicin, an antimicrobial agent extracted from garlic, was widely tried to treat this infection and was believed to be effective (YAO & CHEN, 1989). But cryptosporidiosis is self-limited in immunocompetent hosts, and no conclusion can be made until well-designed controlled clinical trials have been conducted. These limited observations suggest that Cryptosporidium infection is widely distributed in China, and is of significant medical importance. Screening for oocysts in stools should be considered as a routine laboratory test in childhood diarrhoea, particularly when persistent. Further understanding of the epidemiology is needed in order to attempt to control the infection.
in China
Zu, Shu-Yu Zhu and Jin-Fen Li Department of Clinical Epidemiology and Community Medicine, Anhui Medical University, Hefei, Anhui 230032, China Shu-Xian
Cryptosporidium parvum is a common cause of diarrhoeal disease not only in immunocompromised patients but also in immunocompetent humans, particularly in children living in impoverished regions, and livestock throughout the world. Since 1987, clinical reports of human cryptosporidiosis have been accumulated in Chinese journals, but, to our knowledge, there has been only one report in the English literature of the occurrence of Cryptosporidium infection in mainland China (CHEN et al., 1992). This paper recorded the examination of 5089 children in the suburbs of Xuzhou city and in 6 rural areas of Jiangsu province; 89 (1.75%) were excreting Cyptosporidium oocysts, with prevalences of 1.5% in the suburbs and 0.7-5.1% in the rural areas. Of the 89 infected children, 57 (61%) had diarrhoea. In this paper we report our surveys in clinical settings and in children in rural communities of Anhui, and review papers on the prevalences of cryptosporidiosis published in Chinese journals. These data are essential for an overall descriptive epidemiology of this infection. During the summer of 1987, we examined 504 outpatients with diarrhoea for enteropathogenic bacteria and protozoa in a clinic for coal miners in Huaibai, Anhui. Stool specimens were screened for Cryptosporidium by auramine-rhodamine staining for oocysts and confirmed by the modified cold Ziehl-Neelsen method. All the adult patients were negative. Among 105 paediatric patients, a 2 years old girl with persistent diarrhoea was found to be excreting cryptosporidial oocysts with no other pathogen in her stool. Her 4 family members were examined and her 5 years old brother was also found to be infected, but without clinical illness. Her younger brother aged 6 months had mild diarrhoea but no oocyst was found in his stool (ZU & Du, 1987). We also examined 25 adult patients without diarrhoea, receiving chemotherapy and/or radiotherapy for malignant diseases, and therefore considered to be immunocompromised, at the Anhui Table.
Summary
of reports
Regions Huaibai, Anhui Naming, Jiangsu Chongqing, Sichuan Fuzhou, Fujian Xuzhou, Jiangsu Wuhu, Anhui Qingdao, Shandong Huhohaote, Inner Mongolia Jinan, Shandong Anren, Hunan Kunming, Yunnan “There is controversy
of human cryptosporidiosis
Acknowledgements This study was supported by project 3-P-86-0121 from the International Development Research Centre, Canada. We thank Dr Richard L. Guerrant for his continuous encouragement and help. Dr S.-X. Zu is the recipient of a Rockefeller Foundation Fellowship in the Division of Geographic Medicine at the University of Virginia, Charlotesville, VA 22908, USA.
in China
Clinical condition of subjects
No. positive no. examined
Childhood diarrhoea Paediatric acute diarrhoea Acute diarrhoea Healthy individuals Immunocompromised patients Paediatric diarrhoea Paediatric diarrhoea Childhood diarrhoea Acute and persistent diarrhoea Paediatric diarrhoea Diarrhoea patients Infant diarrhoea Paediatric patients
11105 (1%) 91924 (1%) 5150 (10%) 51150 (3.3%) lOi (20%) 161120 (13%) 201478 (4,2%j 53 cases 5 cases
about the validity
of the identification
Medical University Hospital. Ctyptosporidium infection was found in a female patient with digestive tract carcinoma, receiving radiotherapy. In a community-based study in 1989, we investigated 320 apparently healthy children under 10 years of age in rural Tongling, and found cryptosporidial oocysts in 3 children, with no recent record of diarrhoea, a subclinical infection rate of
51140 (3.6%) 1123 (4.3%) 2 cases 9173 (12%)
References Zu & Du, 1987 HAN et al., Luo et al.,
1987; 1988”
HAN,
1989
Su et al., 1989 CHEN & YAO, 1990 ZHU et al., 1990 GONG et al., 1990 GLIAN et al.,
1990 Su et al., 1990 Lu et al., 1990 Zu0 et al., 1990
of the oocysts in this report. References Chen, Y.-G. & Yao, F.-B. (1990). Report of 14 cases of human cryptosporidiosis in Xuzhou. China ZoonosesJournal, 6,28. Chen, Y. G., Yao, F. B., Li, H. S., Shi, W. S., Dai, M. X. & Lu, M. (1992). Cryptosporidium infection and diarrhoea in rural and urban areas of Jiangsu, People’s Republic of China.Joumal of Clinical Microbiology, 30,492-494.
640
Gong, Y.-X. Cao, S.-Q., Shi, X.-Z. & Zhou, S.-C. (1990). Crvotosuoridiosis in Oinedao. China ‘foumal ofParasitic DiseaSeiCo;ltrol,~3,?2.
- -
Guan, J.-S., Cm, S.-L., Yen, X.-L., Zhao, X. H., Zhang, J.Z., Wang, Y.-M. & Tuo, Y. (1990). Preliminary clinical observations of cryptosporidial enteritis. Chinese Jouwtal of Pediatrics, 28,41.
Han, F. (1989). Infant cryptosporidiosis found in China. ChineseJournal of Pediatrics, 27,75-76. Han, F., Tan, W.-X. & Zhou, X.-L. (1987). Human cryptosporidiosis in Naming region-two casereports. Jiangsu Medicine, 12,692. Huang, S.-R., Zhang, H.-J,., Huang, M.-J., Li, S.-Y. & Zhang, L. (1990). Cryptosportdrum infection of calves in the suburbs of Beijing. China ZoonosesJournal, 6,45. Lu, L.-A., Fan, Z.-Z., Li, C.-C. & Liu, B.-Z. (1990). Two casesof infant cryptosporidiosis. HunanMedicine, 7, 106. Luo, M.-Y., Wang, Q.-N. & Zhen,, X.-P. (1988). Cryptosporidial infection and enteritis. Chmese 3ournal of Internal Medicine, 27,686-688. Su, Q.-P., Chen, D.-G., Zhao, Z.-G., Hua, Y., Hua, X.-L., Chen, S., Huang, M. H., Wan, J.-Q., Guo, Y.-H. & Li, S.M. (1989). Cryptosporidiosis from infants in Fuzhou region. China ZoonosesJournal, 5,35-36.
TRANSACTIONS OFTHEROYAL
Su, L.-Y., Chen, X.-C., Li, Y.-H. & Liu, J. H. (1990). Casereport of infant cryptosporidiosis. China Journal of Parasitic : DiseasesControl, 3, 154. Unaar, B. L. I’.. Soave, R., Faver, R. & Nash, T. E. (1986). Enzyme immunoassay detection of immunoglobulin ‘M and G antibodies to Cryptosporidium in immunocompetent and immunocomeromised oersons. 7ouwtal oflnfectious Diseases. lS3,570-578’. ” ’ ’ Yao, F.-B. & Chen, Y.-G. (1989). Preliminary study on the effir cacy of garlicin in treatment of childhood cryptosporidiosis. 3ournal of Xuzhou Medical College, 9,56.
Zhu, F.-C., Hun, Z.-X. & Wang, F.-P. (1990). A case-control studv of childhood crvutosuoridiosis in Wuhu. China Public __ _ Health, 6,503-504.
Zu, S.-X. & Du, M.-W. (1987). Human cryptosporidiosis seen in China.3ouwaal ofAnhuiMedica1 University, 22,276. Zuo, Y.-X., Chen, F.-Q. & Fang, L. (1990). Human and domestic cryptosporidiosis in Yunnan and experimental infection study. China ZoonosesJournal, 6, 34-35. Received 5 February 1992; revised 16 April 1992; accepted for publication 16 April I992
SOCIETY OF TROPICAL MEDICINE ANDHYGIENE (1992) 86, 640
/Short Report 1 Rapid purification and concentration technique for Cryptosporidium parvum oocysts M. Lyagoubi, A. Datry, I. Malet, M. Danis and M. Gentilini Laboratoire, de Parasitologie, Uniti INSERM 313, CHU Piti&Salpet&re, 91 Boulevard de l’H6pita1, 75534 Paris ckdex 13, France Purification of faecal Cyptosporidium oocysts is necessary for infection of cell cultures (in vitro models) and of laboratory animals (in vivo models). For immunological studies purified parasites are essential. C. parzwm oocysts are separated from patients’ stools or from the stools of naturallv or exuerimentallv infected animals. Several techniques are used to isolate the oocysts: PercoB@ gradients (DULSKI & TURNER. 1988). sucrose gradientsu(CURREN? & LONG, 1983) and’saturated NaCl solution (DATRY et al., 1989). In order to obtain a sufficient number of oocysts with these techniques, very long and fastidious manipulations are needed. We propose a technique in which stools are previously filtered through a wide-mesh screen and a Blutter cloth (40 urn pore4 and conserved in potassium bichromate. The stools, diluted 50% in potassium bichromate, are filtered through a moistened hydrophylic cotton column (height 5 cm, diameter 2 cm); 50 ml of this material can be filtered through each column, before it becomes saturated. The filtrate is washed in phosphate-buffered saline (PBS) and centrifuged twice (10 min at 1500 g). The pellet obtained is suspended in 3 ml of PBS and the suspension is placed on a discontinuous angiographine gradient (meglumine sodium amidotrizoate, Radioselectan@, Schering Laboratories). This discon-
tinuous gradient is obtained by pouring 3 ml of a solution containing 3 parts Radioselectan@, 1 part human serum and 3 parts MEM medium on to 3 ml of a solution containing the same ingredients in the proportions 4: 1:2. The tubes are centrifueed at 12 000 P for 10 min in a Beckman horizontal ro’ior centrifuge.“Two dense rings are formed between the 2 liquid-liquid interfaces. We have recovered from the liquid column located above the lower ring, 90% of the oocysts that were introduced; the quality of this purification method is excellent. In the residue, we find bacteria, yeasts and other small debris. The method permits the detection of Cyptosporidium oocysts in a stool-bichromate solution containing as few as 20 oocvstsiml. This technique has been previously used to purify leishmanial amastigotes (MONIOUR et al., 1984). It is useful because of its rapidity and ease compared with the usual techniques and, above all, the quality and yield of the end product. The oocysts obtained are pure, viable and suitable, after excystation and sporozoite liberation, for the infection of cell cultures. References Current, L. W. & Long, I’. L. (1983). Development of human and calf Ctyptosporidium in chicken embryos. 3oumal of Infectious Diseases, 148, 1108-l 113. Datry, A., Danis, M. & Gentilini, M. (1989). Developpement complet de Cryptosporidium en culture cellulaire. Medicine et Sciences, 5, 762-766.
Dulski, I’. & Turner, M. (1988). The purification of sporocysts and sporozoites from Eimena tenella oocysts using PercoIl” density gradients. Avian Diseases, 32, 235-239. Monjour, L., Vouldoukis, I., Brandicourt, 0.) Mazier, D., Alfred, C., Ploton, I. & Gentilini, M. (1984). Rapid large-scale production and isolation of Leishmania amastigotes. Annals of Tropical hfedicine and Parasitology, 78, 423-425. Received 5 December 1990; revised 12 February 1991; accepted for publication 13 February 1992 [subsequently unavoidably delayed by mailing problems]
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1992) 86, 639-640
Human
cryptosporidiosis
0.9%. These 3 children had high absorbence values at a serum dilution of 1:32 in an enzyme-linked immunosorbent assay (ELISA) for specific immunoglobulin (Ig) G and IgM antibodies to Cryptosporidium (the method described by UNCAR et al., 1986). However, in another community-based study in rural Fengyang, no positive stool was found from 239 children under 12 years of age. At the same time, blood samples were collected from the children in these 2 communities and examined by ELISA. About half of the children had IgG antibody against cryptosporidial oocysts (S.-X. Zu et al., to be published). Human cryptosporidiosis has been reported from all regions of China. Most surveys were carried out during the summer on patients presenting with diarrhoea. The cases reported were mainly children living in rural communities. The prevalences are shown in the Table. Sucking lingers seemed to be a risk factor for this infection in children (ZHU et al., 1990). Although no significant zoonotic transmission was found, both clinical and inapparent infections have been reported in 6 of 22 diarrhoeal calves and 1 of 4 kittens in Kunming, Yunnan (ZLJO et al., 1990) and in 28 of 49 healthy calves aged 2-5 months on one dairy farm, but in none of 19 calves aged 4-7 months on another dairy farm in the suburbs of Beijing (HUANG et al., 1990). We examined 28 normal dairy calves about 6 months of age in Hefei in the autumn of 1988, and found none with oocysts in their stool specimens. Garlicin, an antimicrobial agent extracted from garlic, was widely tried to treat this infection and was believed to be effective (YAO & CHEN, 1989). But cryptosporidiosis is self-limited in immunocompetent hosts, and no conclusion can be made until well-designed controlled clinical trials have been conducted. These limited observations suggest that Cryptosporidium infection is widely distributed in China, and is of significant medical importance. Screening for oocysts in stools should be considered as a routine laboratory test in childhood diarrhoea, particularly when persistent. Further understanding of the epidemiology is needed in order to attempt to control the infection.
in China
Zu, Shu-Yu Zhu and Jin-Fen Li Department of Clinical Epidemiology and Community Medicine, Anhui Medical University, Hefei, Anhui 230032, China Shu-Xian
Cryptosporidium parvum is a common cause of diarrhoeal disease not only in immunocompromised patients but also in immunocompetent humans, particularly in children living in impoverished regions, and livestock throughout the world. Since 1987, clinical reports of human cryptosporidiosis have been accumulated in Chinese journals, but, to our knowledge, there has been only one report in the English literature of the occurrence of Cryptosporidium infection in mainland China (CHEN et al., 1992). This paper recorded the examination of 5089 children in the suburbs of Xuzhou city and in 6 rural areas of Jiangsu province; 89 (1.75%) were excreting Cyptosporidium oocysts, with prevalences of 1.5% in the suburbs and 0.7-5.1% in the rural areas. Of the 89 infected children, 57 (61%) had diarrhoea. In this paper we report our surveys in clinical settings and in children in rural communities of Anhui, and review papers on the prevalences of cryptosporidiosis published in Chinese journals. These data are essential for an overall descriptive epidemiology of this infection. During the summer of 1987, we examined 504 outpatients with diarrhoea for enteropathogenic bacteria and protozoa in a clinic for coal miners in Huaibai, Anhui. Stool specimens were screened for Cryptosporidium by auramine-rhodamine staining for oocysts and confirmed by the modified cold Ziehl-Neelsen method. All the adult patients were negative. Among 105 paediatric patients, a 2 years old girl with persistent diarrhoea was found to be excreting cryptosporidial oocysts with no other pathogen in her stool. Her 4 family members were examined and her 5 years old brother was also found to be infected, but without clinical illness. Her younger brother aged 6 months had mild diarrhoea but no oocyst was found in his stool (ZU & Du, 1987). We also examined 25 adult patients without diarrhoea, receiving chemotherapy and/or radiotherapy for malignant diseases, and therefore considered to be immunocompromised, at the Anhui Table.
Summary
of reports
Regions Huaibai, Anhui Naming, Jiangsu Chongqing, Sichuan Fuzhou, Fujian Xuzhou, Jiangsu Wuhu, Anhui Qingdao, Shandong Huhohaote, Inner Mongolia Jinan, Shandong Anren, Hunan Kunming, Yunnan “There is controversy
of human cryptosporidiosis
Acknowledgements This study was supported by project 3-P-86-0121 from the International Development Research Centre, Canada. We thank Dr Richard L. Guerrant for his continuous encouragement and help. Dr S.-X. Zu is the recipient of a Rockefeller Foundation Fellowship in the Division of Geographic Medicine at the University of Virginia, Charlotesville, VA 22908, USA.
in China
Clinical condition of subjects
No. positive no. examined
Childhood diarrhoea Paediatric acute diarrhoea Acute diarrhoea Healthy individuals Immunocompromised patients Paediatric diarrhoea Paediatric diarrhoea Childhood diarrhoea Acute and persistent diarrhoea Paediatric diarrhoea Diarrhoea patients Infant diarrhoea Paediatric patients
11105 (1%) 91924 (1%) 5150 (10%) 51150 (3.3%) lOi (20%) 161120 (13%) 201478 (4,2%j 53 cases 5 cases
about the validity
of the identification
Medical University Hospital. Ctyptosporidium infection was found in a female patient with digestive tract carcinoma, receiving radiotherapy. In a community-based study in 1989, we investigated 320 apparently healthy children under 10 years of age in rural Tongling, and found cryptosporidial oocysts in 3 children, with no recent record of diarrhoea, a subclinical infection rate of
51140 (3.6%) 1123 (4.3%) 2 cases 9173 (12%)
References Zu & Du, 1987 HAN et al., Luo et al.,
1987; 1988”
HAN,
1989
Su et al., 1989 CHEN & YAO, 1990 ZHU et al., 1990 GONG et al., 1990 GLIAN et al.,
1990 Su et al., 1990 Lu et al., 1990 Zu0 et al., 1990
of the oocysts in this report. References Chen, Y.-G. & Yao, F.-B. (1990). Report of 14 cases of human cryptosporidiosis in Xuzhou. China ZoonosesJournal, 6,28. Chen, Y. G., Yao, F. B., Li, H. S., Shi, W. S., Dai, M. X. & Lu, M. (1992). Cryptosporidium infection and diarrhoea in rural and urban areas of Jiangsu, People’s Republic of China.Joumal of Clinical Microbiology, 30,492-494.
640
Gong, Y.-X. Cao, S.-Q., Shi, X.-Z. & Zhou, S.-C. (1990). Crvotosuoridiosis in Oinedao. China ‘foumal ofParasitic DiseaSeiCo;ltrol,~3,?2.
- -
Guan, J.-S., Cm, S.-L., Yen, X.-L., Zhao, X. H., Zhang, J.Z., Wang, Y.-M. & Tuo, Y. (1990). Preliminary clinical observations of cryptosporidial enteritis. Chinese Jouwtal of Pediatrics, 28,41.
Han, F. (1989). Infant cryptosporidiosis found in China. ChineseJournal of Pediatrics, 27,75-76. Han, F., Tan, W.-X. & Zhou, X.-L. (1987). Human cryptosporidiosis in Naming region-two casereports. Jiangsu Medicine, 12,692. Huang, S.-R., Zhang, H.-J,., Huang, M.-J., Li, S.-Y. & Zhang, L. (1990). Cryptosportdrum infection of calves in the suburbs of Beijing. China ZoonosesJournal, 6,45. Lu, L.-A., Fan, Z.-Z., Li, C.-C. & Liu, B.-Z. (1990). Two casesof infant cryptosporidiosis. HunanMedicine, 7, 106. Luo, M.-Y., Wang, Q.-N. & Zhen,, X.-P. (1988). Cryptosporidial infection and enteritis. Chmese 3ournal of Internal Medicine, 27,686-688. Su, Q.-P., Chen, D.-G., Zhao, Z.-G., Hua, Y., Hua, X.-L., Chen, S., Huang, M. H., Wan, J.-Q., Guo, Y.-H. & Li, S.M. (1989). Cryptosporidiosis from infants in Fuzhou region. China ZoonosesJournal, 5,35-36.
TRANSACTIONS OFTHEROYAL
Su, L.-Y., Chen, X.-C., Li, Y.-H. & Liu, J. H. (1990). Casereport of infant cryptosporidiosis. China Journal of Parasitic : DiseasesControl, 3, 154. Unaar, B. L. I’.. Soave, R., Faver, R. & Nash, T. E. (1986). Enzyme immunoassay detection of immunoglobulin ‘M and G antibodies to Cryptosporidium in immunocompetent and immunocomeromised oersons. 7ouwtal oflnfectious Diseases. lS3,570-578’. ” ’ ’ Yao, F.-B. & Chen, Y.-G. (1989). Preliminary study on the effir cacy of garlicin in treatment of childhood cryptosporidiosis. 3ournal of Xuzhou Medical College, 9,56.
Zhu, F.-C., Hun, Z.-X. & Wang, F.-P. (1990). A case-control studv of childhood crvutosuoridiosis in Wuhu. China Public __ _ Health, 6,503-504.
Zu, S.-X. & Du, M.-W. (1987). Human cryptosporidiosis seen in China.3ouwaal ofAnhuiMedica1 University, 22,276. Zuo, Y.-X., Chen, F.-Q. & Fang, L. (1990). Human and domestic cryptosporidiosis in Yunnan and experimental infection study. China ZoonosesJournal, 6, 34-35. Received 5 February 1992; revised 16 April 1992; accepted for publication 16 April I992
SOCIETY OF TROPICAL MEDICINE ANDHYGIENE (1992) 86, 640
/Short Report 1 Rapid purification and concentration technique for Cryptosporidium parvum oocysts M. Lyagoubi, A. Datry, I. Malet, M. Danis and M. Gentilini Laboratoire, de Parasitologie, Uniti INSERM 313, CHU Piti&Salpet&re, 91 Boulevard de l’H6pita1, 75534 Paris ckdex 13, France Purification of faecal Cyptosporidium oocysts is necessary for infection of cell cultures (in vitro models) and of laboratory animals (in vivo models). For immunological studies purified parasites are essential. C. parzwm oocysts are separated from patients’ stools or from the stools of naturallv or exuerimentallv infected animals. Several techniques are used to isolate the oocysts: PercoB@ gradients (DULSKI & TURNER. 1988). sucrose gradientsu(CURREN? & LONG, 1983) and’saturated NaCl solution (DATRY et al., 1989). In order to obtain a sufficient number of oocysts with these techniques, very long and fastidious manipulations are needed. We propose a technique in which stools are previously filtered through a wide-mesh screen and a Blutter cloth (40 urn pore4 and conserved in potassium bichromate. The stools, diluted 50% in potassium bichromate, are filtered through a moistened hydrophylic cotton column (height 5 cm, diameter 2 cm); 50 ml of this material can be filtered through each column, before it becomes saturated. The filtrate is washed in phosphate-buffered saline (PBS) and centrifuged twice (10 min at 1500 g). The pellet obtained is suspended in 3 ml of PBS and the suspension is placed on a discontinuous angiographine gradient (meglumine sodium amidotrizoate, Radioselectan@, Schering Laboratories). This discon-
tinuous gradient is obtained by pouring 3 ml of a solution containing 3 parts Radioselectan@, 1 part human serum and 3 parts MEM medium on to 3 ml of a solution containing the same ingredients in the proportions 4: 1:2. The tubes are centrifueed at 12 000 P for 10 min in a Beckman horizontal ro’ior centrifuge.“Two dense rings are formed between the 2 liquid-liquid interfaces. We have recovered from the liquid column located above the lower ring, 90% of the oocysts that were introduced; the quality of this purification method is excellent. In the residue, we find bacteria, yeasts and other small debris. The method permits the detection of Cyptosporidium oocysts in a stool-bichromate solution containing as few as 20 oocvstsiml. This technique has been previously used to purify leishmanial amastigotes (MONIOUR et al., 1984). It is useful because of its rapidity and ease compared with the usual techniques and, above all, the quality and yield of the end product. The oocysts obtained are pure, viable and suitable, after excystation and sporozoite liberation, for the infection of cell cultures. References Current, L. W. & Long, I’. L. (1983). Development of human and calf Ctyptosporidium in chicken embryos. 3oumal of Infectious Diseases, 148, 1108-l 113. Datry, A., Danis, M. & Gentilini, M. (1989). Developpement complet de Cryptosporidium en culture cellulaire. Medicine et Sciences, 5, 762-766.
Dulski, I’. & Turner, M. (1988). The purification of sporocysts and sporozoites from Eimena tenella oocysts using PercoIl” density gradients. Avian Diseases, 32, 235-239. Monjour, L., Vouldoukis, I., Brandicourt, 0.) Mazier, D., Alfred, C., Ploton, I. & Gentilini, M. (1984). Rapid large-scale production and isolation of Leishmania amastigotes. Annals of Tropical hfedicine and Parasitology, 78, 423-425. Received 5 December 1990; revised 12 February 1991; accepted for publication 13 February 1992 [subsequently unavoidably delayed by mailing problems]