Abstract
Cryptosporidium parvum is an important pathogen that causes diarrhea in virtually all human populations. Improved diagnostic methods are needed to understand the risk factors, modes of transmission, and impact of cryptosporidiosis. In the present study, we fluorescently labeled and counted C. parvum oocysts by flow cytometry (FC) and developed a simple and efficient method of processing human stool samples for FC analysis. Formed stool (suspended in phosphate-buffered saline) from an asymptomatic, healthy individual was seeded with known concentrations of oocysts, and oocysts were labeled with a cell wall-specific monoclonal antibody and detected by FC. The method described herein resulted in a mean oocyst recovery rate of 45% +/- 16% (median, 42%), which consistently yielded a fourfold increase in sensitivity compared to direct fluorescent-antibody assay of seeded stool samples. However, in many instances, FC detected as few as 10(3) oocysts per ml. Thus, FC provides a reproducible and sensitive method for C. parvum oocyst detection.
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- Arrowood M. J., Hurd M. R., Mead J. R. A new method for evaluating experimental cryptosporidial parasite loads using immunofluorescent flow cytometry. J Parasitol. 1995 Jun;81(3):404–409. [PubMed] [Google Scholar]
- Arrowood M. J., Sterling C. R. Comparison of conventional staining methods and monoclonal antibody-based methods for Cryptosporidium oocyst detection. J Clin Microbiol. 1989 Jul;27(7):1490–1495. doi: 10.1128/jcm.27.7.1490-1495.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Current W. L., Garcia L. S. Cryptosporidiosis. Clin Microbiol Rev. 1991 Jul;4(3):325–358. doi: 10.1128/cmr.4.3.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DuPont H. L., Chappell C. L., Sterling C. R., Okhuysen P. C., Rose J. B., Jakubowski W. The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med. 1995 Mar 30;332(13):855–859. doi: 10.1056/NEJM199503303321304. [DOI] [PubMed] [Google Scholar]
- Flanigan T., Whalen C., Turner J., Soave R., Toerner J., Havlir D., Kotler D. Cryptosporidium infection and CD4 counts. Ann Intern Med. 1992 May 15;116(10):840–842. doi: 10.7326/0003-4819-116-10-840. [DOI] [PubMed] [Google Scholar]
- Goldstein S. T., Juranek D. D., Ravenholt O., Hightower A. W., Martin D. G., Mesnik J. L., Griffiths S. D., Bryant A. J., Reich R. R., Herwaldt B. L. Cryptosporidiosis: an outbreak associated with drinking water despite state-of-the-art water treatment. Ann Intern Med. 1996 Mar 1;124(5):459–468. doi: 10.7326/0003-4819-124-5-199603010-00001. [DOI] [PubMed] [Google Scholar]
- Goodgame R. W., Genta R. M., White A. C., Chappell C. L. Intensity of infection in AIDS-associated cryptosporidiosis. J Infect Dis. 1993 Mar;167(3):704–709. doi: 10.1093/infdis/167.3.704. [DOI] [PubMed] [Google Scholar]
- Kehl K. S., Cicirello H., Havens P. L. Comparison of four different methods for detection of Cryptosporidium species. J Clin Microbiol. 1995 Feb;33(2):416–418. doi: 10.1128/jcm.33.2.416-418.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kotler D. P., Francisco A., Clayton F., Scholes J. V., Orenstein J. M. Small intestinal injury and parasitic diseases in AIDS. Ann Intern Med. 1990 Sep 15;113(6):444–449. doi: 10.7326/0003-4819-113-6-444. [DOI] [PubMed] [Google Scholar]
- Mac Kenzie W. R., Hoxie N. J., Proctor M. E., Gradus M. S., Blair K. A., Peterson D. E., Kazmierczak J. J., Addiss D. G., Fox K. R., Rose J. B. A massive outbreak in Milwaukee of cryptosporidium infection transmitted through the public water supply. N Engl J Med. 1994 Jul 21;331(3):161–167. doi: 10.1056/NEJM199407213310304. [DOI] [PubMed] [Google Scholar]
- MacPherson D. W., McQueen R. Cryptosporidiosis: multiattribute evaluation of six diagnostic methods. J Clin Microbiol. 1993 Feb;31(2):198–202. doi: 10.1128/jcm.31.2.198-202.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mølbak K., Højlyng N., Gottschau A., Sá J. C., Ingholt L., da Silva A. P., Aaby P. Cryptosporidiosis in infancy and childhood mortality in Guinea Bissau, west Africa. BMJ. 1993 Aug 14;307(6901):417–420. doi: 10.1136/bmj.307.6901.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Roberts W. G., Green P. H., Ma J., Carr M., Ginsberg A. M. Prevalence of cryptosporidiosis in patients undergoing endoscopy: evidence for an asymptomatic carrier state. Am J Med. 1989 Nov;87(5):537–539. doi: 10.1016/s0002-9343(89)80610-2. [DOI] [PubMed] [Google Scholar]
- Vesey G., Hutton P., Champion A., Ashbolt N., Williams K. L., Warton A., Veal D. Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water. Cytometry. 1994 May 1;16(1):1–6. doi: 10.1002/cyto.990160102. [DOI] [PubMed] [Google Scholar]
- Vesey G., Slade J. S., Byrne M., Shepherd K., Dennis P. J., Fricker C. R. Routine monitoring of Cryptosporidium oocysts in water using flow cytometry. J Appl Bacteriol. 1993 Jul;75(1):87–90. doi: 10.1111/j.1365-2672.1993.tb03413.x. [DOI] [PubMed] [Google Scholar]
- Weber R., Bryan R. T., Bishop H. S., Wahlquist S. P., Sullivan J. J., Juranek D. D. Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J Clin Microbiol. 1991 Jul;29(7):1323–1327. doi: 10.1128/jcm.29.7.1323-1327.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]