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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1989 Aug;27(8):1787–1792. doi: 10.1128/jcm.27.8.1787-1792.1989

Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction.

J L Burg 1, C M Grover 1, P Pouletty 1, J C Boothroyd 1
PMCID: PMC267672  PMID: 2768467

Abstract

We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

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Selected References

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  1. Abbott M. A., Poiesz B. J., Byrne B. C., Kwok S., Sninsky J. J., Ehrlich G. D. Enzymatic gene amplification: qualitative and quantitative methods for detecting proviral DNA amplified in vitro. J Infect Dis. 1988 Dec;158(6):1158–1169. doi: 10.1093/infdis/158.6.1158. [DOI] [PubMed] [Google Scholar]
  2. Armstrong D., Gold J. W., Dryjanski J., Whimbey E., Polsky B., Hawkins C., Brown A. E., Bernard E., Kiehn T. E. Treatment of infections in patients with the acquired immunodeficiency syndrome. Ann Intern Med. 1985 Nov;103(5):738–743. doi: 10.7326/0003-4819-103-5-738. [DOI] [PubMed] [Google Scholar]
  3. Bhagavati S., Ehrlich G., Kula R. W., Kwok S., Sninsky J., Udani V., Poiesz B. J. Detection of human T-cell lymphoma/leukemia virus type I DNA and antigen in spinal fluid and blood of patients with chronic progressive myelopathy. N Engl J Med. 1988 May 5;318(18):1141–1147. doi: 10.1056/NEJM198805053181801. [DOI] [PubMed] [Google Scholar]
  4. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  5. Conley F. K., Jenkins K. A., Remington J. S. Toxoplasma gondii infection of the central nervous system. Use of the peroxidase-antiperoxidase method to demonstrate toxoplasma in formalin fixed, paraffin embedded tissue sections. Hum Pathol. 1981 Aug;12(8):690–698. doi: 10.1016/s0046-8177(81)80170-0. [DOI] [PubMed] [Google Scholar]
  6. Cornelissen A. W., Overdulve J. P., van der Ploeg M. Determination of nuclear DNA of five eucoccidian parasites, Isospora (Toxoplasma) gondii, Sarcocystis cruzi, Eimeria tenella, E. acervulina and Plasmodium berghei, with special reference to gamontogenesis and meiosis in I. (T.) gondii. Parasitology. 1984 Jun;88(Pt 3):531–553. doi: 10.1017/s0031182000054792. [DOI] [PubMed] [Google Scholar]
  7. Daffos F., Forestier F., Capella-Pavlovsky M., Thulliez P., Aufrant C., Valenti D., Cox W. L. Prenatal management of 746 pregnancies at risk for congenital toxoplasmosis. N Engl J Med. 1988 Feb 4;318(5):271–275. doi: 10.1056/NEJM198802043180502. [DOI] [PubMed] [Google Scholar]
  8. Demmler G. J., Buffone G. J., Schimbor C. M., May R. A. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis. 1988 Dec;158(6):1177–1184. doi: 10.1093/infdis/158.6.1177. [DOI] [PubMed] [Google Scholar]
  9. Desmonts G., Daffos F., Forestier F., Capella-Pavlovsky M., Thulliez P., Chartier M. Prenatal diagnosis of congenital toxoplasmosis. Lancet. 1985 Mar 2;1(8427):500–504. doi: 10.1016/s0140-6736(85)92096-3. [DOI] [PubMed] [Google Scholar]
  10. Duggan D. B., Ehrlich G. D., Davey F. P., Kwok S., Sninsky J., Goldberg J., Baltrucki L., Poiesz B. J. HTLV-I-induced lymphoma mimicking Hodgkin's disease. Diagnosis by polymerase chain reaction amplification of specific HTLV-I sequences in tumor DNA. Blood. 1988 Apr;71(4):1027–1032. [PubMed] [Google Scholar]
  11. Farkash A. E., Maccabee P. J., Sher J. H., Landesman S. H., Hotson G. CNS toxoplasmosis in acquired immune deficiency syndrome: a clinical-pathological-radiological review of 12 cases. J Neurol Neurosurg Psychiatry. 1986 Jul;49(7):744–748. doi: 10.1136/jnnp.49.7.744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farzadegan H., Polis M. A., Wolinsky S. M., Rinaldo C. R., Jr, Sninsky J. J., Kwok S., Griffith R. L., Kaslow R. A., Phair J. P., Polk B. F. Loss of human immunodeficiency virus type 1 (HIV-1) antibodies with evidence of viral infection in asymptomatic homosexual men. A report from the Multicenter AIDS Cohort Study. Ann Intern Med. 1988 Jun;108(6):785–790. doi: 10.7326/0003-4819-108-6-785. [DOI] [PubMed] [Google Scholar]
  13. Fauci A. S., Masur H., Gelmann E. P., Markham P. D., Hahn B. H., Lane H. C. NIH conference. The acquired immunodeficiency syndrome: an update. Ann Intern Med. 1985 Jun;102(6):800–813. doi: 10.7326/0003-4819-102-6-800. [DOI] [PubMed] [Google Scholar]
  14. Frenkel J. K., Nelson B. M., Arias-Stella J. Immunosuppression and toxoplasmic encephalitis: clinical and experimental aspects. Hum Pathol. 1975 Jan;6(1):97–111. doi: 10.1016/s0046-8177(75)80111-0. [DOI] [PubMed] [Google Scholar]
  15. Koppel B. S., Wormser G. P., Tuchman A. J., Maayan S., Hewlett D., Jr, Daras M. Central nervous system involvement in patients with acquired immune deficiency syndrome (AIDS). Acta Neurol Scand. 1985 May;71(5):337–353. doi: 10.1111/j.1600-0404.1985.tb03211.x. [DOI] [PubMed] [Google Scholar]
  16. Kwok S., Mack D. H., Mullis K. B., Poiesz B., Ehrlich G., Blair D., Friedman-Kien A., Sninsky J. J. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J Virol. 1987 May;61(5):1690–1694. doi: 10.1128/jvi.61.5.1690-1694.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Li H. H., Gyllensten U. B., Cui X. F., Saiki R. K., Erlich H. A., Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature. 1988 Sep 29;335(6189):414–417. doi: 10.1038/335414a0. [DOI] [PubMed] [Google Scholar]
  18. Loche M., Mach B. Identification of HIV-infected seronegative individuals by a direct diagnostic test based on hybridisation to amplified viral DNA. Lancet. 1988 Aug 20;2(8608):418–421. doi: 10.1016/s0140-6736(88)90412-6. [DOI] [PubMed] [Google Scholar]
  19. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McCabe R. E., Remington J. S. The diagnosis and treatment of toxoplasmosis. Eur J Clin Microbiol. 1983 Apr;2(2):95–104. doi: 10.1007/BF02001573. [DOI] [PubMed] [Google Scholar]
  21. Mills J. Pneumocystis carinii and Toxoplasma gondii infections in patients with AIDS. Rev Infect Dis. 1986 Nov-Dec;8(6):1001–1011. doi: 10.1093/clinids/8.6.1001. [DOI] [PubMed] [Google Scholar]
  22. Morris B. J., Flanagan J. L., McKinnon K. J., Nightingale B. N. Papillomavirus screening of cervical lavages by polymerase chain reaction. Lancet. 1988 Dec 10;2(8624):1368–1368. doi: 10.1016/s0140-6736(88)90906-3. [DOI] [PubMed] [Google Scholar]
  23. Murakawa G. J., Zaia J. A., Spallone P. A., Stephens D. A., Kaplan B. E., Wallace R. B., Rossi J. J. Direct detection of HIV-1 RNA from AIDS and ARC patient samples. DNA. 1988 May;7(4):287–295. doi: 10.1089/dna.1988.7.287. [DOI] [PubMed] [Google Scholar]
  24. Navia B. A., Petito C. K., Gold J. W., Cho E. S., Jordan B. D., Price R. W. Cerebral toxoplasmosis complicating the acquired immune deficiency syndrome: clinical and neuropathological findings in 27 patients. Ann Neurol. 1986 Mar;19(3):224–238. doi: 10.1002/ana.410190303. [DOI] [PubMed] [Google Scholar]
  25. Ou C. Y., Kwok S., Mitchell S. W., Mack D. H., Sninsky J. J., Krebs J. W., Feorino P., Warfield D., Schochetman G. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science. 1988 Jan 15;239(4837):295–297. doi: 10.1126/science.3336784. [DOI] [PubMed] [Google Scholar]
  26. Pfefferkorn E. R., Pfefferkorn L. C., Colby E. D. Development of gametes and oocysts in cats fed cysts derived from cloned trophozoites of Toxoplasma gondii. J Parasitol. 1977 Feb;63(1):158–159. [PubMed] [Google Scholar]
  27. Pfefferkorn E. R., Pfefferkorn L. C. Toxoplasma gondii: isolation and preliminary characterization of temperature-sensitive mutants. Exp Parasitol. 1976 Jun;39(3):365–376. doi: 10.1016/0014-4894(76)90040-0. [DOI] [PubMed] [Google Scholar]
  28. Rayfield M., De Cock K., Heyward W., Goldstein L., Krebs J., Kwok S., Lee S., McCormick J., Moreau J. M., Odehouri K. Mixed human immunodeficiency virus (HIV) infection in an individual: demonstration of both HIV type 1 and type 2 proviral sequences by using polymerase chain reaction. J Infect Dis. 1988 Dec;158(6):1170–1176. doi: 10.1093/infdis/158.6.1170. [DOI] [PubMed] [Google Scholar]
  29. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  30. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  31. Shibata D. K., Arnheim N., Martin W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med. 1988 Jan 1;167(1):225–230. doi: 10.1084/jem.167.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shibata D., Martin W. J., Appleman M. D., Causey D. M., Leedom J. M., Arnheim N. Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis. 1988 Dec;158(6):1185–1192. doi: 10.1093/infdis/158.6.1185. [DOI] [PubMed] [Google Scholar]
  33. Snider W. D., Simpson D. M., Nielsen S., Gold J. W., Metroka C. E., Posner J. B. Neurological complications of acquired immune deficiency syndrome: analysis of 50 patients. Ann Neurol. 1983 Oct;14(4):403–418. doi: 10.1002/ana.410140404. [DOI] [PubMed] [Google Scholar]
  34. Twigg A. J., Sherratt D. Trans-complementable copy-number mutants of plasmid ColE1. Nature. 1980 Jan 10;283(5743):216–218. doi: 10.1038/283216a0. [DOI] [PubMed] [Google Scholar]
  35. Ware P. L., Kasper L. H. Strain-specific antigens of Toxoplasma gondii. Infect Immun. 1987 Mar;55(3):778–783. doi: 10.1128/iai.55.3.778-783.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilson C. B., Remington J. S. Activity of human blood leukocytes against Toxoplasma gondii. J Infect Dis. 1979 Dec;140(6):890–895. doi: 10.1093/infdis/140.6.890. [DOI] [PubMed] [Google Scholar]
  37. Wilson C. B., Tsai V., Remington J. S. Failure to trigger the oxidative metabolic burst by normal macrophages: possible mechanism for survival of intracellular pathogens. J Exp Med. 1980 Feb 1;151(2):328–346. doi: 10.1084/jem.151.2.328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Young L. S. Management of opportunistic infections complicating the acquired immunodeficiency syndrome. Med Clin North Am. 1986 May;70(3):677–692. doi: 10.1016/s0025-7125(16)30946-4. [DOI] [PubMed] [Google Scholar]

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