• 1.

    Dubey JP, 2010. Toxoplasma gondii infections in chickens (Gallus domesticus): prevalence, clinical disease, diagnosis and public health significance. Zoonoses Public Health 57: 6073.

    • Search Google Scholar
    • Export Citation
  • 2.

    Hill DE, Chirukandoth S, Dubey JP, 2005. Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev 6: 4161.

  • 3.

    Weiss LM, Dubey JP, 2009. Toxoplasmosis: a history of clinical observations. Int J Parasitol 39: 895901.

  • 4.

    Tenter AM, Heckeroth AR, Weiss LM, 2000. Toxoplasma gondii: from animals to humans. Int J Parasitol 30: 12171258.

  • 5.

    Nissapatorn V, Lee C, Quek KF, Leong CL, Mahmud R, Abdullah KA, 2004. Toxoplasmosis in HIV/AIDS patients: a current situation. Jpn J Infect Dis 57: 160165.

    • Search Google Scholar
    • Export Citation
  • 6.

    Levine GI, 1991. Parasitic diseases. Diseases associated with acquired immunodeficiency syndrome. Prim Care 18: 129152.

  • 7.

    Holliman RE, 1988. Toxoplasmosis and the acquired immune deficiency syndrome. J Infect 16: 121128.

  • 8.

    Machala L, Kodym P, Malý M, Geleneky M, Beran O, Jilich D, 2015. Toxoplasmosis in immunocompromised patients. Epidemiol Mikrobiol Imunol 64: 5965.

  • 9.

    Hide G et al. 2009. Evidence for high levels of vertical transmission in Toxoplasma gondii. Parasitology 136: 18771885.

  • 10.

    Balsari A, Poli G, Molina V, Dovis M, Petruzzelli E, Boniolo A, Rolleri E, 1980. ELISA for toxoplasma antibody detection: a comparison with other serodiagnostic tests. J Clin Pathol 33: 640643.

    • Search Google Scholar
    • Export Citation
  • 11.

    Piergili Fioretti D, 2004. Problems and limitations of conventional and innovative methods for the diagnosis of toxoplasmosis in humans and animals [article in Italian]. Parassitologia 46: 177181.

    • Search Google Scholar
    • Export Citation
  • 12.

    Liu Q, Wang ZD, Huang SY, Zhu XQ, 2015. Diagnosis of toxoplasmosis and typing of Toxoplasma gondii. Parasit Vectors 8: 292.

  • 13.

    Wallon M, Franck J, Thulliez P, Huissoud C, Peyron F, Garcia-Meric P, Kieffer F, 2010. Accuracy of real-time polymerase chain reaction for Toxoplasma gondii in amniotic fluid. Obstet Gynecol 115: 727733.

    • Search Google Scholar
    • Export Citation
  • 14.

    Switaj K, Master A, Skrzypczak M, Zaborowski P, 2005. Recent trends in molecular diagnostics for Toxoplasma gondii infections. Clin Microbiol Infect 11: 170176.

    • Search Google Scholar
    • Export Citation
  • 15.

    Hohlfeld P, Daffos F, Costa JM, Thulliez P, Forestier F, Vidaud M, 1994. Prenatal diagnosis of congenital toxoplasmosis with a polymerase-chain-reaction test on amniotic fluid. N Engl J Med 331: 695699.

    • Search Google Scholar
    • Export Citation
  • 16.

    Reischl U, Bretagne S, Krüger D, Ernault P, Costa J-M, 2003. Comparison of two DNA targets for the diagnosis of toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infect Dis 3: 7.

    • Search Google Scholar
    • Export Citation
  • 17.

    Burg JL, Grover CM, Pouletty P, Boothroyd JC, 1989. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol 27: 17871792.

    • Search Google Scholar
    • Export Citation
  • 18.

    Homan WL, Vercammen M, De Braekeleer J, Verschueren H, 2000. Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol 30: 6975.

    • Search Google Scholar
    • Export Citation
  • 19.

    Cassaing S, Bessières MH, Berry A, Berrebi A, Fabre R, Magnaval JF, 2006. Comparison between two amplification sets for molecular diagnosis of toxoplasmosis by real-time PCR. J Clin Microbiol 44: 720724.

    • Search Google Scholar
    • Export Citation
  • 20.

    Belaz S, Gangneux J-P, Dupretz P, Guiguen C, Robert-Gangneux F, 2015. A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR. J Clin Microbiol 53: 12941300.

    • Search Google Scholar
    • Export Citation
  • 21.

    Bourdin C, Busse A, Kouamou E, Touafek F, Bodaghi B, Le Hoang P, Mazier D, Paris L, Fekka A, 2014. PCR-based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis. J Clin Microbiol 52: 39873991.

    • Search Google Scholar
    • Export Citation
  • 22.

    Dupon M, Cazenave J, Pellegrin JL, Ragnaud JM, Cheyrou A, Fischer I, Leng B, Lacut JY, 1995. Detection of Toxoplasma gondii by PCR and tissue culture in cerebrospinal fluid and blood of human immunodeficiency virus-seropositive patients. J Clin Microbiol 33: 24212426.

    • Search Google Scholar
    • Export Citation
  • 23.

    Montoya JG, 2002. Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J Infect Dis 185 (Suppl 1): S73S82.

  • 24.

    Lin MH, Chen TC, Kuo TT, Tseng CC, Tseng CP, 2000. Real-time PCR for quantitative detection of Toxoplasma gondii. J Clin Microbiol 38: 41214125.

  • 25.

    Fitzwater S, Calderon M, Lafuente C, Galdos-Cardenas G, Ferrufino L, Verastegui M, Gilman RH, Bern C; Chagas Disease Working Group in Peru and Bolivia, 2008. Polymerase chain reaction for chronic Trypanosoma cruzi infection yields higher sensitivity in blood clot than buffy coat or whole blood specimens. Am J Trop Med Hyg 79: 768770.

    • Search Google Scholar
    • Export Citation
  • 26.

    McCulloch E, Ramage G, Jones B, Warn P, Kirkpatrick WR, Patterson TF, Williams C, 2009. Don’t throw your blood clots away: use of blood clot may improve sensitivity of PCR diagnosis in invasive aspergillosis. J Clin Pathol 62: 539541.

    • Search Google Scholar
    • Export Citation
  • 27.

    Duffy T, Bisio M, Altcheh J, Burgos JM, Diez M, Levin MJ, Favaloro RR, Freilij H, Schijman AG, 2009. Accurate real-time PCR strategy for monitoring bloodstream parasitic loads in Chagas disease patients. PLoS Negl Trop Dis 3: e419.

    • Search Google Scholar
    • Export Citation
  • 28.

    Sterkers Y et al. 2010. Multicentric comparative analytical performance study for molecular detection of low amounts of Toxoplasma gondii from simulated specimens. J Clin Microbiol 48: 32163222.

    • Search Google Scholar
    • Export Citation
  • 29.

    Wahab T, Edvinsson B, Palm D, Lindh J, 2010. Comparison of the AF146527 and B1 repeated elements, two real-time PCR targets used for detection of Toxoplasma gondii. J Clin Microbiol 48: 591592.

    • Search Google Scholar
    • Export Citation
  • 30.

    Alfonso Y et al. 2009. Molecular diagnosis of Toxoplasma gondii infection in cerebrospinal fluid from AIDS patients. Cerebrospinal Fluid Res 6: 2.

    • Search Google Scholar
    • Export Citation
  • 31.

    Grigg ME, Boothroyd JC, 2001. Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene. J Clin Microbiol 39: 398400.

    • Search Google Scholar
    • Export Citation
  • 32.

    Kompalic-Cristo A, Frotta C, Suárez-Mutis M, Fernandes O, Britto C, 2007. Evaluation of a real-time PCR assay based on the repetitive B1 gene for the detection of Toxoplasma gondii in human peripheral blood. Parasitol Res 101: 619625.

    • Search Google Scholar
    • Export Citation
  • 33.

    Bastien P, 2002. Molecular diagnosis of toxoplasmosis. Trans R Soc Trop Med Hyg 96: S205S215.

  • 34.

    Bustin SA et al. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611622.

    • Search Google Scholar
    • Export Citation
 
 
 
 

 

 
 

 

 

 

 

 

 

Development of a Novel Protocol Based on Blood Clot to Improve the Sensitivity of qPCR Detection of Toxoplasma gondii in Peripheral Blood Specimens

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  • 1 Laboratorio de Investigación en Enfermedades Infecciosas, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia, Lima, Peru;
  • | 2 Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina;
  • | 3 Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland;
  • | 4 Tulane University Medical Center, New Orleans, Louisiana;
  • | 5 Hospital Regional de Loreto “Felipe Santiago Arriola Iglesias,” Iquitos, Peru;
  • | 6 Infectious Diseases and Tropical Medicine Unit, Hospital Nacional Dos de Mayo, Lima, Peru;
  • | 7 School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, Arizona

Quantitative polymerase chain reaction (qPCR) for Toxoplasma gondii multicopy genes has emerged as a promising strategy for sensitive detection of parasite DNA. qPCR can be performed from blood samples, which are minimally invasive to collect. However, there is no consensus about what type of blood specimen yields the best sensitivity. The development of a novel protocol for qPCR detection of T. gondii using blood clot, involving an appropriate DNA extraction method and the use of an internal amplification control to monitor the reaction is presented in the current study. Assays directed to the B1 and REP529 genes were performed in spiked specimens of whole blood, guanidine–ethylenediaminetetraacetic acid blood, and clot. The clot-based qPCR was shown to be more sensitive when compared with other types of specimens, detecting five and 0.05 T. gondii genomes, using B1 and REP529 targets, respectively. Finally, a comparative analysis with samples from HIV patients with clinical suspicion of toxoplasmosis was performed, demonstrating the detection of four positive suspected cases with clots compared with only one using guanidine–ethylenediaminetetraacetic acid blood. The high analytical sensitivity and the cost-effective advantages offered by clot supports this methodology as a good laboratory tool to monitor parasite burden.

Author Notes

Address correspondence to Renzo Gutierrez-Loli, Laboratorio de Investigación en Enfermedades Infecciosas, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia, Av. Honorio Delgado 430, SMP 15102, Lima, Peru. E-mail: renzo.gutierrez@upch.pe

Financial support: This work was partially funded with the support of “Programa Nacional de Innovación para la Productividad y Competitividad” (Innóvate Perú), contract No. 137-PNICP-PIAP-2015. R. G. is supported by a training grant from NIH-Fogarty (2D43TW007120-11A1). Dr. Gilman’s NIH grant 1D43TW010074-01 supported the training of many of the Peruvian and Bolivian authors and working group members.

Prior presentation of findings: A poster titled “Real-time PCR strategy for detection of Toxoplasma gondii from peripheral blood clot” (Abstract Number: 3331-1882) was presented in November 2017 at the Annual Meeting of the American Society of Tropical Medicine and Hygiene in Baltimore, MD.

Authors’ addresses: Renzo Gutierrez-Loli, Cusi Ferradas, Andrea Diestra, Aliki Traianou, Holger Mayta, Maritza Calderon, and Jaeson S. Calla-Choque, Laboratorio de Investigación en Enfermedades Infecciosas, Laboratorios de Investigación y Desarrollo, Universidad Peruana Cayetano Heredia, Lima, Peru, E-mails: renzo.gutierrez@upch.pe, cusi.ferradas@upch.pe, andreadiestra13@gmail.com, aliki.traianou@gmail.com, holger.mayta@upch.pe, mmcalderons@yahoo.es, and jcalla@ucsd.edu. Natalie Bowman, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, E-mail: natalie.bowman@gmail.com. Jeroen Bok, Hannah Steinberg, and Robert H. Gilman, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, E-mails: jeroenbok87@gmail.com, hannahsteinberg08@gmail.com, and gilmanbob@gmail.com. Melissa Reimer-McAtee, Tulane University Medical Center, New Orleans, LA, E-mail: mreimer2@tulane.edu. Cesar Ramal, Hospital Regional de Loreto “Felipe Santiago Arriola Iglesias,” Iquitos, Peru, E-mail: ramalasayag@yahoo.fr. Eduardo Ticona, Infectious Diseases and Tropical Medicine Unit, Hospital Nacional Dos de Mayo, Lima, Peru, E-mail: eticonacrg@gmail.com. Charles Sterling, School of Animal and Comparative Biomedical Sciences, University of Arizona, Tucson, AZ, E-mail: csterlin@email.arizona.edu.

Working Group: Linda Chanamé Pinedo, Gaston Valencia, Lenny Sanchez, Edith Málaga, Deanna Zhu, Juan Jiménez, Caryn Bern, Noelia Angulo, Francesca Schiaffino, Janet Acosta, Meredith Holtz, Daniel Clark, Taryn Clark, Grace Trompeter, Jeong Choi, Omar Gandarilla, Mauricio Dorn, Enzo Fortuny, Gerson Galdos, and Roni Colanzi.

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