PCR-based Diagnosis of Toxoplasma Parasite in Ocular Infections Having Clinical Indications of Toxoplasmosis
Background: The diagnosis of ocular toxoplasmosis is mainly based on clinical features. However, ocular fluid testing by PCR may be very helpful for approval or rejection of this etiology. In this study, we utilized a nested-PCR technique, targeting the B1 partial sequence to analyze the aqueous and vitreous samples for evaluating the presence of the Toxoplasma DNA.
Methods: Fifty aqueous or vitreous humor samples were obtained from patients with clinical features of ocular toxoplasmosis admitted to ophthalmology hospitals and clinics in Iran, within 2014. The samples were subsequently subjected to DNA extraction and purification. For nested amplification of the Toxoplasma B1 gene, two primer pairs were used. The outer and inner primers are expected to produce a 193 bp and a 96 bp fragments, respectively.
Results: The first-round PCR resulted in the detection of T. gondii in 58% of samples by amplification of the expected 193bp DNA fragment. The nested-PCR using the inner primers, detected 15 additional samples from those with negative amplicons in the first round PCR (overall positivity of 88%). In addition, vitreous samples showed relatively more positive cases than aqueous humor in detection of the infection.Conclusion: The nested-PCR protocol using the B1 gene, with the high detection power, could be a useful complimentary method to clinical diagnose of ocular toxoplasmosis.
Robert-Gangneux F, Dardé ML. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev. 2012; 25(2): 264–96.
Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet. 2004; 363(9425):1965-76.
Butler NJ, Furtado JM, Winthrop KL, Smith JR. Ocular toxoplasmosis II: clinical features, pathology and management. Clin Exp Ophthalmol. 2013; 41(1): 95-108.
Jones JL, Holland GN. Annual burden of ocular toxoplasmosis in the United States. Am J Trop Med Hyg. 2010; 82(3): 464-465.
Soheilian M, Heidari K, Yazdani S, Shahsavari M, Ahmadieh H, Dehghan M. Patterns of uveitis in a tertiary eye care center in Iran. Ocul Immunol Inflamm. 2004; 12(4): 297-310.
Kianersi F. Clinical patterns of uveitis in an Iranian tertiary eye care center. Bina J Oph-thalmol. 2005; 10 (2): 147-154.
Holland GN. Ocular toxoplasmosis: the influence of patient age. Mem Inst Oswaldo Cruz. 2009; 104(2): 351-7.
Ivovic V, Vujanic M, Zivkovic T, Klun I, Djurkovic-Djakovic O. (2012). Molecular detection and genotyping of Toxoplasma gondii from clinical samples. In: Djurković Djaković O editor. Toxoplasmosis-recent advances. InTech, DOI: 10.5772/50830.
Burg JL, Grover CM, Pouletty P, Boothroyd JC. Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol. 1989; 27(8): 1787-92.
Jones CD, Okhravi N, Adamson P, Tasker S, Lightman S. Comparison of PCR detection methods for B1, P30, and 18S rDNA genes of T. gondii in aqueous humor. Invest Ophthalmol Vis Sci 2000; 41(3): 634-44.
Calderaro A, Piccolo G, Gorrini C, Peruzzi S, Zerbini L, Bommezzadri S, Dettori G, Chezzi C. Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii. Acta Biomed. 2006; 77(2): 75-80.
Homan WL, Vercammen M, De Braekeleer J, Verschueren H. 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. 2000; 30(1): 69-75.
Wahab T, Edvinsson B, Palm D, Lindh J. Comparison of the AF146527 and B1 repeated elements, two real-time PCR targets used for detection of Toxoplasma gondii. J Clin Microbiol. 2010; 48(2): 591-2.
Costa JM, Bretagne S. Variation of B1 gene and AF146527 repeat element copy numbers according to Toxoplasma gondii strains assessed using real-time quantitative PCR. J Clin Microbiol. 2012; 50(4): 1452-54.
15- Okay TS, Yamamoto L, Oliveira LC, Manuli ER, Andrade Jr HF, Del Negro GMB. Significant performance variation among PCR systems in diagnosing congenital toxoplasmosis in São Paulo, Brazil: analysis of 467 amniotic fluid samples. Clinics. 2009; 64(3): 171-6.
Vidal JE, Colombo FA, Penalva de Oliveira AC, Focaccia R, Pereira-Chioccola VL. PCR assay using cerebrospinal fluid for diagnosis of cerebral toxoplasmosis in Brazilian AIDS patients. J Clin Microbiol. 2004; 42(10): 4765–68.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed., Cold Spring Harbor Laboratory Press, New York. 1989; 9.14–9.23.
Tabatabaei S, Soleimani M, Foroutan A, Ahmadabadi M, Zarei R, Piri N, Gordiz A. Ocular toxoplasmosis in Iran: 40 cases analysis. Int J Ophthalmol. 2011; 4(2): 199-201.
Garweg JG, de Groot-Mijnes JDF, Montoya JG. Diagnostic approach to ocular toxoplasmosis. Ocul Immunol Inflamm. 2011; 19(4): 255-61.
Talabani H, Asseraf M, Yera H, Delair E, Ancelle T, Thulliez P, Brezin AP, Dupouy-Camet J. Contributions of immunoblotting, real-time PCR, and the Goldmann-Witmer coefficient to diagnosis of atypical toxoplasmic retinochoroiditis. J Clin Microbiol. 2009; 47(7): 2131-35.
Fekkar A, Bodaghi B, Touafek F, Le Hoang P, Mazier D, Paris L. Comparison of immunoblotting, calculation of the Goldmann-Witmer coefficient, and real-time PCR using aqueous humor samples for diagnosis of ocular toxoplasmosis. J Clin Microbiol. 2008: 46(6): 1965-67.
Montoya JG, Parmley S, Liesenfeld O, Jaffe GJ, Remington JS. Use of the polymerase chain reaction for diagnosis of ocular toxoplasmosis. Ophthalmology. 1999; 106(8):1554–63.
Harper TW, Miller D, Schiffman JC, Davis JL. Polymerase chain reaction analysis of aqueous and vitreous specimens in the diagnosis of posterior segment infectious uveitis. Am J Ophthalmol. 2009; 147(1): 140–7.
|Issue||Vol 12 No 1 (2017)|
|Ocular toxoplasmosis Nested-PCR Aqueous Vitreous Iran|
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