ABSTRACT

Purpose:

To evaluate the ability of real-time quantitative PCR (qPCR) for detectingToxoplasma gondii DNA in the peripheral blood and aqueous humor of patients with toxoplasmic active focal necrotizing retinochoroiditis.

Methods:

Fifty-five patients with infectious uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were enrolled in this study. Forty-three patients had toxoplasmic active focal necrotizing retinochoroiditis, and the remaining 12 had non-toxoplasmic infectious uveitis and served as controls. qPCR analysis forT. gondii DNA was performed on the patients' peripheral blood and aqueous humor samples.

Results:

The qPCR was positive for T. gondii DNA in 37.21% (16/43) of the aqueous humor samples and 2.33% (1/43) of the peripheral blood samples; further, 16.27% (7/43) of the patients had positive results in both their blood and aqueous humor samples.

Conclusion:

qPCR was able to detect T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis in the blood as well as the aqueous humor and can help with the diagnosis of the disease.

Keywords:
Toxoplasmosis, ocular/diagnosis; Toxoplasma; Blood/parasitology; Choroiditis; Real-time polymerase techniques/methods; Aqueous humor

RESUMO

Objetivo:

Analisar o uso do PCR em tempo real (qPCR) na detecção do DNA do T. gondii no sangue periférico e no humor aquoso de pacientes com lesões de retinocoroidite focal, ativa por toxoplasmose.

Métodos:

Cinquenta e cinco pacientes com uveite infecciosa foram incluídos neste estudo. Os pacientes foram atendidos entre 2009 a 2013, no Departamento de Oftalmologia e Ciências Visuais da Universidade Federal de São Paulo. Quarenta e três pacientes tiveram o diagnóstico de lesões de retinocoroidite focal, ativa por toxoplasmose e, os outros 12 tiveram o diagnóstico de uveíte infecciosa não toxoplásmica e, por isso foram usados como grupo controle. A técnica de qPCR foi utilizada na detecção de DNA do T. gondii em amostras de sangue periférico e humor aquoso.

Resultados:

O qPCR foi positivo para o DNA do T. gondii em 37,21% (16/43) das amostras de humor aquoso, 2,33% (1/43) nas amostras de sangue periférico e, 16,27% (7/43) em ambas amostras simultaneamente.

Conclusão:

O qPCR foi capaz de detectar o DNA do T. gondii em pacientes com lesões de retinocoroidite focal, ativa por Toxoplasmose, no sangue bem como, no humor aquoso, podendo ajudar no diagnostico.

Descritores:
Toxoplasmose ocular/diagnóstico; Toxoplasma; Sangue/parasitologia; Coriodite; Reação em cadeia da polimerase em tempo real; Humor aquoso

INTRODUCTION

Toxoplasma gondii infection is the most common cause of posterior uveitis worldwide and is an important cause of ocular disease in both immunocompromised and immunocompetent individuals⁽¹1 Cordeiro CA, Moreira PR, Dutra WO, Young L, Campos WR, Oréfice F, et al. Imunologia da retinocoroidite toxoplásmica. Arq Bras Oftalmol. 2010;73(6):548-51. Comment in: Arq Bras Oftalmol. 2011;74(3):227.⁾, accounting for 30% to 50% of uveitis in different countries⁽²2 de-la-Torre A, Sauer A, Pfaff AW, Bourcier T, Brunet J, Speeg-Schatz C, et al. Severe South American ocular toxoplasmosis is associated with decreased Ifn-γ/Il-17a and increased Il-6/Il-13 intraocular levels. PLoS Negl Trop Dis. 2013;7(11):e2541.⁾.

The prevalence of T. gondii infection in adults in Brazil ranges from 50% to 80% depending on the region studied⁽¹1 Cordeiro CA, Moreira PR, Dutra WO, Young L, Campos WR, Oréfice F, et al. Imunologia da retinocoroidite toxoplásmica. Arq Bras Oftalmol. 2010;73(6):548-51. Comment in: Arq Bras Oftalmol. 2011;74(3):227.^,33 Garcia JL, Navarro IT, Ogawa L, de Oliveira RC, de Faria Garcia SM, Leite J. Soroepidemiologia da toxoplasmose e avaliacao ocular pela Tela de Amsler, em pacientes da zona rural, atendidos na unidade de saude do municipio de Jaguapitã, PR, Brasil. Rev Soc Bras de Med Trop. 1999;32(6):671-6.

4 Kompalic-Cristo A, Britto C, Fernandes O. Diagnóstico molecular da toxoplasmose: revisao. J Bras Patol Med Lab. 2005;41(4):229-35.^-55 Khan A, Jordan C, Muccioli C, Vallochi AL, Rizzo LV, Belfort Jr R, et al. Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis. Brazil. Emerg Infect Dis. 2006;12(6):942-9.⁾. In Erechim, a city located in the south of Brazil, 88% of the population is seropositive for T. gondii, with 18% developing ocular toxoplasmosis⁽⁵5 Khan A, Jordan C, Muccioli C, Vallochi AL, Rizzo LV, Belfort Jr R, et al. Genetic divergence of Toxoplasma gondii strains associated with ocular toxoplasmosis. Brazil. Emerg Infect Dis. 2006;12(6):942-9.^,66 Silveira C, Belfort Jr R, Muccioli C, Abreu MT, Martins MC, Victoria C, et al. A Follow-up study of Toxoplasma gondii infection in Southern Brazil. Am J Ophthalmol. 2001; 131(3):351-4.⁾.

Clinical diagnosis of ocular toxoplasmosis is usually made by ophthalmic examination. Molecular biology techniques can be successfully used to detect T. gondii DNA⁽⁷7 Park YH, Nam HW. Clinical features and treatment of ocular toxoplasmosis. Korean J Parasitol. 2013;51(4):393-9.⁾ when the clinical diagnosis is unclear. Polymerase chain reaction (PCR) can detect microorganism DNA and is a rapid method with high sensitivity and specificity that has been used to detect T. gondiiDNA in different biological samples⁽⁸8 Cantos GA, Prando MD, Siqueira MV, Teixeira RM. Toxoplasmose: ocorrência de anticorpos antitoxoplasma gondii e diagnóstico. Rev Assoc Med Bras. 2000;46(4):335-41.^,99 Bou G, Figueroa MS, Martí-Belda P, Navas E, Guerrero A. Value of PCR for detection of toxoplasma gondii in aqueous humor and blood samples from immunocompetent patients with ocular toxoplasmosis. J Clin Microbiol. 1999;37(11):3465-8.⁾. With the recent advances in molecular biology, the use of real-time PCR makes quantitative measurement of T. gondii DNA possible in patients with ocular toxoplasmosis⁽¹⁰10 Costa JG, Carneiro AC, Tavares AT, Andrade GM, Vasconcelos-Santos DV, Januário JN, et al. Real-time PCR as a prognostic tool for human congenital toxoplasmosis. J Clin Microbiol. 2013;51(8):2766-8.

11 Santos FF, Commodaro AG, Souza AV, Pinho JR, Sitnik R, Garcia C, et al. Real-time PCR in infectious uveitis as an alternative diagnosis. Arq Bras Oftalmol. 2011;74(4):258-61.^-1212 Novais EA, Commodaro AG, Santos F, Muccioli C, Maia A, Nascimento H, et al. Patients with diffuse uveitis and inactive toxoplasmic retinitis lesions test PCR positive for Toxoplasma gondii in their vitreous and blood. Br J Ophthalmol. 2014;98(7):937-40.⁾. Rapid recognition of specific infections is important for adequate management of uveitis⁽¹³13 Aouizerate F, Cazenave J, Poirier L, Verin P, Cheyrou A, Begueret J, et al. Detection of Toxoplasma gondii in aqueous humour by the polymerase chain reaction. Br J Ophthalmol. 1993;77(2):107- 9.⁾.

The aim of this study was to evaluate the ability of real-time quantitative PCR (qPCR) for the detection of T. gondii DNA in peripheral blood and aqueous humor samples of patients with toxoplasmic active focal necrotizing retinochoroiditis.

METHODS

Patients

Fifty-five patients with uveitis seen from 2009 to 2013 at the Department of Ophthalmology and Visual Sciences of the Federal University of São Paulo were included in this study. From the 55 patients, 43 were diagnosed with toxoplasmic active focal necrotizing retinochoroiditis. The remaining 12 were diagnosed with non-toxoplasmic infectious uveitis (toxocariasis=6, tuberculosis=3, and herpes virus=3), and served as controls.

The study was approved by the local ethics committee investigational review board (0094/09), and informed consent was obtained from all patients.

Samples

The samples were collected by an ophthalmologist. Peripheral blood (5 ml) was collected in EDTA tubes. Approximately 0.1 ml of the aqueous humor was collected by anterior chamber paracentesis with a 30-gauge needle. Aqueous samples were obtained from patients who had at least two anterior chamber cells.

A total of 43 peripheral blood and 43 aqueous humor samples were collected from 43 patients with toxoplasmic necrotizing active focal retinochoroiditis and two or more inflammatory cells in the aqueous humor. Twelve non-toxoplasmic patients with active uveitis and two or more inflammatory cells in the aqueous humor (12 peripheral blood and 12 aqueous humor samples) were used as controls.

Dna extraction

Total DNA was extracted from the peripheral blood and aqueous humor with a commercially available DNA mini column kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The DNA samples were stored at -20°C until use.

Real-time qPCR

Real-time qPCR was performed using TaqMan 10 µl, with sense and antisense primers at a concentration of 10 mM, 0.4 µl of probe, 6.8 µl of nuclease- and DEPC-free water (Invitrogen, Carlsbad, CA, USA), and 2 µl of DNA, with a total reaction volume of 20 µl. qPCR was performed on an ABI Prism 7500 DNA sequence detection system (Applied Biosystems, Waltham, MA, USA) and targeted the T. gondii 529-bp repetitive genomic sequence (rep529)⁽¹⁴14 Danise A, Cinque P, Vergani S, Candino M, Racca S, De Bona A, et al. Use of polymerase chain reaction assays of aqueous humor in the differential diagnosis of retinitis in patients infected with human immunodeficiency virus. Clin Infec Dis. 1997;24(6):1100-6.^,1515 Hierl T, Reischl U, Lang P, Hebart H, Stark M, Kyme P, et al. Preliminary evaluation of one conventional nested and two real time PCR assays for the detection of Toxoplasma gondii in immunocompromised patients. J Med Microbiol. 2004;53(Pt 7):629-32.⁾ and the B1 gene⁽¹⁶16 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):1965-7.⁾. β-globin qPCR was performed in parallel for each sample as described previously⁽¹⁷17 Bispo PJ, de Melo GB, Hofling-Lima AL, Pignatari AC. Detection and gram discrimination of bacterial pathogens from aqueous and vitreous humor using real-time PCR assays. Invest Ophthalmol Vis Sci. 2011;52(2):873-81.⁾ in order to confirm the integrity of the DNA and to verify the PCR inhibitors.

RESULTS

Real-time qPCR analysis detected T. gondii DNA in 37.21% (16/43) of the aqueous humor samples and 2.33% (1/43) of the peripheral blood samples; further, 16.27% (7/43) of the patients had positive results in both the peripheral blood and aqueous humor samples (Table 1).

Further, among the 43 patients, the qPCR analysis showed negative results in both the peripheral blood and aqueous humor samples in 19 patients (data not shown).

In the control group (n=12), all samples analyzed showed negative qPCR results forT. gondii (data not shown).

DISCUSSION

Blindness and visual impairment caused by infectious uveitis can be prevented at least partially by early identification of the pathogen and the subsequent administration of appropriate treatment⁽¹⁸18 London NJ, Rathinam SR, Cunningham ET Jr. The epidemiology of uveitis in developing countries. Int Clin Ophthalmol. 2010;50(2):1-17.⁾. The use of real-time PCR as a diagnostic tool for infectious uveitis has been demonstrated by many groups⁽¹¹11 Santos FF, Commodaro AG, Souza AV, Pinho JR, Sitnik R, Garcia C, et al. Real-time PCR in infectious uveitis as an alternative diagnosis. Arq Bras Oftalmol. 2011;74(4):258-61.^,1919 Matos K, Muccioli C, Belfort Jr. R, Rizzo LV. Correlation between clinical diagnosis and PCR analysis of serum, aqueous, and vitreous samples in patients with inflammatory eye disease. Arq Bras Oftalmol. 2007;70(1):109-14.

20 Oahalou A, Schellekens PA, de Groot-Mijnes JD, Rothova A. Diagnostic pars plana vitrectomy and aqueous analyses in patients with uveitis of unknown cause. Retina. 2014;34(1):108-14.^-2121 Sugita S, Ogawa M, Shimizu N, Morio T, Ohguro N, Nakai K, et al. Use of a comprehensive polymerase chain reaction system for diagnosis of ocular infectious diseases. Ophthalmology. 2013;120(9):1761-8.⁾.

In this study, qPCR identified T. gondii infection in 37.21% of the aqueous humor samples, confirming the clinical hypothesis of toxoplasmic active focal necrotizing retinochoroiditis. A previous study reported that aqueous PCR analyses are useful in AIDS patients with ocular toxoplasmosis (sensitivity of 75%)⁽¹⁹19 Matos K, Muccioli C, Belfort Jr. R, Rizzo LV. Correlation between clinical diagnosis and PCR analysis of serum, aqueous, and vitreous samples in patients with inflammatory eye disease. Arq Bras Oftalmol. 2007;70(1):109-14.⁾ and demonstrated that the aqueous humor is the best source for identifying T. gondii infection. Another study also detected T. gondii DNA (38%) by PCR in aqueous humor samples⁽¹⁶16 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):1965-7.⁾.

PCR has been found to detect T. gondii DNA more effectively in aqueous humor (25%) than in peripheral blood (5%) samples⁽²²22 Lee SE, Hong SH, Lee SH, Jeong YI, Lim SJ, Kwon OW, et al. Detection of ocular Toxoplasma gondii infection in chronic irregular recurrent uveitis by PCR. Korean J Parasitol. 2012;50(3):229-31.⁾. Recently, a study demonstrated that the sensitivity for blood was poor (4.1%) compared with that for ocular samples (35.9%)⁽²³23 Bourdin C, Busse A, Kouamou E, Touafek F, Bodaghi B, Le Hoang P, et al. PCR-based detection of Toxoplasma gondii DNA in blood and ocular samples for diagnosis of ocular toxoplasmosis. J Clin Microbiol. 2014;52(11):3987-91.⁾. These findings corroborate our results, which showed that the ability of PCR to detectT. gondii DNA was more sensitive in aqueous humor (37.21%) than in peripheral blood samples (2.33%). Positive results in the peripheral blood were found in only one patient (2.3%), while positive results were found in both peripheral blood and aqueous humor samples in 16.27% of the patients. This finding confirms that qPCR can identify parasites circulating in the peripheral blood of patients. T. gondii has been found in the blood of acutely and chronically infected patients with ocular toxoplasmosis⁽²⁴24 Silveira C, Vallochi AL, Rodrigues da Silva L, Muccioli C, Holland GN, Nussenblatt RB, et al. Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis. Br J Opththalmol. 2011;95(3):396-400.⁾.

CONCLUSION

qPCR is a useful tool for detecting T. gondii DNA in patients with toxoplasmic active focal necrotizing retinochoroiditis and may help in the establishment of appropriate treatment, with the exception of noninfectious uveitis or infectious uveitis caused by other pathogens.

REFERENCES

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