Application of qPCR in conjunctival swab samples for the evaluation of canine leishmaniasis in borderline cases or disease relapse and correlation with clinical parameters

Particle and Fibre Toxicology

Mauro Magnani²  Barbara Bianchi¹  Davide Sisti²  Luca Galluzzi²  Marcello Ceccarelli² 

[1] Veterinary Clinic ¿Santa Teresa¿, via Piave 23, Fano, 61032, PU, Italy;Department of Biomolecular Sciences, University of Urbino ¿Carlo Bo¿, via Saffi 2, Urbino, 61029, PU, Italy

关键词: Globulins;    Haemoglobin;    IFAT;    Canine leishmaniasis;    Dog;    qPCR;    Conjunctival swabs;    Leishmania infantum;   

Others  :  1149735
DOI  :  10.1186/s13071-014-0460-3

 received in 2014-07-22, accepted in 2014-09-26,  发布年份 2014

【 摘 要 】

Background

In leishmaniasis caused by Leishmania infantum, the dog acts as the main reservoir for the disease. Non-invasive sampling for Leishmania detection is pivotal for rapid and affordable diagnosis. Recently, the use of conjunctival swab (CS) has been evaluated as a non-invasive sampling technique for quantitative real-time PCR (qPCR). However, few investigations have been made on the applicability of CS qPCR in particular cases such as dogs with borderline IFAT titres, suspected disease relapse with comorbidity and therapy monitoring. The aims of this study were i) to confirm the efficacy of CS, comparing these samples to buffy coat (BC) samples, as effective non-invasive samples for Leishmania quantitative detection by qPCR and ii) to verify the usefulness of qPCR compared to conventional laboratory and clinical parameters to assist in therapeutic decision making regarding dogs with complex clinical cases.

Methods

Eighty dogs were divided into 4 groups based on their IFAT titres and clinical histories. Two qPCR assays were performed both on CS raw lysates and on purified DNA from BC samples. The assays were then compared. Z tests for difference of proportion, with Bonferroni correction, were carried out to evaluate the qPCR results. Logistic regression with backward stepwise elimination was performed to detect the subset of haematochemical variables significantly associated with PCR positivity.

Results

The qPCR performed on CS samples showed better sensitivity (87%) and specificity (96%) than assays carried out using BC samples, regardless of the primers used. The haematochemical parameters haemoglobin and globulins were found to be significantly associated with qPCR positivity. Pearson correlations between Leishmania kDNA load in CS and body condition scores or IFAT titres were calculated in dogs with new leishmaniasis diagnoses. The Leishmania kDNA load in CS correlated moderately with IFAT titres (R?=?0.59) but a very weak correlation (R?=?0.37) with body condition score (BCS) was found.

Conclusions

The applicability of CS for Leishmania detection in dogs was confirmed, revealing the usefulness of raw lysates for quantitative purposes. Moreover, the qPCR was found to be particularly useful in cases lacking a clear clinical diagnosis, where the haematochemical values cannot be predictive.

【 授权许可】

   
2014 Ceccarelli et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den BM: Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE 2012, 7:e35671.
• [2]Working to Overcome the Global Impact of Neglected Tropical Diseases: First WHO Report on Neglected Tropical Diseases. 2010.
• [3]Marty P, Le FY, Giordana D, Brugnetti A: Leishmanin reaction in the human population of a highly endemic focus of canine leishmaniasis in Alpes-Maritimes, France. Trans R Soc Trop Med Hyg 1992, 86:249-250.
• [4]Semiao-Santos SJ, El HA, Ferreira E, Pires CA, Sousa C, Gusmao R: Evora district as a new focus for canine leishmaniasis in Portugal. Parasitol Res 1995, 81:235-239.
• [5]Cabral M, O¿Grady JE, Gomes S, Sousa JC, Thompson H, Alexander J: The immunology of canine leishmaniosis: strong evidence for a developing disease spectrum from asymptomatic dogs. Vet Parasitol 1998, 76:173-180.
• [6]Sideris V, Papadopoulou G, Dotsika E, Karagouni E: Asymptomatic canine leishmaniasis in Greater Athens area, Greece. Eur J Epidemiol 1999, 15:271-276.
• [7]Rodriguez-Cortes A, Ojeda A, Francino O, Lopez-Fuertes L, Timon M, Alberola J: Leishmania infection: laboratory diagnosing in the absence of a ¿gold standard¿. Am J Trop Med Hyg 2010, 82:251-256.
• [8]Solano-Gallego L, Miro G, Koutinas A, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P, Oliva G, Baneth G: LeishVet guidelines for the practical management of canine leishmaniosis. Parasit Vectors 2011, 4:86. BioMed Central Full Text
• [9]Gradoni L, Gramiccia M: Leishmaniasis. In OIE Manual of Standards for Diagnostic Tests and Vaccine. 4th edition. Office International des Epizooties, Paris, France; 2000:802-812.
• [10]Iniesta L, Fernandez-Barredo S, Bulle B, Gomez MT, Piarroux R, Gallego M, Alunda JM, Portus M: Diagnostic techniques to detect cryptic leishmaniasis in dogs. Clin Diagn Lab Immunol 2002, 9:1137-1141.
• [11]Coura-Vital W, Ker HG, Roatt BM, Guiar-Soares RD, Leal GG, Moreira N, Oliveira LA, de Menezes Machado EM, Morais MH, Correa-Oliveira R, Carneiro M, Reis AB: Evaluation of change in canine diagnosis protocol adopted by the visceral leishmaniasis control program in Brazil and a new proposal for diagnosis. PLoS ONE 2014, 9:e91009.
• [12]Gradoni L, Gramiccia M, Khoury C, Maroli M: Guidelines for the Control of the Canine Reservoir of Zoonotic Visceral Leishmaniasis in Italy. ISTISAN 04/12 Edition. ISTITUTO SUPERIORE DI SANITÀ, Rome [in Italian]; 2004.
• [13]Di Muccio T, Veronesi F, Antognoni MT, Onofri A, Piergili FD, Gramiccia M: Diagnostic value of conjunctival swab sampling associated with nested PCR for different categories of dogs naturally exposed to Leishmania infantum infection. J Clin Microbiol 2012, 50:2651-2659.
• [14]Sudarshan M, Weirather JL, Wilson ME, Sundar S: Study of parasite kinetics with antileishmanial drugs using real-time quantitative PCR in Indian visceral leishmaniasis. J Antimicrob Chemother 2011, 66:1751-1755.
• [15]Manna L, Reale S, Vitale F, Picillo E, Pavone LM, Gravino AE: Real-time PCR assay in Leishmania-infected dogs treated with meglumine antimoniate and allopurinol. Vet J 2008, 177:279-282.
• [16]Mary C, Faraut F, Lascombe L, Dumon H: Quantification of Leishmania infantum DNA by a real-time PCR assay with high sensitivity. J Clin Microbiol 2004, 42:5249-5255.
• [17]Lachaud L, Chabbert E, Dubessay P, Reynes J, Lamothe J, Bastien P: Comparison of various sample preparation methods for PCR diagnosis of visceral leishmaniasis using peripheral blood. J Clin Microbiol 2001, 39:613-617.
• [18]Lachaud L, Marchergui-Hammami S, Chabbert E, Dereure J, Dedet JP, Bastien P: Comparison of six PCR methods using peripheral blood for detection of canine visceral leishmaniasis. J Clin Microbiol 2002, 40:210-215.
• [19]Pita-Pereira D, Lins R, Oliveira MP, Lima RB, Pereira BA, Moreira OC, Brazil RP, Britto C: SYBR Green-based real-time PCR targeting kinetoplast DNA can be used to discriminate between the main etiologic agents of Brazilian cutaneous and visceral leishmaniases. Parasit Vectors 2012, 5:15. BioMed Central Full Text
• [20]Solca MS, Bastos LA, Guedes CE, Bordoni M, Borja LS, Larangeira DF, da Silva Estrela Tuy PG, Amorim LD, Nascimento EG, de Sa Oliveira GG, Dos-Santos WL, Fraga DB, Veras PS: Evaluating the accuracy of molecular diagnostic testing for canine visceral leishmaniasis using latent class analysis. PLoS ONE 2014, 9:e103635.
• [21]Maia C, Campino L: Methods for diagnosis of canine leishmaniasis and immune response to infection. Vet Parasitol 2008, 158:274-287.
• [22]Berrahal F, Mary C, Roze M, Berenger A, Escoffier K, Lamouroux D, Dunan S: Canine leishmaniasis: identification of asymptomatic carriers by polymerase chain reaction and immunoblotting. Am J Trop Med Hyg 1996, 55:273-277.
• [23]Strauss-Ayali D, Jaffe CL, Burshtain O, Gonen L, Baneth G: Polymerase chain reaction using noninvasively obtained samples, for the detection of Leishmania infantum DNA in dogs. J Infect Dis 2004, 189:1729-1733.
• [24]Pena MT, Naranjo C, Klauss G, Fondevila D, Leiva M, Roura X, Davidson MG, Dubielzig RR: Histopathological features of ocular leishmaniosis in the dog. J Comp Pathol 2008, 138:32-39.
• [25]Lombardo G, Pennisi MG, Lupo T, Migliazzo A, Capri A, Solano-Gallego L: Detection of Leishmania infantum DNA by real-time PCR in canine oral and conjunctival swabs and comparison with other diagnostic techniques. Vet Parasitol 2012, 184:10-17.
• [26]de Almeida FS, Leite RS, Ituassu LT, Almeida GG, Souza DM, Fujiwara RT, de Andrade AS, Melo MN: Canine skin and conjunctival swab samples for the detection and quantification of Leishmania infantum DNA in an endemic urban area in Brazil. PLoS Negl Trop Dis 2012, 6:e1596.
• [27]Geisweid K, Weber K, Sauter-Louis C, Hartmann K: Evaluation of a conjunctival swab polymerase chain reaction for the detection of Leishmania infantum in dogs in a non-endemic area. Vet J 2013, 198:187-192.
• [28]Ikonomopoulos J, Kokotas S, Gazouli M, Zavras A, Stoitsiou M, Gorgoulis VG: Molecular diagnosis of leishmaniosis in dogs. Comparative application of traditional diagnostic methods and the proposed assay on clinical samples. Vet Parasitol 2003, 113:99-113.
• [29]Gramiccia M, Di MT, Fiorentino E, Scalone A, Bongiorno G, Cappiello S, Paparcone R, Foglia MV, Maroli M, Gradoni L, Oliva G: Longitudinal study on the detection of canine Leishmania infections by conjunctival swab analysis and correlation with entomological parameters. Vet Parasitol 2010, 171:223-228.
• [30]Reale S, Maxia L, Vitale F, Glorioso NS, Caracappa S, Vesco G: Detection of Leishmania infantum in dogs by PCR with lymph node aspirates and blood. J Clin Microbiol 1999, 37:2931-2935.
• [31]Ceccarelli M, Galluzzi L, Migliazzo A, Magnani M: Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA. PLoS ONE 2014, 9:e88845.
• [32]Kraemer HC, Kupfer DJ: Size of treatment effects and their importance to clinical research and practice. Biol Psychiatry 2006, 59:990-996.
• [33]User Bulletin #2 Relative Quantitation of Gene Expression. Dec. 11, 1997 (updated 10/2001). 1997.
• [34]Galletti E, Bonilauri P, Bardasi L, Fontana MC, Ramini M, Renzi M, Dosa G, Merialdi G: Development of a minor groove binding probe based real-time PCR for the diagnosis and quantification of Leishmania infantum in dog specimens. Res Vet Sci 2011, 91:243-245.
• [35]dos Santos Marques LH, Gomes LI, da Rocha ICM, da Silva TA, Oliveira E, Morais MH, Rabello A, Carneiro M: Low parasite load estimated by qPCR in a cohort of children living in urban area endemic for visceral leishmaniasis in Brazil. PLoS Negl Trop Dis 2012, 6:e1955.
• [36]Mary C, Faraut F, Drogoul MP, Xeridat B, Schleinitz N, Cuisenier B, Dumon H: Reference values for Leishmania infantum parasitemia in different clinical presentations: quantitative polymerase chain reaction for therapeutic monitoring and patient follow-up. Am J Trop Med Hyg 2006, 75:858-863.
• [37]Mohammadiha A, Haghighi A, Mohebali M, Mahdian R, Abadi AR, Zarei Z, Yeganeh F, Kazemi B, Taghipour N, Akhoundi B, Barati M, Mahmoudi MR: Canine visceral leishmaniasis: a comparative study of real-time PCR, conventional PCR, and direct agglutination on sera for the detection of Leishmania infantum infection. Vet Parasitol 2013, 192:83-90.
• [38]Solano-Gallego L, Koutinas A, Miro G, Cardoso L, Pennisi MG, Ferrer L, Bourdeau P, Oliva G, Baneth G: Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis. Vet Parasitol 2009, 165:1-18.
• [39]Pilatti MM, Ferreira SA, de Melo MN, de Andrade AS: Comparison of PCR methods for diagnosis of canine visceral leishmaniasis in conjunctival swab samples. Res Vet Sci 2009, 87:255-257.
• [40]Prina E, Roux E, Mattei D, Milon G: Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR. Microbes Infect 2007, 9:1307-1315.