Leishmaniasis şüpheli örneklerin kültür ve PCR sonuçlarının değerlendirilmesi

Amaç: Bu çalışmada leishmaniasis şüphesi ile laboratuvarımıza gönderilen kan, kemik iliği ve/veya doku biyopsisi ve yara aspiratı örneklerinin NovyMacNeal-Nicolle NNN besiyerine ekimi ve moleküler yöntemlerle inceleme sonuçlarının değerlendirilmesi amaçlanmıştır. Ülkemizde yerli olguların görülme sıklığı düşük olmasına rağmen son yıllarda artan göçmen sayısı nedeniyle leishmaniasis tüm dünyada olduğu gibi ülkemizde de yeniden önem kazanmıştır. Visseral leishmaniasis tanısında özellikle kemik iliği ve buffy coat örnekleri; kütanöz leishmaniasis tanısında ise yara aspiratı veya doku biyopsi örneklerinden yararlanılmaktadır. Kemik iliği ve doku biyopsi örneklerinin alınması hasta açısından oldukça zahmetli olmakta, ayrıca doğru şekilde alınmadığında yanlış tanıya yol açabilmektedir. Bu nedenle mümkün olduğunca birden fazla yöntemin bir arada kullanılması ile tanı koyma şansının artırılması önerilmektedir.Yöntem: Ocak 2015 ile Temmuz 2018 tarihleri arasında leishmaniasis şüphesiyle Halk Sağlığı Genel Müdürlüğü Ulusal Parazitoloji Referans Laboratuvarı’na gönderilen 271 adet kan, kemik iliği, doku ve/veya yara aspiratı örneğinin NNN besiyerine ekimi yapılmış ve ticari kit kullanılarak Genesig, Primer Design, UK realDesign, UK for real-time PCR method. Results: While 45 16.60% of the samples were evaluated as positive by any method, 226 83.40% were evaluated as negative. Of the 174 64.20% samples evaluated by PCR alone, 22 12.64% were positive and 152 87.36% were negative. Of the 52 19.20% samples evaluated by culture method only, 7 13.46% were positive and 45 86.54% were negative. And finally, of the 45 16.60% samples evaluated by both methods, 29 64.45% were negative by both PCR and culture, 10 22.22% were positive by PCR and culture, 6 13.33% were positive by PCR and negative by culture. There were no samples detected negative by PCR and positive by culture. There were no samples detected negative by PCR and positive by culture. A moderate concordance was found between the two methods studied κ= 0.545 . Conclusion: Since the sensitivity and specificity of the two diagnostic methods are different, it is concluded that the use of a combination of culture and PCR methods that allow the growth of the parasite by culture, especially in the case of low parasitemia and amplification of DNA by PCR, will increase the chance of diagnosis of the disease

Anahtar Kelimeler:

Leishmaniasis, kültür, NNN besiyeri, real-time PCR

Evaluation of culture and PCR results of leishmaniasis suspected samples

Objective: In this study, we aimed to evaluate the results of blood, bone marrow and / or tissue biopsy and wound aspirate samples sent to our laboratory with suspicion of leishmaniasis on Novy-MacNeal-Nicolle NNN medium and the results of molecular examination. Although incidence of indigenous cases in our country is low, due to increasing number of migrants in recent years, leishmaniasis has regained importance in our country as in the whole world. Especially bone marrow and buffy coat samples for the diagnosis of visceral leishmaniasis and wound aspirate or tissue biopsy specimens for the diagnosis of cutaneous leishmaniasis are used. Obtaining bone marrow and tissue biopsy specimens is very painful for the patient and may lead to misdiagnosis if not obtained correctly. Therefore, it is recommended to increase the chance of diagnosis by using more than one method together.Methods: Two hundred seventy one blood, bone marrow, tissue and / or wound aspirate specimens between January 2015 and July 2018 were sent to the National Parasitology Reference Laboratory of the General Directorate of Public Health on suspicion of leishmaniasis and cultured in NNN medium were then processed by using a commercial kit Genesig, Primer Design, UK for real-time PCR method. Results: While 45 16.60% of the samples were evaluated as positive by any method, 226 83.40% were evaluated as negative. Of the 174 64.20% samples evaluated by PCR alone, 22 12.64% were positive and 152 87.36% were negative. Of the 52 19.20% samples evaluated by culture method only, 7 13.46% were positive and 45 86.54% were negative. And finally, of the 45 16.60% samples evaluated by both methods, 29 64.45% were negative by both PCR and culture, 10 22.22% were positive by PCR and culture, 6 13.33% were positive by PCR and negative by culture. There were no samples detected negative by PCR and positive by culture. There were no samples detected negative by PCR and positive by culture. A moderate concordance was found between the two methods studied κ= 0.545 . Conclusion: Since the sensitivity and specificity of the two diagnostic methods are different, it is concluded that the use of a combination of culture and PCR methods that allow the growth of the parasite by culture, especially in the case of low parasitemia and amplification of DNA by PCR, will increase the chance of diagnosis of the disease

Keywords:

Leishmaniasis, culture, NNN medium, real-time PCR,

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1. Singh S, New developments in diagnosis of leishmaniasis, Indian J Med Res 2006; 123:311-30.
2. Öztoprak N, Aydemir H, Pişkin N, Seremet Keskin A, Araslı M, Gökmen A, Çelebi G, Külekçi Uğur A, Taylan Özkan A, Zonguldak’ta Erişkin Viseral Leyşmaniyaz Olgusu, Mikrobiyol Bul 2010; 44: 671- 7.
3. Sayili A, Taylan Ozkan A, Schallig HDFH, Case Report: Pediatric Visceral Leishmaniasis Caused by Leishmania infantum in Northern Cyprus, Am J Trop Med Hyg 2016; 95(6): 1386–8.
4. Dinçer D, Arca E, Koç E, Topal Y, Taylan Özkan A, Çelebi B, Ülkemizin Endemik Olmayan Bir İlinde (Ankara) Saptanan Leishmania infantum’a Bağlı Bir Kütanöz Leyşmanyazis Olgusu, Mikrobiyol Bul 2012; 46(3): 499-506.
5. Malatyalı E, Özçelik S, Gürsoy N, Kekik (Thymus vulgaris), kimyon (Cuminum cyminum) ve mersin (Myrtus communis) bitkilerinden elde edilen yağların invitro antileishmanial etkileri, Turk Hij Den Biyol Derg 2009; 66 (1): 7-13.
6. Çulha G, Doğramacı ÇA, Gülkan B, Savaş N, Kutanöz leishmaniasis ve Hatay İlindeki durumu, Turk Hij Den Biyol Derg 2014; 71(4): 171-8.
7. Fraga TL, Brustoloni YM, Lima RB, Cavalheiros Dorval ME, Teruya Oshiro E, Oliveira J, Lyrio de Oliveira AL, Pirmez C, Polymerase Chain Reaction of Peripheral Blood as a Tool for the Diagnosis of Visceral Leishmaniasis in Children, Mem Inst Oswaldo Cruz, Rio de Janeiro 2010;105(3):310-3.
8. Rahi AA, Nsaif S, Hassoni JJ, Ali MA, Hamza HA, Comparison of Diagnostic Methods in Cutaneous Leishmaniasis in Iraq, Am J BioSci 2013;1(1):1-5.
9. Ertabaklar H, Çalışkan SÖ, Boduç E, Ertuğ S, Kutanöz Leyşmanyazis Tanısında Direkt Mikroskopi, Kültür ve Polimeraz Zincir Reaksiyonu Yöntemlerinin Karşılaştırılması, Mikrobiyol Bul 2015;49(1):77-84.
10. Nsaif AL-Hucheimi S, Sultan BA, Al-Dhalimi MA, Abdullah Mahmood T, Tracking of Ceotaneous Leishmaniasis by Parasitological, Molecular and Biochemical Analysis, Kufa J Nursing Sci 2015;5(1):1-10.
11. ILemrani M, Hamdi S, Laamrani A, Hassar M, PCR Detection of Leishmania in Skin Biopsies, J Infect Developing Countries 2009; 3(2):115-22.
12. Zakai HA, Cutaneous Leishmaniasis (CL) in Saudi Arabia: Current Status, J Adv Lab Res Biol 2014;5(2):29-34
13. El Hassan AM, Cutaneous Leishmaniasis in Al-Ahsa Oasis in Saudi Arabia and in Sudan: A Comparative Study, Saudi J Med Med Sci 2013;1(2):64-71
14. Georgiadou SP, Makaritsis KP, Dalekos GN, Leishmaniasis Revisited: Current Aspects on Epidemiology, Diagnosis and Treatment, J Translat Intern Med 2015;3(2):43-50
15. Imran AL-Mosa MA, The best method for Diagnosis of Cutaneous Leishmaniasis and Identification of the Causative Leishmania Species in Al-Najaf Governorate by Using PCR Assay, Int J Adv Res 2015;3(5):226-33
16. Pourmohammadi B, Motazedian MH, Hatam GR, Kalantari M, Habibi P, Sarkari B, Comparison of Three Methods for Diagnosis of Cutaneous Leishmaniasis, Iranian J Parasitol 2010;5(4):1-8
17. Abda IB, de Monbrison F, Bousslimi N, Aoun K, Bouratbine A, Picot S, Advantages and Limits of Real-time PCR Assay and PCR-Restriction Fragment Length Polymorphism for the Identification of Cutaneous Leishmania Species in Tunisia, Trans R Soc Trop Med Hyg 2011;105(1):17-22
18. Daldal N, Taylan Özkan A, Etkene Yönelik Tanı Yöntemleri, Korkmaz M, Ok ÜZ (eds) In: Parazitolojide Laboratuvar Yöntem, Yorum, Akreditasyon, Meta Basım, İzmir, 2011:87-117
19. Profeta Luz ZM, da Silva AR, de Oliveira Silva F, Caligiorne RB, Oliveira E, Rabello A, Lesion aspirate culture for the diagnosis and isolation of Leishmania spp. from patients with cutaneous leishmaniasis, Mem Inst Oswaldo Cruz, Rio de Janeiro 2009;104(1):62-6
20. Thomaz-Soccol A, Mocellin M, Mulinari F, de Castro EA, de Queiroz-Telles F, de Souza Alcântara F, Bavaresco MT, Hennig L, Andraus A, Luz E, Thomaz-Soccol V, Clinical Aspects and Relevance of Molecular Diagnosis in Late Mucocutaneous Leishmaniasis Patients in Paraná, Brazil, Braz Arch Biol Technol 2011, 54(3): 487-94
21. Qader AM, Abood MK, Bakir TY, Identification of Leishmania Parasites in Clinical Samples Obtained from Cutaneous Leishmaniasis Patients Using PCR Technique in Iraq, Iraqi J Sci 2009;50(1):32-6