Method Verification of Inhouse Real-time Polymerase Chain Reaction for Detection of Leishmania Species
Öz
Objective: Leishmaniasis is a vector-borne disease caused by many Leishmania
species which can infect both humans and other mammals. Turkey has special
epidemiologic importance in terms of this disease due to being located at the
junction of Asia and Europe and containing seven geographical regions with
environmental and ecologic differences. Microscopic evaluation may miss
diagnosis in cases with low levels of parasitemia. The culture method is not
chosen by many laboratories due to being laborious preparation of media and
results taking a long time. Molecular methods may assist in diagnosis
especially with low parasitemia levels and provide results in a short time, so
they have begun to be chosen more often currently. This study aimed to verify
the method for inhouse real-time PCR using primers for ITS-1 gene region for
routine molecular diagnosis of leishmaniasis.
Methods: After parasite counting from Leishmania infantum
strain, which was carried out on Novy-MacNeal-Nicolle (NNN) medium in our
laboratory, DNA extraction was performed with a commercial kit (QIAamp DNA
Blood Mini kit, Qiagen, Germany) according to the manufacturer's
recommendations. The number of copies in the reaction was determined from DNA
sample and serial dilutions of 1/10 were prepared. Then, inhouse real-time PCR
method was applied using primers targeting the ITS-1 gene region to determine
limit of detection and high and low positivity rates. Then accuracy and
precision studies were performed with these samples for method verification.
Amplification was performed with a Light Cycler 96 (Roche, France) device. Results
were evaluated with amplification curve analysis. The variation coefficients
were calculated via accuracy and precision studies from the obtained results.
Results: The parasite counts of Leishmania infantum strain were determined as
17,000 promastigote/ml. With using 400 µl of this sample (6,800 copies), DNA
obtained in 100 µl elution buffer. Using inhouse real-time PCR amplification,
the detection limit for 2 µl DNA (136 copies) was determined as 10-3
dilution (0.136 copies/reaction). The high positivity rate was determined as
dilution above 2 log10 of limit of detection's and the low positivity rate was
determined as dilution above 1 log10 of limit of detections. Efficiency of the
method was measured with a regression curve for CT values. The standard curve
obtained according to CT values and equivalent promastigote counts was linear
(slope: -4.097). There was a significant correlation coefficient found between
mean CT values and Leishmania infantum DNA concentrations (R=0.99).
Conclusion: Our study determined the variation coefficient for the inhouse real-time
PCR method was below 15%, confirming it is appropriate for use in our
laboratory for routine molecular diagnosis of Leishmania spp.
Anahtar Kelimeler
Kaynakça
- Akhlagh A, Salehzadeh A, Zahirnia AH and Davari B, 10-Year Trends in Epidemiology, Diagnosis, and Treatment of Cutaneous Leishmaniasis in Hamadan Province, West of Iran (2007–2016), Front Public Health, 2019; 7:27.
- Antonia AL, Wang L, Ko DC. 2018. A Real-time PCR Assay for Quantification of Parasite Burden in Murine Models of Leishmaniasis, Peer J, 6:e5905: 1-17.
- Clark RB, Lewinski MA, Loeffelholz MJ, Tibbetts RJ, 2009. Cumitech 31A, verification and validation of procedures in the clinical microbiology laboratory. Coordinating ed., S. E. Sharp. ASM Press, Washington, DC.
- Deepachandi B, Weerasinghe S, Soysa P, Karunaweera N, Siriwardana Y, A Highly Sensitive Modified Nested PCR to Enhance Case Detection in Leishmaniasis, BMC Infect Dis, 2019; 19:623.
- de Vries HJC, Reedijk SH, Schallig HDFH, Cutaneous Leishmaniasis: Recent Developments in Diagnosis and Management, Am J Clin Dermatol, 2015;16: 99–109.
- Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M, Real-time PCR applications for diagnosis of leishmaniasis, Parasites & Vectors, 2018;11(273): 1-13.
- Hernández C, Alvarez C, González C, Ayala MS, León CM, Ramírez JD, Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay, Parasit Vectors, 2014; 7: 501, p. 1-7.
- Hossain F, Ghosh P, Khan M.AA, Duthie MS, Vallur AC, Picone A, Howard RF, Reed SG, Mondal D, Real-time PCR in Detection and Quantitation of Leishmania donovani for the Diagnosis of Visceral Leishmaniasis Patients and the Monitoring of Their Response to Treatment, PLoS ONE, 2017;12(9): e0185606. https://www.gu.se/digitalAssets/1125/1125331_ABI_Guide_Relative_Quantification_using_realtime_PCR.pdf
- León CM, Muñoz M, Hernández C, Ayala MS, Flórez C, Teherán A, Cubides JR, Ramírez JD, Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia, Front Microbiol, 2017;8(1907): 1-13.
- 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(11): 5249–5255.
- Moreira OC, Yadon ZE, Cupolillo E, The applicability of real-time PCR in the diagnostic of cutaneous leishmaniasis and parasite quantification for clinical management: Current status and perspectives, Acta Tropica, 2018;184: 29-37.
- Ozensoy Toz S, Culha G, Yildiz Zeyrek F, Ertabaklar H, Alkan MZ, Tetik Vardarlı A, Gunduz C, Ozbel Y, A Real-Time ITS1-PCR Based Method in the Diagnosis and Species Identification of Leishmania Parasite from Human and Dog Clinical Samples in Turkey, PLoS Negl Trop Dis, 2013;7(5): e2205.
- Paiva-Cavalcanti M, Regis-da-Silva CG, Gomes YM, Comparison of Real-time PCR and Conventional PCR for Detection of Leishmania (Leishmania) infantum Infection: A Mini-Review, J Venom Anim Toxins incl Trop Dis, 2010;16(4): 537-542.
- Rezvan H, Nourian AR, Hamoon Navard S, An Overview on Leishmania Diagnosis, J Med Microbiol Infec Dis, 2017;5 (1-2): 1-11.
- Sirekbasan S, Polat E, Kutlubay Z, Engin B, Leishmania infantum’un Etken Olduğu Bir Kutanöz Leishmaniasis Olgusu, Türkiye Parazitol Derg, 2019;(43)1:41-3.
- Soofi Abadi MF, Fekri M, Moradabadi AR, Vahidi R, Shamsi‑Meymandi S, Dabiri D, Dabiri S, Ability of Real‑time PCR for Differential Diagnosis of Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology, BMC Res Notes, 2019; 12:615.-365.
Öz
Kaynakça
- Akhlagh A, Salehzadeh A, Zahirnia AH and Davari B, 10-Year Trends in Epidemiology, Diagnosis, and Treatment of Cutaneous Leishmaniasis in Hamadan Province, West of Iran (2007–2016), Front Public Health, 2019; 7:27.
- Antonia AL, Wang L, Ko DC. 2018. A Real-time PCR Assay for Quantification of Parasite Burden in Murine Models of Leishmaniasis, Peer J, 6:e5905: 1-17.
- Clark RB, Lewinski MA, Loeffelholz MJ, Tibbetts RJ, 2009. Cumitech 31A, verification and validation of procedures in the clinical microbiology laboratory. Coordinating ed., S. E. Sharp. ASM Press, Washington, DC.
- Deepachandi B, Weerasinghe S, Soysa P, Karunaweera N, Siriwardana Y, A Highly Sensitive Modified Nested PCR to Enhance Case Detection in Leishmaniasis, BMC Infect Dis, 2019; 19:623.
- de Vries HJC, Reedijk SH, Schallig HDFH, Cutaneous Leishmaniasis: Recent Developments in Diagnosis and Management, Am J Clin Dermatol, 2015;16: 99–109.
- Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M, Real-time PCR applications for diagnosis of leishmaniasis, Parasites & Vectors, 2018;11(273): 1-13.
- Hernández C, Alvarez C, González C, Ayala MS, León CM, Ramírez JD, Identification of Six New World Leishmania species through the implementation of a High-Resolution Melting (HRM) genotyping assay, Parasit Vectors, 2014; 7: 501, p. 1-7.
- Hossain F, Ghosh P, Khan M.AA, Duthie MS, Vallur AC, Picone A, Howard RF, Reed SG, Mondal D, Real-time PCR in Detection and Quantitation of Leishmania donovani for the Diagnosis of Visceral Leishmaniasis Patients and the Monitoring of Their Response to Treatment, PLoS ONE, 2017;12(9): e0185606. https://www.gu.se/digitalAssets/1125/1125331_ABI_Guide_Relative_Quantification_using_realtime_PCR.pdf
- León CM, Muñoz M, Hernández C, Ayala MS, Flórez C, Teherán A, Cubides JR, Ramírez JD, Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia, Front Microbiol, 2017;8(1907): 1-13.
- 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(11): 5249–5255.
- Moreira OC, Yadon ZE, Cupolillo E, The applicability of real-time PCR in the diagnostic of cutaneous leishmaniasis and parasite quantification for clinical management: Current status and perspectives, Acta Tropica, 2018;184: 29-37.
- Ozensoy Toz S, Culha G, Yildiz Zeyrek F, Ertabaklar H, Alkan MZ, Tetik Vardarlı A, Gunduz C, Ozbel Y, A Real-Time ITS1-PCR Based Method in the Diagnosis and Species Identification of Leishmania Parasite from Human and Dog Clinical Samples in Turkey, PLoS Negl Trop Dis, 2013;7(5): e2205.
- Paiva-Cavalcanti M, Regis-da-Silva CG, Gomes YM, Comparison of Real-time PCR and Conventional PCR for Detection of Leishmania (Leishmania) infantum Infection: A Mini-Review, J Venom Anim Toxins incl Trop Dis, 2010;16(4): 537-542.
- Rezvan H, Nourian AR, Hamoon Navard S, An Overview on Leishmania Diagnosis, J Med Microbiol Infec Dis, 2017;5 (1-2): 1-11.
- Sirekbasan S, Polat E, Kutlubay Z, Engin B, Leishmania infantum’un Etken Olduğu Bir Kutanöz Leishmaniasis Olgusu, Türkiye Parazitol Derg, 2019;(43)1:41-3.
- Soofi Abadi MF, Fekri M, Moradabadi AR, Vahidi R, Shamsi‑Meymandi S, Dabiri D, Dabiri S, Ability of Real‑time PCR for Differential Diagnosis of Various Forms of Cutaneous Leishmaniasis: A Comparative Study with Histopathology, BMC Res Notes, 2019; 12:615.-365.
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Sağlık Kurumları Yönetimi |
| Bölüm | Araştırma Makaleleri |
| Yazarlar | |
| Yayımlanma Tarihi | 31 Aralık 2019 |
| Yayımlandığı Sayı | Yıl 2019 Cilt: 5 Sayı: 3 |
