Keywords
PCR
vaccine
yellow fever
How to Cite
Abstract
Among the vaccines produced by Bio-Manguinhos, a major centre for manufacturing the immunobiological products in Latin America, stands out the yellow fever (YF) vaccine. To guarantee the excellence and safety of the YF vaccine, the quality control tests has to be performed throughout its production. The World Health Organization (WHO) demands the producers to guarantee the absence of Mycoplasma orale, M. pneumoniae, M. gallisepticum and M. synoviae in the biological products. Mycoplasma is a fastidious microorganism, requiring about 35 days for attaining the conclusive culturing test. In this study PCR methods were selected for amplifying 16SrRNA gene fragments for detecting mycoplasma in the intermediate products of YF vaccine. This standardized methodology was specific and sensitive to detect the low concentrations of mycoplasma in spiked intermediary vaccine products; and the absence of unspecific amplification was also demonstrated. The detection rates ranged from 3.1 to 12.5 colony forming units and showed 100 % of sensitivity and specificity in the tested samples. The PCR protocol for detecting mycoplasmal DNA in YF vaccine was validated by analysing 286 samples. Bio-Manguinhos produces annually 10,000,000 YF vaccine doses, and this method has been successfully employed, complementing the traditional approach in the mycoplasma detection since 2008.
References
1. Heinz FX, stiasny k. Flaviviruses and flavivirus vaccines. Vaccine. 2012;30:4301-6. [DoI: https://dx.doi.org/10.1016/j.vaccine.2011.09.114].2.
2. Querec tD, Pulendran B. understanding the role of innate immunity in the mechanism of action of the live attenuated Yellow Fever Vaccine 17D. Adv exp med Biol. 2007;590:43-53.[DoI: https://dx.doi.org/10.1007/978-0-387-34814-8_3].3.
3. Frierson JG. The Yellow Fever Vaccine: A history. Yale J Biol med. 2010;83:77-85.
4. World health organization – who. Requirements for yellow fever vaccine. technical Report series 872, who, Geneva; 1998.p.30-68.
5. World health organization – who. General requirements for the sterility of biological substances. technical Report series 872, who, Geneva; 1998.p.69-74.
6. Razin s, hayflick L. highlights of mycoplasma research - An historical perspective. Biologicals 2010;38:183-190. [DoI: https://dx.doi.org/10.1016/j.biologicals.2009.11.008].
7. Armstrong se, mariano JA, Lundin DJ. The scope of mycoplasma contamination within the biopharmaceutical industry. Biologicals. 2010;38:211-3. [DoI: https://dx.doi.org/10.1016/j.biologicals.2010.03.002].
8. Volokhov DV, Graham LJ, Brorson kA, Chizhikov. mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques. mol Cell Probes. 2011;25:69-77. [DoI: https://dx.doi.org/10.1016/j.mcp.2011.01.002].
9. International organization for standardization - Iso. Iso/IeC 17025: General requirements for the competence of testing and calibration laboratories. London; 2005.p.28.
10. United states Pharmacopeia – usP. Volume 1, <1223> Validation of Alternative microbiological methods, 30th ed., The united states Pharmacopeial Convention, Rockville; 2007. p.677-0.
11. Frey mL, hanson RP, Anderson DP. A medium for the isolation of avian mycoplasmas. Am J Vet Res. 1968;29(11):2163-2171.
12. Van kuppeveld FJm, van der Logt Jtm, Angulo AF, van Zoest mJ, Quint wGV, niesters hGm, et al. Genus- and species specific identification of mycoplasmas by 16s rRnA amplification. Appl environ microbiol. 1992;58(8):2606-2615.
13. Uphoff CC, Drexler hG. Detection of mycoplasma in leukemia-lymphoma cell lines using polymerase chain reaction. Leukemia. 2002;16(2):289-293. [DoI: https://dx.doi.org/10.1038/sj.leu.2402365].
14. Bruchmuller I, Pirkl e, herrman R, stoermer m, eichler h, kluter h, et al. Introduction of a validation concept for a PCR-based mycoplasma detection assay.Cytother. 2006;8(1):62-9. [DoI: https://dx.doi.org/10.1080/14653240500518413].
15. Japanese Pharmacopoeia. mycoplasma testing for Cell substrates used for the Production of Biotechnological/Biological Products, 15th ed. society of Japanese Pharmacopoeia, tokyo; 2006. p.1717-1724.
16. Conference report. who working Group on technical specifications for manufacture and evaluation of Yellow Fever Vaccines, Geneva, switzerland, 13–14 may 2009. Vaccine. 2010;28:8236–8245. [DoI: https://dx.doi.org/10.1016/j.vaccine.2010.10.070].
17. Bernet C, Garret m, de Barbeyrac B, Bebear C, Bonnet J. Detection of mycoplasma pneumoniae by using the polymerase chain reaction. J Clin microbiol. 1989;27(11):2492-6.
18. Teyssou R, Poutiers F, saillard C, Grau o, Laigret F, Bove Jm, et al . Detection of mollicute Contamination in Cell Cultures by 16s rDnA Amplification. mol Cell Probes. 1993;7(3):209-216. [DoI: https://dx.doi.org/10.1006/mcpr.1993.1030].
19. Eldering JA, Felten C, Veilleux CA, Potts BJ. Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell culture used in the manufacture of recombinant therapeutic proteins. Biologicals. 2004;32:183-193. [DoI: https://dx.doi.org/10.1016/j.biologicals.2004.08.005].
20. Sung h, kang sh, Bae YJ, hong Jt, Chung YB, Lee Ck, et al. PCR-Based Detection of mycoplasma species. J microbiol. 2006;4(1):42-9.
21. Deutschmann sm, kavermann h, knack Y. Validation of a nAt-based mycoplasma assay according european Pharmacopoiea. Biologicals. 2010;38:238–248. [DoI: https://dx.doi.org/10.1016/j.biologicals.2009.11.004].
22. Jurstrand m, Jensen Js, Fredlund h, Falk L, molling P. Detection of mycoplasma genitalium in urogenital specimens by real-time PCR and by conventional PCR assay. J med microbiol. 2005;54:23-9. [DoI: https://dx.doi.org/10.1099/jmm.0.45732-0].
23. Bereczki L, kis G, Bagdi e, krenacs L. optimization of PCR amplifcation for B- and t-cell clonality analysis on formalin-fixed and paraffin-embedded samples. Pathol oncol Res. 2007;13(3):209-214. [DoI: https://dx.doi.org/10.1007/BF02893501].
24. Markoulatos P, mangana-Vougiouka o, koptopoulos G, nomikou k, Papadopoulos o. Detection of sheep poxvirus in skin biopsy samples by a multiplex polymerase chain reaction. J Virol methods. 2000;84:161-7. [DoI: https://dx.doi.org/10.1016/s0166-0934(99)00141-X].
25. Radstrom P, knutsson R, wolffs P, Lovenklev m, Lofstrom C. Pre-PCR Processing: strategies to Generate PCR-Compatible samples. mol Biotechnol. 2004;26:133-146. [DoI: https://dx.doi.org/10.1385/mB:26:2:133].
26. Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl environ microbiol. 1997;63(10):3741-3751.
27. Al-soud wA, Radstrom P. effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin microbiol.2000;38(12):4463-4470.
28. Al-soud wA, Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin microbiol. 2001;39(2):485-493. [DoI: https://dx.doi.org/10.1128/JCm.39.2.485-493.2001].
29. Malorny B, hoorfar J, Bunge C, helmuth R. multicenter Validation of the Analytical Accuracy of salmonella PCR: towards an International standard. Appl environ microbiol. 2003;69(1):290–6. [DoI: https://dx.doi.org/10.1128/Aem.69.1.290-296.2003].
30. Conraths FJ, schares G. Validation of molecular-diagnostic techniques in the parasitological laboratory. Vet Parasitol. 2006;136:91-8. [DoI: https://dx.doi.org/10.1016/j.vetpar.2005.12.004].
31. United states Pharmacopeia – usP. Volume 1, <71> sterility tests, 30th ed., The united states Pharmacopeial Convention, Rockville; 2007. p.97-102.
32. Jimenez-Coello m, Poot-Cob m, ortega-Pacheco A, Guzman-marin e, Ramos-Ligonio A, sauri-Arceo Ch, et al. American trypanosomiasis in dogs from an urban and rural area of Yucatan, mexico. Vector Borne Zoonotic Dis. 2008;8(6):755-61. [DoI: https://dx.doi.org/10.1089/vbz.2007.0224].
33. Sutton s. Accuracy of Plate Counts. J Val technol. 2011; (summer):42-6.
34. American type Culture Collection - AtCC. Culture method. [accessed 2015 oct 26]. Available at:
a. [ h t t p : / / w w w. a t c c . o r g / P r o d u c t s / A l l / 1 5 3 0 2 .aspx#culturemethod]
b. [ h t t p : / / w w w. a t c c . o r g / P r o d u c t s / A l l / 1 5 4 9 2 .aspx#culturemethod]
c. [ h t t p : / / w w w. a t c c . o r g / P r o d u c t s / A l l / 2 3 7 1 4 .aspx#culturemethod]
d. [ h t t p : / / w w w. a t c c . o r g / P r o d u c t s / A l l / 2 5 2 0 4 .aspx#culturemethod]
35. European Pharmacopoeia - Ph eur. supplement 6.1, 2.6.7 mycoplasmas, 6th ed., Council of europe, strasbourg; 2008. p.3317-3321.
36. Milne C, Daas A. establishment of european Pharmacopoeia mycoplasma Reference strains. Pharmeuropa Bio. 2006;(1):57-72.
37. Asarnow D, warford A, Fernandez L, hom J, sandhu G, Candichoy Z, et al. Validation and international regulatory experience for a mycoplasma touchdown PCR assay. Biologicals. 2010;38:224-231. [DoI: https://dx.doi.org/10.1016/j.biologicals.2009.11.006].
38. Dabrazhynetskaya A, Volokhov DV, David sw, Ikonomi P, Brewer A, Chang A, et al Preparation of reference strains for validation and comparison of mycoplasma testing methods. J Appl microbiol. 2011;111:904–914. [DoI: https://dx.doi.org/10.1111/j.1365-2672.2011.05108.x].
39. Chen X, Finch LR. novel Arrangement of rRnA Genes in mycoplasma gallisepticum: separation of the 16s Gene of one set from the 23s and 5s Genes. J Bacteriol. 1989;171(5):2876-8.
40. Skamrov A, Goldman m, klasova J, Beabealashvilli R. mycoplasma gallisepticum 16s rRnA genes. Fems microbiol Lett. 1995;128:321–325. [DoI: https://dx.doi.org/10.1111/j.1574-6968.1995.tb07543.x].
This work is licensed under a Creative Commons Attribution 4.0 International License.
Copyright (c) 2016 Instituto Adolfo Lutz Journal