Overcoming the Challenges of Lyme Disease Diagnosis: The Role of Phage-based Testing

Main Article Content

Ying Jia Tatjana Mijatovic Louis Teulières Martha Clokie Jinyu Shan

Abstract

Tick-borne diseases are a growing concern worldwide, affecting both human and animal populations. Ticks are known to harbour a wide range of pathogens and are considered one of the most important vectors of diseases. Lyme disease, caused by Borrelia burgdorferi sensu lato, is the most common tick-borne disease in the US and Europe. However, accurate diagnosis of Lyme disease can be challenging due to the complex immune evasion strategies employed by Borrelia species and the limitations of existing diagnostic tests. To address this issue, researchers are exploring novel approaches, including the use of bacteriophages as diagnostic tools. Bacteriophages are highly specific and offer advantages over traditional methods for detecting bacteria, including Borrelia. In particular, the use of multicopy bacteriophages as molecular markers for Borrelia detection is a promising approach that may provide greater sensitivity than targeting single-copy bacterial genes. Nonetheless, the task of identifying trace amounts of bacteriophages in blood samples necessitates attention, and scientists are devising innovative techniques to surmount this hurdle. In summary, employing bacteriophages as a diagnostic tool for Lyme disease, by specifically targeting free circulating bacteriophages in blood samples, offers significant potential for enhancing patient outcomes and public health. However, additional rigorous scientific validation is required to definitively ascertain the efficacy and accuracy of using a phage-based methodology for detecting Borrelia in blood samples.

Keywords: Tick-borne diseases, Lyme disease, Diagnostic methods, Polymerase chain reaction (PCR), Bacteriophages (phages), Borrelia, Detection limit

Article Details

How to Cite
JIA, Ying et al. Overcoming the Challenges of Lyme Disease Diagnosis: The Role of Phage-based Testing. Medical Research Archives, [S.l.], v. 11, n. 11, nov. 2023. ISSN 2375-1924. Available at: <https://esmed.org/MRA/mra/article/view/4650>. Date accessed: 13 apr. 2024. doi: https://doi.org/10.18103/mra.v11i11.4650.
Section
Research Articles

References

1. Rochlin I, Toledo A. Emerging tick-borne pathogens of public health importance: a mini-review. J Med Microbiol. Jun 2020;69(6):781-791. doi:10.1099/jmm.0.001206
2. Johnson L, Wilcox S, Mankoff J, Stricker RB. Severity of chronic Lyme disease compared to other chronic conditions: a quality of life survey. PeerJ. 2014;2:e322. doi:10.7717/peerj.322
3. Marques AR. Laboratory diagnosis of Lyme disease: advances and challenges. Research Support, N I H , Intramural Review. Infect Dis Clin North Am. 2015;29(2):295-307.
4. Pegalajar-Jurado A, Schriefer ME, Welch RJ, et al. Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Laboratory Diagnosis Using Well-Characterized Serum Samples. J Clin Microbiol. 2018;56(8):e01943-17. doi:doi:10.1128/JCM.01943-17
5. Branda JA, Body BA, Boyle J, et al. Advances in Serodiagnostic Testing for Lyme Disease Are at Hand. Clin Infect Dis. 2017;66(7):1133-1139. doi:10.1093/cid/cix943
6. Pritt BS, Mead PS, Johnson DKH, et al. Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. Lancet Infect Dis. May 2016;16(5):556-564. doi:10.1016/s1473-3099(15)00464-8
7. Nepal R, Houtak G, Wormald P-J, Psaltis AJ, Vreugde S. Prophage: a crucial catalyst in infectious disease modulation. The Lancet Microbe. 2022;3(3):e162-e163. doi:10.1016/S2666-5247(21)00354-2
8. Shan J, Clokie MR, Teulières L, inventors; Borrelia phage. UK patent application PCT/GB2017/053323. 2018.
9. Shan J, Patel KV, Hickenbotham PT, Nale JY, Hargreaves KR, Clokie MR. Prophage carriage and diversity within clinically relevant strains of Clostridium difficile. Research Support, Non-U S Gov't. Appl Environ Microbiol. 2012;78(17):6027-34.
10. Beinhauerova M, Slana I. Phage Amplification Assay for Detection of Mycobacterial Infection: A Review. Microorganisms. Jan 23 2021;9(2)doi:10.3390/microorganisms9020237
11. Fuente Jdl, Estrada-Pena A, Venzal JM, Kocan KM, Sonenshine DE. Overview: Ticks as vectors of pathogens that cause disease in humans and animals. FBL. 2008-05-01 2008;13(18):6938-6946. doi:10.2741/3200
12. Parola P, Raoult D. Ticks and Tickborne Bacterial Diseases in Humans: An Emerging Infectious Threat. Clin Infect Dis. 2001;32(6):897-928. doi:10.1086/319347
13. Parola P. Tick-borne rickettsial diseases: emerging risks in Europe. Comp Immunol Microbiol Infect Dis. Sep 2004;27(5):297-304. doi:10.1016/j.cimid.2004.03.006
14. Kugeler K, Schwartz A, Delorey M, Mead P, Hinckley A. Estimating the Frequency of Lyme Disease Diagnoses, United States, 2010–2018. Emerg Infect Dis. 2021;27(2):616. doi:10.3201/eid2702.202731
15. Sykes RA, Makiello P. An estimate of Lyme borreliosis incidence in Western Europe. J Public Health (Oxf). Mar 1 2017;39(1):74-81. doi:10.1093/pubmed/fdw017
16. Organization WH. Vector-borne diseases. https://www.who.int/news-room/fact-sheets/detail/vector-borne-diseases
17. Gray JS, Dautel H, Estrada-Peña A, Kahl O, Lindgren E. Effects of climate change on ticks and tick-borne diseases in Europe. Interdiscip Perspect Infect Dis. 2009;2009:593232. doi:10.1155/2009/593232
18. Murray TS, Shapiro ED. Lyme disease. Clin Lab Med. Mar 2010;30(1):311-28. doi:10.1016/j.cll.2010.01.003
19. Dubrey SW, Bhatia A, Woodham S, Rakowicz W. Lyme disease in the United Kingdom. Postgrad Med J. Jan 2014;90(1059):33-42. doi:10.1136/postgradmedj-2012-131522
20. Stanek G, Wormser GP, Gray J, Strle F. Lyme borreliosis. Review. Lancet. 2012;379(9814):461-73.
21. Margos G, Gofton A, Wibberg D, et al. The genus Borrelia reloaded. Research Support, Non-U S Gov't. PLoS One. 2018;13(12)
22. Margos G, Hepner S, Mang C, Sing A, Liebl B, Fingerle V. Completed genome sequences of Borrelia burgdorferi sensu stricto B31(NRZ) and closely related patient isolates from Europe. Genome Announc. 2017;5(28):e00637-17. doi:10.1128/genomeA.00637-17
23. Fukunaga M, Takahashi Y, Tsuruta Y, et al. Genetic and Phenotypic Analysis of Borrelia miyamotoi sp. nov., Isolated from the Ixodid Tick Ixodes persulcatus, the Vector for Lyme Disease in Japan. Int J Syst Evol Microbiol. 1995;45(4):804-810. doi:https://doi.org/10.1099/00207713-45-4-804
24. Xu G, Luo CY, Ribbe F, Pearson P, Ledizet M, Rich SM. Borrelia miyamotoi in Human-Biting Ticks, United States, 2013-2019. Emerg Infect Dis. Dec 2021;27(12):3193-3195. doi:10.3201/eid2712.204646
25. Platonov AE, Karan LS, Kolyasnikova NM, et al. Humans infected with relapsing fever spirochete Borrelia miyamotoi, Russia. Emerg Infect Dis. Oct 2011;17(10):1816-23. doi:10.3201/eid1710.101474
26. Wormser GP, Dattwyler RJ, Shapiro ED, et al. The clinical assessment, treatment, and prevention of lyme disease, human granulocytic anaplasmosis, and babesiosis: clinical practice guidelines by the Infectious Diseases Society of America. Practice Guideline. Clin Infect Dis. 2006;43(9):1089-134.
27. Lantos PM, Rumbaugh J, Bockenstedt LK, et al. Clinical Practice Guidelines by the Infectious Diseases Society of America (IDSA), American Academy of Neurology (AAN), and American College of Rheumatology (ACR): 2020 Guidelines for the Prevention, Diagnosis and Treatment of Lyme Disease. Clin Infect Dis. 2020;72(1):e1-e48. doi:10.1093/cid/ciaa1215
28. Marques A. Chronic Lyme Disease: A Review. Infect Dis Clin N Am. 2008/06/01/ 2008;22(2):341-360. doi:https://doi.org/10.1016/j.idc.2007.12.011
29. Lantos PM. Chronic Lyme disease. Infect Dis Clin North Am. 2015;29(2):325-340. doi:10.1016/j.idc.2015.02.006
30. Aucott JN, Rebman AW, Crowder LA, Kortte KB. Post-treatment Lyme disease syndrome symptomatology and the impact on life functioning: is there something here? Qual Life Res. 2013/02/01 2013;22(1):75-84. doi:10.1007/s11136-012-0126-6
31. Horowitz RI, Freeman PR. Precision medicine: retrospective chart review and data analysis of 200 patients on dapsone combination therapy for chronic Lyme disease/post-treatment Lyme disease syndrome: part 1. Int J Gen Med. 2019;12:101-119. doi:10.2147/ijgm.S193608
32. Moore A, Nelson C, Molins C, Mead P, Schriefer M. Current guidelines, common clinical pitfalls, and future directions for laboratory diagnosis of lyme disease, United States. Review. Emerg Infect Dis. 2016;22(7):1169-77.
33. Schutzer SE, Body BA, Boyle J, et al. Direct diagnostic tests for Lyme disease. Research Support, N I H , Extramural Research Support, N I H , Intramural Research Support, Non-U S Gov't. Clin Infect Dis. 2019;68(6):1052-1057.
34. Coulter P, Lema C, Flayhart D, et al. Two-year evaluation of Borrelia burgdorferi culture and supplemental tests for definitive diagnosis of Lyme disease. J Clin Microbiol. Oct 2005;43(10):5080-4. doi:10.1128/jcm.43.10.5080-5084.2005
35. Klempner MS, Schmid CH, Hu L, et al. Intralaboratory reliability of serologic and urine testing for Lyme disease. Am J Med. Feb 15 2001;110(3):217-9. doi:10.1016/s0002-9343(00)00701-4
36. Angel TE, Luft BJ, Yang X, et al. Proteome analysis of Borrelia burgdorferi response to environmental change. PLoS One. Nov 2 2010;5(11):e13800. doi:10.1371/journal.pone.0013800
37. Bil-Lula I, Matuszek P, Pfeiffer T, Woźniak M. Lyme Borreliosis--the Utility of Improved Real-Time PCR Assay in the Detection of Borrelia burgdorferi Infections. Adv Clin Exp Med. Jul-Aug 2015;24(4):663-70. doi:10.17219/acem/28625
38. Lohr B, Fingerle V, Norris DE, Hunfeld K-P. Laboratory diagnosis of Lyme borreliosis: Current state of the art and future perspectives. Crit Rev Clin Lab Sci. 2018/05/19 2018;55(4):219-245. doi:10.1080/10408363.2018.1450353
39. Eshoo MW, Crowder CC, Rebman AW, et al. Direct molecular detection and genotyping of Borrelia burgdorferi from whole blood of patients with early Lyme disease. PLoS One. 2012;7(5):e36825-e36825. doi:10.1371/journal.pone.0036825
40. Snyder JL, Giese H, Bandoski-Gralinski C, et al. T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples. J Clin Microbiol. Aug 2017;55(8):2453-2461. doi:10.1128/jcm.00510-17
41. Shan J, Jia Y, Teulières L, Patel F, Clokie MRJ. Targeting Multicopy Prophage Genes for the Increased Detection of Borrelia burgdorferi Sensu Lato (s.l.), the Causative Agents of Lyme Disease, in Blood. Original Research. Front Microbiol. 2021-March-15 2021;12 doi:10.3389/fmicb.2021.651217
42. Kuhar U, Barlič-Maganja D, Grom J. Development and validation of TaqMan probe based real time PCR assays for the specific detection of genotype A and B small ruminant lentivirus strains. BMC Vet Res. 2013/09/03 2013;9(1):172. doi:10.1186/1746-6148-9-172
43. Wei B, Chen L, Kibukawa M, Kang J, Waskin H, Marton M. Development of a PCR Assay to detect low level Trypanosoma cruzi in blood specimens collected with PAXgene blood DNA tubes for clinical trials treating chagas disease. PLoS Negl Trop Dis. Dec 2016;10(12):e 0005146. doi:10.1371/journal.pntd.0005146
44. O'Rourke M, Traweger A, Lusa L, et al. Quantitative detection of Borrelia burgdorferi sensu lato in erythema migrans skin lesions using internally controlled duplex real time PCR. PLoS One. 2013;8(5):e63968. doi:10.1371/journal.pone.0063968
45. Primus S, Akoolo L, Schlachter S, Gedroic K, Rojtman AD, Parveen N. Efficient detection of symptomatic and asymptomatic patient samples for Babesia microti and Borrelia burgdorferi infection by multiplex qPCR. PloS one. 2018;13(5):e0196748-e0196748. doi:10.1371/journal.pone.0196748
46. Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol. Oct 1997;63(10):3741-51. doi:10.1128/aem.63.10.3741-3751.1997
47. Akane A, Matsubara K, Nakamura H, Takahashi S, Kimura K. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci. Mar 1994;39(2):362-72.
48. Al-Soud WA, Rådström P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol. Feb 2001;39(2):485-93. doi:10.1128/jcm.39.2.485-493.2001
49. Taylor SC, Laperriere G, Germain H. Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data. Sci Rep. 2017/05/25 2017;7(1):2409. doi:10.1038/s41598-017-02217-x
50. Leth TA, Joensen SM, Bek-Thomsen M, Møller JK. Establishment of a digital PCR method for detection of Borrelia burgdorferi sensu lato complex DNA in cerebrospinal fluid. Sci Rep. Nov 21 2022;12(1):19991. doi:10.1038/s41598-022-24041-8
51. Das S, Hammond_McKibben D, Guralski D, Lobo S, Fiedler PN. Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from blood of Lyme disease patients by digital PCR. bioRxiv. 2020:2020.06.16.154336. doi:10.1101/2020.06.16.154336
52. Metzker ML. Sequencing technologies — the next generation. Nature Reviews Genetics. 2010/01/01 2010;11(1):31-46. doi:10.1038/nrg2626
53. Caboche S, Audebert C, Hot D. High-Throughput Sequencing, a VersatileWeapon to Support Genome-Based Diagnosis in Infectious Diseases: Applications to Clinical Bacteriology. Pathogens. Apr 2 2014;3(2):258-79. doi:10.3390/pathogens3020258
54. Forshew T, Murtaza M, Parkinson C, et al. Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA. Sci Transl Med. 2012;4(136):136ra68-136ra68. doi:doi:10.1126/scitranslmed.3003726
55. Madugundu AK, Muthusamy B, Sreenivasamurthy SK, et al. A Next-Generation Sequencing-Based Molecular Approach to Characterize a Tick Vector in Lyme Disease. Omics. Aug 2018;22(8):565-574. doi:10.1089/omi.2018.0089
56. Abril MK, Barnett AS, Wegermann K, et al. Diagnosis of Capnocytophaga canimorsus Sepsis by Whole-Genome Next-Generation Sequencing. Open Forum Infect Dis. Sep 2016;3(3):ofw144. doi:10.1093/ofid/ofw144
57. Handel AS, Ho C, Hollemon DD, Hong DK, Beneri C. 231. Microbial cell-free DNA Sequencing to Detect Borrelia burgdorferi DNA in the Plasma of Pediatric Patients with Lyme Disease. Open Forum Infect Dis. 2019;6(Supplement_2):S133-S133. doi:10.1093/ofid/ofz360.306
58. van Oorschot RA, Ballantyne KN, Mitchell RJ. Forensic trace DNA: a review. Investig Genet. Dec 1 2010;1(1):14. doi:10.1186/2041-2223-1-14
59. Butler JM. The future of forensic DNA analysis. Philos Trans R Soc Lond, B, Biol Sci. 2015;370(1674):20140252. doi:doi:10.1098/rstb.2014.0252
60. Hill CR, Duewer DL, Kline MC, et al. Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex® ESX 17 and ESI 17 Systems. Forensic Sci Int Genet. 2011/08/01/ 2011;5(4):269-275. doi:https://doi.org/10.1016/j.fsigen.2010.03.014
61. Chatfield L. Forensic DNA Typing: Biology and Technology behind STR Markers. Heredity. 2002/10/01 2002;89(4):327-327. doi:10.1038/sj.hdy.6800124
62. Gill P, Whitaker J, Flaxman C, Brown N, Buckleton J. An investigation of the rigor of interpretation rules for STRs derived from less than 100 pg of DNA. Forensic Sci Int Genet. 2000/07/24/ 2000;112(1):17-40. doi:https://doi.org/10.1016/S0379-0738(00)00158-4
63. Abu Al-Soud W, Rådström P. Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol. Dec 2000;38(12):4463-70. doi:10.1128/jcm.38.12.4463-4470.2000
64. Pratama AA, Chaib De Mares M, van Elsas JD. Evolutionary history of bacteriophages in the genus Paraburkholderia. Original Research. Front Microbiol. 2018-May-11 2018;9(835)doi:10.3389/fmicb.2018.00835
65. Carrias A, Welch TJ, Waldbieser GC, Mead DA, Terhune JS, Liles MR. Comparative genomic analysis of bacteriophages specific to the channel catfish pathogen Edwardsiella ictaluri. Virol J. 2011;8:6-6. doi:10.1186/1743-422X-8-6
66. Xu J, Chau Y, Lee YK. Phage-based Electrochemical Sensors: A Review. Micromachines (Basel). Dec 6 2019;10(12) doi:10.3390/mi10120855
67. Hussain W, Ullah MW, Farooq U, Aziz A, Wang S. Bacteriophage-based advanced bacterial detection: Concept, mechanisms, and applications. Biosens Bioelectron. Apr 1 2021;177:112973. doi:10.1016/j.bios.2021.112973
68. Richter Ł, Janczuk-Richter M, Niedziółka-Jönsson J, Paczesny J, Hołyst R. Recent advances in bacteriophage-based methods for bacteria detection. Drug Discov Today. Feb 2018;23(2):448-455. doi:10.1016/j.drudis.2017.11.007
69. Shield CG, Swift BMC, McHugh TD, Dedrick RM, Hatfull GF, Satta G. Application of Bacteriophages for Mycobacterial Infections, from Diagnosis to Treatment. Microorganisms. Nov 16 2021;9(11) doi:10.3390/microorganisms9112366
70. Shkoporov AN, Hill C. Bacteriophages of the Human Gut: The “Known Unknown” of the Microbiome. Cell Host & Microbe. 2019/02/13/ 2019;25(2):195-209. doi:https://doi.org/10.1016/j.chom.2019.01.017
71. Clokie MR, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage. Jan 2011;1(1):31-45. doi:10.4161/bact.1.1.14942
72. Haddock NL, Barkal LJ, Ram-Mohan N, et al. The circulating phageome reflects bacterial infections. bioRxiv. 2023:2022.08.15.504009. doi:10.1101/2022.08.15.504009
73. Waller AS, Yamada T, Kristensen DM, et al. Classification and quantification of bacteriophage taxa in human gut metagenomes. The ISME Journal. 2014/07/01 2014;8(7):1391-1402. doi:10.1038/ismej.2014.30
74. Bakhshinejad B, Ghiasvand S. Bacteriophages in the human gut: Our fellow travelers throughout life and potential biomarkers of heath or disease. Review. Virus Res. 2017;240:47-55.
75. Londono-Avendano MA, Libreros G, Osorio L, Parra B. A Rapid RT-LAMP Assay for SARS-CoV-2 with Colorimetric Detection Assisted by a Mobile Application. Diagnostics (Basel). Mar 29 2022;12(4)doi:10.3390/diagnostics12040848
76. Dao Thi VL, Herbst K, Boerner K, et al. A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples. Sci Transl Med. Aug 12 2020;12(556)doi:10.1126/scitranslmed.abc7075
77. Chan BK, Turner PE, Kim S, Mojibian HR, Elefteriades JA, Narayan D. Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol Med Public Health. 2018;2018(1):60-66. doi:10.1093/emph/eoy005
78. Manrique P, Bolduc B, Walk ST, van der Oost J, de Vos WM, Young MJ. Healthy human gut phageome. Proc Natl Acad Sci U S A. Sep 13 2016;113(37):10400-5. doi:10.1073/pnas.1601060113
79. Canchaya C, Proux C, Fournous G, Bruttin A, Brüssow H. Prophage genomics. Microbiol Mol Biol Rev. 2003;67(2):238-276. doi:10.1128/MMBR.67.2.238-276.2003
80. Argov T, Sapir SR, Pasechnek A, et al. Coordination of cohabiting phage elements supports bacteria–phage cooperation. Nat Commun. 2019/11/21 2019;10(1):5288. doi:10.1038/s41467-019-13296-x
81. Gaidelyte A, Vaara M, Bamford DH. Bacteria, phages and septicemia. PloS one. 2007;2(11):e1145-e1145. doi:10.1371/journal.pone.0001145
82. Kwon HJ, Seong WJ, Kim JH. Molecular prophage typing of avian pathogenic Escherichia coli. Research Support, Non-U S Gov't. Vet Microbiol. 2013;162(2-4):785-792.
83. Mccarthy AJ, Witney AA, Lindsay JA. Staphylococcus aureus lysogenic bacteriophage: carriage and horizontal gene transfer (HGT) is lineage associated. Original Research. Front Cell Inf Microbio. 2012-February-8 2012;2(6) doi:10.3389/fcimb.2012.00006.
84. Baker AC, Goddard VJ, Davy J, Schroeder DC, Adams DG, Wilson WH. Identification of a diagnostic marker to detect freshwater cyanophages of filamentous cyanobacteria. Appl Environ Microbiol. Sep 2006;72(9):5713-9. doi:10.1128/aem.00270-06
85. Damman CJ, Eggers CH, Samuels DS, Oliver DB. Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system. Research Support, Non-U S Gov't Research Support, U S Gov't, Non-P H S Research Support, U S Gov't, P H S. J Bacteriol. 2000;182(23):6791-7.
86. Eggers C, Samuels DS. Molecular Evidence for a New Bacteriophage of Borrelia burgdorferi. J Bacteriol. 2000;181:7308-13.
87. Eggers CH, Kimmel BJ, Bono JL, Elias AF, Rosa P, Samuels DS. Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi. J Bacteriol. 2001;183(16):4771-4778. doi:10.1128/jb.183.16.4771-4778.2001
88. Liang L, Wang J, Schorter L, et al. Rapid clearance of Borrelia burgdorferi from the blood circulation. Parasit Vectors. 2020/04/21 2020;13(1):191. doi:10.1186/s13071-020-04060-y
89. Rosa PA, Tilly K, Stewart PE. The burgeoning molecular genetics of the Lyme disease spirochaete. Nat Rev Microbiol. Feb 2005;3(2):129-43. doi:10.1038/nrmicro1086
90. Łusiak-Szelachowska M, Weber-Dąbrowska B, Żaczek M, Borysowski J, Górski A. The Presence of Bacteriophages in the Human Body: Good, Bad or Neutral? Microorganisms. Dec 16 2020;8(12)doi:10.3390/microorganisms8122012
91. Zuckert WR. Laboratory maintenance of Borrelia burgdorferi. Curr Protoc Microbiol. 2007;12(1)
92. Barbour AG, Hayes SF. Biology of Borrelia species. Microbiol Rev. 1986;50(4):381-400. doi:doi:10.1128/mr.50.4.381-400.1986
93. Metz CE. Basic principles of ROC analysis. Semin Nucl Med. 1978/10/01/ 1978;8(4):283-298. doi:https://doi.org/10.1016/S0001-2998(78)80014-2
94. Hanley JA, McNeil BJ. The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology. Apr 1982;143(1):29-36. doi:10.1148/radiology.143.1.7063747
95. Brettschneider S, Bruckbauer H, Klugbauer N, Hofmann H. Diagnostic value of PCR for detection of Borrelia burgdorferi in skin biopsy and urine samples from patients with skin borreliosis. J Clin Microbiol. 1998;36(9):2658-2665.
96. Ellison SLR, English CA, Burns MJ, Keer JT. Routes to improving the reliability of low level DNA analysis using real-time PCR. BMC Biotechnol. 2006;6:33-33. doi:10.1186/1472-6750-6-33
97. Bachmann LM, Jüni P, Reichenbach S, Ziswiler HR, Kessels AG, Vögelin E. Consequences of different diagnostic "gold standards" in test accuracy research: Carpal Tunnel Syndrome as an example. Int J Epidemiol. Aug 2005;34(4):953-5. doi:10.1093/ije/dyi105
98. Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. Apr 2009;55(4):611-22. doi:10.1373/clinchem.2008.112797