1. Introduction
More than 500,000 new cases of Lyme disease are diagnosed each year in the USA, making it the most common vector-borne disease of North America [1] [2]. If the acute spirochetal infection is not adequately addressed due to variable symptoms, poor diagnostic test sensitivity and a lack of clinical biomarkers, patients may develop chronic Lyme disease (CLD), which remains a controversial illness [3]. At the heart of this controversy lies a profound disagreement over the existence of persistent infection with the Lyme spirochete, Borrelia burgdorferi, and the ability of this persistent infection to cause chronic symptoms in patients who are untreated or undertreated for the spirochetal disease. CLD constitutes a significant health care burden, costing the US healthcare system nearly 1.3 billion dollars annually [4].
CLD is a multisystem illness with diverse musculoskeletal, neuropsychiatric and/or cardiovascular manifestations [3]. The disease is associated with pathogenic members of the Borrelia spirochete complex often in combination with other tickborne disease (TBD) pathogens. To qualify for the diagnosis of CLD, patients must have Lyme-compatible symptoms and signs that are either consistently or variably present for six or more months. Two subcategories of CLD include untreated chronic Lyme disease (CLD-U) and chronic Lyme disease following a limited course of antibiotic treatment (CLD-T), as defined elsewhere [3].
Although some earlier infectious disease articles maintain that there is no “credible scientific evidence” for persistent infection with B. burgdorferi following 2 - 4 weeks of antibiotic therapy [5], a number of animal and human studies provide evidence for persistent infection as a cause of chronic symptoms in Lyme disease patients, thereby contradicting the earlier infectious disease point of view [1] [6] (Table A1 and Table A2). Alternative explanations for persistent symptoms in CLD include infection-induced immune dysfunction, inflammation due to persistent bacteria, bacterial “debris” and genetic and other mechanisms [7].
Unfortunately, the CLD controversy often results in misdiagnosis, inadequate treatment, and medical gaslighting that contribute to patient suffering and persistent infection [6] [8]. The failure to recognize persistent infection as a cause of CLD has a detrimental health impact on patients and society because these patients are often denied antibiotic treatment that may restore their health. These patients have worse quality of life than many other chronic disease conditions, including diabetes, multiple sclerosis, congestive heart failure, and arthritis [6].
This literature review presents the evidence for persistent infection with B. burgdorferi and provides a resource for academics and clinicians who require further validation for CLD.
2. Methods
We conducted a review of the medical literature to identify studies demonstrating persistent Borrelia infection in both animal models and humans. Two data bases (PubMed and Google Scholar) were searched using key words such as Borrelia, B. burgdorferi, Lyme disease, chronic, persistent, and infection. The literature was reviewed and categorized based on whether the subjects were animal or human and subcategories were created based on animal type. Animal studies were included if direct or functional testing techniques for B. burgdorferi were used, including culture, histology, xenodiagnosis, polymerase chain reaction (PCR) and/or RNA in-situ hybridization. Antibiotic therapy was not a requirement for this group, and length and sites of infection were noted. Human studies were only included if direct detection of Borrelia sequences or organisms was noted using culture, histology, xenodiagnosis, PCR and/or fluorescent in situ hybridization (FISH) following antibiotic treatment.
3. Results
We identified 24 animal studies (13 rodent, 2 canine, 7 monkey, and 2 horse) and 32 human studies supporting persistence of Borrelia infection (Table A1 and Table A2). Borrelia was identified in various sample sites from 60 days to 46 months following infection. Persistent detection of Borrelia sequences following antibiotic treatment was found in 13 of 24 animal studies and 31 of 32 human studies. In 10 animal studies and 25 human studies viable spirochetes were demonstrated by culture, histology and/or xenodiagnosis following antibiotic treatment. The primary methods used to identify Borrelia in the animal and human studies included culture (13 animal, 15 human), histology (18 animal, 11 human), xenodiagnosis (4 animal, 1 human), and PCR (15 animal, 12 human). Additional modes of detection can be found in Table A1 and Table A2. Detection of Borrelia organisms was described in 21 (88%) animal studies and 26 (81%) human studies. The remaining studies only used PCR for molecular detection of spirochete sequences.
4. Discussion
After performing a literature review, we identified 56 studies showing that B. burgdorferi was detectable in longterm culture (Table A1 and Table A2). Viable spirochetes were identified by culture, histology and/or xenodiagnosis following antibiotic treatment in 10 animal studies and 25 human studies, demonstrating that live organisms can persist following this treatment. These findings contradict the earlier contention that the Lyme spirochete cannot survive antibiotics. Further evidence for evasion of the immune response and antibiotic therapy is described below.
A monkey study by Embers et al. published in 2012 provides the best animal evidence for persistent infection as a mechanism for CLD [9]. The study was conceived as an animal counterpart to the human trial by Klempner et al. that was published in 2001 [10], and the monkeys were treated with a regimen of intravenous ceftriaxone followed by oral doxycycline that was identical to the protocol used in the human trial. The results of this study showed that three-quarters of the monkeys failed treatment, and these animals had evidence of persistent infection in various tissues at necropsy using culture, immunofluorescence and PCR techniques [9]. Equally important, the study showed that 25% of treated monkeys cleared their infection, thereby demonstrating antibiotic efficacy in some animals. This finding contradicts the negative treatment results reported by Klempner et al. in humans to support the conclusion that antibiotics are not effective in treating patients with persistent Lyme disease symptoms [11]. In short, Embers was able to demonstrate persistence using an invasive approach (necropsy) that could not be used in human clinical trials [9] [12].
In another study, Bockenstedt et al. presented a mouse model of B. burgdorferi infection that on the surface appears to contradict the monkey study [13]. Following infection, the mice were treated with subcutaneous ceftriaxone or doxycycline administered in drinking water. The authors arrive at the conclusion that non-infectious spirochetal “debris” gets deposited around the joints of these mice, and instead of being cleared by the reticuloendothelial system this “debris” is responsible for persistent inflammation in mouse tissues [13]. The “debris”, which contained both DNA and protein particles, could not be cultured, transmitted to other mice via ear transplants or to ticks that were allowed to feed on the mice (xenodiagnosis).
This novel hypothesis of non-infectious persistence of B. burgdorferi “debris” including the presence of DNA contradicts previous experimental results. For example, Malawista et al. showed that B. burgdorferi DNA is rapidly cleared from culture-negative ear and bladder tissues of mice following prompt antibiotic treatment [14], and Lazarus et al. demonstrated that DNA from dead spirochetes is routinely cleared from mouse skin within several hours [15]. The “debris” hypothesis fails to explain persistence of viable spirochetes in culture, histology and xenodiagnosis experiments following antibiotic therapy. Furthermore, the study methods of Bockenstedt et al. may have been insufficient to rule out persistent spirochetal forms of B. burgdorferi, since ear transplants are often negative following antibiotic treatment, and using an insufficient number of animals for xenodiagnosis may fail to demonstrate transmissible infection [16] [17]. Of greater importance, there appear to be two alternative mechanisms of B. burgdorferi persistence that merit consideration in these mice: the persistence of cysts (L-forms) and the inability to detect spirochetes in biofilms.
In a commentary on the mouse study, Alan Barbour proposed the alternative hypothesis that cell-wall deficient cysts (L-forms) may be responsible for B. burgdorferi persistence in these animals [18]. He noted that these cystic structures, which Bockenstedt et al. observed in their infected animals, have been described as a persister mechanism employed by many bacteria, including B. burgdorferi [19]-[27]. Bockenstedt et al. claim that these are not true cysts because they form too fast, appearing in minutes rather than hours or days. However, Brorson and Brorson have demonstrated that cysts of B. burgdorferi may develop in minutes under appropriate culture conditions [28]. Thus the observation of Bockenstedt et al. supports B. burgdorferi cyst formation in their mouse model, and this cyst formation appears to be a better explanation for spirochetal persistence compared to the “debris” that the authors postulate.
As noted above, the methods employed by Bockenstedt et al. may not have been sufficient to exclude other persistent spirochetal forms such as cysts (L-forms) in their animals. Persistent viable organisms also may have been hidden in biofilms, the adherent polysaccharide-based matrices that protect bacteria against the host immune system and antibiotic therapy [1]. Biofilms of B. burgdorferi have been demonstrated in vitro by Sapi et al. [29]. These biofilms may take the form of “debris” on intravital microscopy, and they may contain organisms that are non-cultivable but still viable and prone to reactivation [29]-[31]. Biofilms of B. burgdorferi would also be consistent with the “amber hypothesis” proposed as a mechanism of persistent Lyme disease symptoms due to “introduction into the joint space of non-viable spirochetes or spirochetal debris enmeshed in a host-derived fibrinous or collagenous matrix” [32]. Like the “debris” hypothesis, the “amber” hypothesis fails to explain live Borrelia persistence after antibiotic therapy. Persister spirochetes in biofilms could explain the experimental results of Bockenstedt et al. and would offer a more plausible explanation than the “debris” and “amber” hypotheses for the reasons outlined above.
Recently McClune et al. presented evidence that B. burgdorferi peptidoglycan (PG) can persist in synovial fluid after murine infection and may cause symptoms compatible with CLD [33]. McCausland et al. demonstrated unusual properties of the spirochete-derived PG that allow it to persist in mouse liver for weeks [34]. Although these observations may support the “debris” hypothesis of CLD, they do not rule out persistent B. burgdorferi infection in cysts (L-forms) and biofilms. Further work is needed to examine the relationship between PG-induced inflammation and persistent spirochete infection in CLD.
Like most aspects of Lyme disease, the role of cysts (L-forms) and biofilms in persistent B. burgdorferi infection has been controversial [1] [30] [35]. These spirochetal forms are resistant to common antibiotics due to reduced metabolic activity or protective matrices. Whether CLD arises from persisting spirochetal forms hidden in biofilms (as suggested by the monkey studies of Embers et al. and the work of Sapi et al.) or from cell wall-deficient cysts (L-forms) of B. burgdorferi (as suggested by the mouse study observations of Bockenstedt et al. and the interpretation of Barbour), persisting forms of bacteria require treatment. To date the treatment options for these bacterial persisters are extremely limited, but their recognition dictates a more aggressive approach to eradication of Lyme disease using combination antibiotic therapy modeled on treatment regimens for tuberculosis and HIV disease [2]. The fact that B. burgdorferi shares cyst (L-form) properties, biofilm configurations and resistance genes with pathogenic mycobacteria supports the need for this therapeutic approach [36] [37]. It remains to be seen which forms of B. burgdorferi are the true culprits in CLD and which treatments are most efficacious in clearing infection from patients [38]-[48].
5. Strengths and Limitations
The strengths of this review lie in the demonstration of longterm Borrelia infection in animal models and persistent infection following antibiotic therapy in animals and humans. Although various detection techniques were used, the culture, histology and xenodiagnosis testing identified viable spirochetes that persisted for long periods in animal models and survived antibiotics in animal and human cases. Although conventional PCR is a useful detection method, it can only detect fragments of the spirochete, leaving the door open for the “debris” hypothesis. Overall, this review confirms that Borrelia can persist despite treatment, but the exact mechanism for this observation remains to be determined. Although this study was not a systematic review of the literature, we sought to include as many studies as possible that demonstrate viable Borrelia persistence through direct detection techniques following antibiotic therapy. Future more robust reviews are required to strengthen the level of evidence for the conclusions drawn from this study.
6. Conclusion
In this article, we summarize evidence from animal models and human studies that support persistent spirochetal infection as the cause of CLD. Specifically, direct and functional testing using culture, histology and xenodiagnosis has shown viable organisms following antibiotic therapy, and the role of cysts (L-forms) and biofilms in this process is highlighted. Determining the mechanism behind Borrelia persistence may hold the key to development of targeted treatments for CLD.
Authors’ Contributions
Raphael B. Stricker, Melissa C. Fesler and Lorraine Johnson meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE). All authors made substantial contributions to the conception, design and revisions of the current article and were involved in the analysis and interpretation of data. All authors have approved the final version.
Acknowledgements
The authors thank Joseph Burrascano, Michael Cook, Christine Green, Steven Harris, Erica Lehman, Ken Liegner, Marianne Middelveen, Eva Sapi, John Scott, Jyotsna Shah, Carl Tuttle, Karen Vanderhoof-Forschner and Edward Winger for helpful discussion. This article is dedicated to the memory of Pat Smith and Alan MacDonald.
Appendix
Table A1. Evidence for persistent infection in animal models of Lyme disease*.
Study/Year/Reference |
Animal Origin |
Persistence of B. burgdorferi Shown by |
B. burgdorferi Detection** |
Sample Source |
1. Rodents |
|
|
|
|
Preac-Mursic et al., 19901 |
Gerbils |
Culture, Histology |
6 months |
Joints, Skin
Spleen |
Duray & Johnson, 19862 |
Hamsters |
Culture, Histology |
9 months |
Spleen, Kidney
Eye |
Goodman et al.,
19913 |
Hamsters |
Culture, Histology |
3 months |
Heart, Bladder |
Schmitz et al., 19914 |
Hamsters |
Culture, Histology |
16 months |
Synovium, Spleen |
Moody et al., 19905 |
Rats |
Culture, Histology |
12 months |
Spleen, Kidney
Joints |
Sonnesyn et al., 19946 |
Guinea
Pigs |
Culture, Histology |
16 weeks |
Bladder, Heart Spleen, Joints
Muscles |
Malawista et al., 19947 |
Mice |
Culture, PCR |
60 days† |
Ear, Bladder |
Moody et al., 19948 Bockenstedt et al., 20029 |
Mice
Mice |
Histology
PCR
Xenodiagnosis |
90 days† 12 weeks† |
Joints, Heart Joints, Bladder |
Hodzic et al., 200810 |
Mice |
PCR, Histology,Xenodiagnosis |
12 weeks† |
Joints, Heart |
Yrjänäinen et al., 201011 |
Mice |
PCR |
30 weeks† |
Joints |
Barthold et al., 201012 |
Mice |
PCR, Histology,Xenodiagnosis |
12 weeks† |
Joints, HeartMuscle |
Bockenstedt et al., 201213 |
Mice |
PCR, Histology |
12 weeks† |
Joints |
2. Dogs |
|
|
|
|
Straubinger et al., 199714 |
Dogs |
PCR, Histology |
3 - 6 months† |
Skin, LNJoints |
Straubinger, 200015 |
Dogs |
PCR |
500 days† |
Skin, Muscle Joints |
3. Monkeys |
|
|
|
|
Roberts et al., 199516 |
Monkeys |
Culture, PCR,Histology |
6 months |
Joints, Nerve |
Roberts et al., 199817 |
Monkeys |
Culture, PCR,Histology |
46 months |
Nerve |
Pachner et al., 200118 |
Monkeys |
Culture, PCR, Histology, |
3 months |
Brain, Nerve,Heart |
Cadavid et al., 200419 |
Monkeys |
Culture, PCR, Histology |
32 months |
Heart |
Miller et al., 200520 |
Monkeys |
PCR |
3 months |
Brain, Nerve, |
|
|
|
|
Heart, Muscle,
Skin, Bladder |
Embers et al., 201221 |
Monkeys |
Culture, Histology, |
6 - 12 months† |
Skin, Heart Bladder Joints |
|
|
PCR, Xenodiagnosis |
|
Tendon,
Spleen |
Crossland et al., 201822 |
Monkeys |
Histology, RNA ISH |
5 - 13 months† |
Nervous
System, |
|
|
|
|
Heart, |
|
|
|
|
Muscle, |
|
|
|
|
Synovium |
4. Horses |
|
|
|
|
Chang et al., 200523 |
Ponies |
Culture |
5 months† |
LN, Joints, Muscle |
Imai et al., 201124 |
Horses |
Histology, PCR |
1 - 4 years† |
Brain, Nerve |
*PCR, polymerase chain reaction; LN, lymph node; ISH, in situ hybridization, **Time from initial infection to final positive testing point. †Detectable B. burgdorferi following antibiotic treatment.
Table A1 References
1. Preac-Mursic V, Patsouris E, Wilske B, Reinhardt S, Gross B, Mehraein P. Persistence of Borrelia burgdorferi and histopathological alterations in experimentally infected animals. A comparison with histopathological findings in human Lyme disease. Infection 1990; 18: 332-41.
2. Duray PH, Johnson RC. The histopathology of experimentally infected hamsters with the Lyme disease spirochete, Borrelia burgdorferi. Proc Soc Exp Biol Med. 1986; 181: 263-9.
3. Goodman JL, Jurkovich P, Kodner C, Johnson RC. Persistent cardiac and urinary tract infections with Borrelia burgdorferi in experimentally infected Syrian hamsters. J Clin Microbiol. 1991; 29: 894-6.
4. Schmitz JL, Schell RF, Lovrich SD, Callister SM, Coe JE. Characterization of the protective antibody response to Borrelia burgdorferi in experimentally infected LSH hamsters. Infect Immun. 1991; 59: 1916-21.
5. Moody KD, Barthold SW, Terwilliger GA. Lyme borreliosis in laboratory animals: effect of host species and in vitro passage of Borrelia burgdorferi. Am J Trop Med Hyg. 1990; 43: 87-92.
6. Sonnesyn SW, Manivel JC, Johnson RC, Goodman JL. A guinea pig model for Lyme disease. Infect Immun. 1993; 61: 4777-84.
7. Malawista SE, Barthold SW, Persing DH. Fate of Borrelia burgdorferi DNA in tissues of infected mice after antibiotic treatment. J Infect Dis. 1994; 170: 1312-6.
8. Moody KD, Adams RL, Barthold SW. Effectiveness of antimicrobial treatment against Borrelia burgdorferi infection in mice. Antimicrob Agents Chemother. 1994; 38: 1567-72.
9. Bockenstedt LK, Mao J, Hodzic E, Barthold SW, Fish D. Detection of attenuated, noninfectious spirochetes in Borrelia burgdorferi-infected mice after antibiotic treatment. J Infect Dis. 2002; 186: 1430-7.
10. Hodzic E, Feng S, Holden K, Freet KJ, Barthold SW. Persistence of Borrelia burgdorferi following antibiotic treatment in mice. Antimicrob Agents Chemother. 2008; 52: 1728-36.
11. Yrjänäinen H, Hytönen J, Hartiala P, Oksi J, Viljanen MK. Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment. APMIS. 20101; 118: 665-73.
12. Barthold SW, Hodzic E, Imai DM, Feng S, Yang X, Luft BJ. Ineffectiveness of tigecycline against persistent Borrelia burgdorferi. Antimicrob Agents Chemother. 2010; 54: 643-51.
13. Bockenstedt LK, Gonzalez DG, Haberman AM, Belperron AA. Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy. J Clin Invest. 2012; 122: 2652-60.
14. Straubinger RK, Summers BA, Chang YF, Appel MJ. Persistence of Borrelia burgdorferi in experimentally infected dogs after antibiotic treatment. J Clin Microbiol. 1997; 35: 111-6.
15. Straubinger RK. PCR-Based quantification of Borrelia burgdorferi organisms in canine tissues over a 500-Day postinfection period. J Clin Microbiol. 2000; 38: 2191-9.
16. Roberts ED, Bohm RP Jr, Cogswell FB, Lanners HN, Lowrie RC Jr, Povinelli L, Piesman J, Philipp MT. Chronic Lyme disease in the rhesus monkey. Lab Invest. 1995; 72: 146-60.
17. Roberts ED, Bohm RP Jr, Lowrie RC Jr, Habicht G, Katona L, Piesman J, Philipp MT. Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system. J Infect Dis. 1998; 178: 722-32.
18. Pachner AR, Cadavid D, Shu G, Dail D, Pachner S, Hodzic E, Barthold SW. Central and peripheral nervous system infection, immunity, and inflammation in the NHP model of Lyme borreliosis. Ann Neurol. 2001; 50: 330-8.
19. Cadavid D, Bai Y, Hodzic E, Narayan K, Barthold SW, Pachner AR. Cardiac involvement in non- human primates infected with the Lyme disease spirochete Borrelia burgdorferi. Lab Invest. 2004; 84: 1439-50.
20. Miller JC, Narayan K, Stevenson B, Pachner AR. Expression of Borrelia burgdorferi erp genes during infection of non-human primates. Microb Pathog. 2005; 39: 27-33.
21. Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, Hodzic E, Jacobs MB, Hasenkampf NR, Martin DS, Narasimhan S, Phillippi-Falkenstein KM, Purcell JE, Ratterree MS, Philipp MT. Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection. PLOS One. 2012; 7: e29914.
22. Crossland NA, Alvarez X, Embers ME. Late disseminated Lyme disease: associated pathology and spirochete persistence posttreatment in rhesus macaques. Am J Pathol. 2018; 188(3): 672-682.
23. Chang YF, Ku YW, Chang CF, Chang CD, McDonough SP, Divers T, Pough M, Torres A. Antibiotic treatment of experimentally Borrelia burgdorferi-infected ponies. Vet Microbiol. 2005; 107: 285-94.
24. Imai DM, Barr BC, Daft B, Bertone JJ, Feng S, Hodzic E, Johnston JM, Olsen KJ, Barthold SW. Lyme neuroborreliosis in 2 horses. Vet Pathol. 2011; 48: 1151-7.
Table A2. Evidence for persistent human infection following treatment of Lyme disease*†.
Study/Year/Reference |
Study Origin |
Persistence of B. burgdorferi Shown by |
Sample Source |
Weber et al., 19881 |
Europe |
Histology |
Brain, liver (Autopsy)** |
Schmidli et al., 19882 |
Europe |
Culture |
Synovial Fluid |
Cimmino et al., 19893 |
Europe |
Histology |
Spleen |
Preac-Mursic et al., 19894 |
Europe |
Culture |
Skin Bx, CSF |
Pfister et al., 19915 |
Europe |
Culture |
CSF |
Strle et al., 19936 |
Europe |
Culture |
Skin Bx |
Preac-Mursic et al., 19937 |
Europe |
Culture |
Iris Bx |
Haupl et al., 19938 |
Europe |
Culture |
Ligament Bx |
Strle et al., 19969 |
Europe |
Culture |
Skin Bx |
Preac-Mursic et al., 199610 |
Europe |
Culture |
Skin Bx, CSF |
Oksi et al., 199611 |
Europe |
Culture |
CSF |
|
|
PCR |
Brain Bx |
|
|
PCR |
Brain (Autopsy) |
Priem et al., 199812 |
Europe |
PCR |
Synovial Bx/Fluid |
Oksi et al., 199913 |
Europe |
Culture, PCR |
Blood |
Breier et al., 200114 |
Europe |
Culture |
Skin Bx |
Hunfeld et al., 200515 |
Europe |
Culture |
Skin Bx |
Svecova et al., 200816 |
Europe |
PCR |
Blood |
Hudson et al., 199817 |
Australia |
Culture, PCR |
Skin Bx |
Steere et al., 198818 |
USA |
Histology |
Synovial Bx |
Kirsch et al., 198819 |
USA |
Histology |
LN (Autopsy) |
Liegner et al., 199320 |
USA |
Histology |
Skin Bx |
|
|
PCR |
Blood |
Battafarano et al., 199321 |
USA |
Histology, PCR |
Synovial Bx/Fluid |
Chancellor et al., 199322 |
USA |
Histology |
Bladder Bx |
Nocton et al., 199423 |
USA |
PCR |
Synovial Fluid |
Shadick et al., 199424 |
USA |
Histology |
Brain (Autopsy) |
Masters et al., 199425 |
USA |
Culture |
Blood |
Lawrence et al., 199526 |
USA |
PCR |
CSF |
Bayer et al., 199627 |
USA |
PCR |
Urine |
Nocton et al., 199628 |
USA |
PCR |
CSF |
Marques et al., 201429 |
USA |
Xenodiagnosis |
Tick*** |
Middelveen et al., 201830 |
USA |
Culture, Histology |
Blood, Genital |
|
|
PCR |
Secretions, Skin |
Sapi et al., 201931 |
USA |
PCR, Histology, FISH, Confocal microscopy |
Liver, Heart, Kidney, Brain (Autopsy) |
Bransfield et al., 202432 |
USA |
Histology, FISH |
Pancreas, Heart, Brain (Autopsy) |
†Adapted from Stricker RB, Johnson L. Lyme disease: the next decade. Infect Drug Resist. 2011; 4:1-9. *Except for case of Weber et al. (see below), all patients received a minimum of 10 days of antibiotic therapy. PCR, polymerase chain reaction; Bx, biopsy; CSF, cerebrospinal fluid; LN, lymph node. FISH, fluorescent in-situ hybridization; **Mother treated with antibiotics for one week during pregnancy; newborn died; ***B. burgdorferi DNA recovered from ticks fed on human Lyme patients.
Table A2 References
1. Weber K, Bratzke HJ, Neubert U, Wilske B, Duray PH. Borrelia burgdorferi in a newborn despite oral penicillin for Lyme borreliosis during pregnancy. Pediatr Infect Dis J. 1988; 7: 286-9.
2. Schmidli J, Hunziker T, Moesli P, Schaad UB. Cultivation of Borrelia burgdorferi from joint fluid three months after treatment of facial palsy due to Lyme borreliosis. J Infect Dis. 1988; 158: 905-6.
3. Cimmino MA, Azzolini A, Tobia F, Pesce CM. Spirochetes in the spleen of a patient with chronic Lyme disease. Am J Clin Pathol. 1989; 91: 95-7.
4. Preac-Mursic V, Weber K, Pfister HW, Wilske B, Gross B, Baumann A, Prokop J. Survival of Borrelia burgdorferi in antibiotically treated patients with Lyme borreliosis. Infection 1989; 17: 355-9.
5. Pfister HW, Preac-Mursic V, Wilske B, Schielke E, Sorgel F, Einhaupl KMJ. Randomized comparison of ceftriaxone and cefotaxime in Lyme neuroborreliosis. Infect Dis. 1991; 163: 311-8.
6. Strle F, Preac-Mursic V, Cimperman J, Ruzic E, Maraspin V, Jereb M. Azithromycin versus doxycycline for treatment of erythema migrans: clinical and microbiological findings. Infection 1993; 21: 83-8.
7. Preac-Mursic V, Pfister HW, Spiegel H, Burk R, Wilske B, Reinhardt S, Böhmer R. First isolation of Borrelia burgdorferi from an iris biopsy. J Clin Neuroophthalmol. 1993; 13: 155-61
8. Haupl T, Hahn G, Rittig M, Krause A, Schoerner C, Schonherr U, Kalden JR, Burmester GR. Persistence of Borrelia burgdorferi in ligamentous tissue from a patient with chronic Lyme borreliosis. Arthritis Rheum. 1993; 36: 1621-6.
9. Strle F, Maraspin V, Lotric-Furlan S, Ruziç-Sabljiç E, Cimperman J. Azithromycin and doxycycline for treatment of Borrelia culture-positive erythema migrans. Infection 1996; 24: 64-8.
10. Preac-Mursic V, Marget W, Busch U, Pleterski Rigler D, Hagl S. Kill kinetics of Borrelia burgdorferi and bacterial findings in relation to the treatment of Lyme borreliosis. Infection 1996; 24: 9-16.
11. Oksi J, Kalimo H, Marttila RJ, Marjamaki M, Sonninen P, Nikoskelainen J, Viljanen MK. Inflammatory brain changes in Lyme borreliosis. A report on three patients and review of literature. Brain 1996; 119: 2143-54.
12. Priem S, Burmester GR, Kamradt T, Wolbart K, Rittig MG, Krause A. Detection of Borrelia burgdorferi by polymerase chain reaction in synovial membrane, but not in synovial fluid from patients with persisting Lyme arthritis after antibiotic therapy. Ann Rheum Dis. 1998; 57: 118-21.
13. Oksi J, Marjamaki M, Nikoskelainen J, Viljanen MK. Borrelia burgdorferi detected by culture and PCR in clinical relapse of disseminated Lyme borreliosis. Ann Med. 1999; 31: 225-232.
14. Breier F, Khanakah G, Stanek G, Kunz G, Aberer E, Schmidt B, Tappeiner G. Isolation and polymerase chain reaction typing of Borrelia afzelii from a skin lesion in a seronegative patient with generalized ulcerating bullous lichen sclerosus et atrophicus. Br J Dermatol. 2001; 144: 387-92.
15. Hunfeld KP, Ruzic-Sabljic E, Norris DE, Kraiczy P, Strle F. In vitro susceptibility testing of Borrelia burgdorferi sensu lato isolates cultured from patients with erythema migrans before and after antimicrobial chemotherapy. Antimicrob Agents Chemother. 2005; 49: 1294-301.
16. Svecová D, Gavornik P. Recurent erythema migrans as a persistent infection. Epidemiol Mikrobiol Imunol. 2008; 57: 97-100.
17. Hudson BJ, Stewart M, Lennox VA, Fukunaga M, Yabuki M, Macorison H, Kitchener-Smith J. Culture-positive Lyme borreliosis. Med J Aust. 1998; 168: 500-2.
18. Steere AC, Duray PH, Butcher EC. Spirochetal antigens and lymphoid cell surface markers in Lyme synovitis. Comparison with rheumatoid synovium and tonsillar lymphoid tissue. Arthritis Rheum. 1988; 31: 487-95.
19. Kirsch M, Ruben FL, Steere AC, Duray PH, Norden CW, Winkelstein A. Fatal adult respiratory distress syndrome in a patient with Lyme disease. JAMA 1988; 259: 2737-9.
20. Liegner KB, Shapiro JR, Ramsay D, Halperin AJ, HogrefeW, Kong L. Recurrent erythema migrans despite extended antibiotic treatment with minocycline in a patient with persisting Borrelia burgdorferi infection. J Am Acad Dermatol. 1993; 28: 312-4.
21. Battafarano DF, Combs JA, Enzenauer RJ, Fitzpatrick JE. Chronic septic arthritis caused by Borrelia burgdorferi. Clin Orthop, 1993; 297: 238-41.
22. Chancellor MB, McGinnis DE, Shenot PJ, Kiilholma P, Hirsch IH. Urinary dysfunction in Lyme disease. J Urol. 1993; 149: 26-30.
23. Nocton JJ; Dressler F; Rutledge BJ; Rys PN; Persing DH; Steere AC. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in synovial fluid from patients with Lyme arthritis N Engl J Med. 1994; 330: 229-34.
24. Shadick NA, Phillips CB, Logigian EL, Steere AC, Kaplan RF, Berardi VP, Duray PH, Larson MG, Wright EA, Ginsburg KS, Katz JN, Liang MH. The long-term clinical outcomes of Lyme disease. A population-based retrospective cohort study. Ann Intern Med. 1994; 121: 560-7.
25. Masters E, Lynxwiler P, Rawlings J. Spirochetemia after continuous high-dose oral amoxicillin therapy. Infect Dis Clin Prac. 1994; 3: 207–208.
26. Lawrence C, Lipton RB, Lowy FD, Coyle PK. Seronegative chronic relapsing neuroborreliosis. Eur Neurol. 1995; 35: 113-7.
27. Bayer ME, Zhang L, Bayer MH. Borrelia burgdorferi DNA in the urine of treated patients with chronic Lyme disease symptoms. A PCR study of 97 cases. Infection 1996; 24: 347–353.
28. Nocton JJ, Bloom BJ, Rutledge BJ, Persing DH, Logigian EL, Schmid CH, Steere AC. Detection of Borrelia burgdorferi DNA by polymerase chain reaction in cerebrospinal fluid in Lyme neuroborreliosis. J Infect Dis. 1996; 174: 623-7.
29. Marques A, Telford SR 3rd, Turk SP, Chung E, Williams C, Dardick K, Krause PJ, Brandeburg C, Crowder CD, Carolan HE, Eshoo MW, Shaw PA, Hu LT. Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study. Clin Infect Dis. 2014; 58: 937-45.
30. Middelveen MJ, Sapi E, Burke J, Filush KR, Franco A, Fesler MC, Stricker RB. Persistent Borrelia infection in patients with ongoing symptoms of Lyme disease. Healthcare (Basel). 2018; 6: 33.
31. Sapi E, Kasliwala RS, Ismail H, Torres JP, Oldakowski M, Markland S, Gaur G, Melillo A, Eisendle K, Liegner KB, et al. The long-term persistence of Borrelia burgdorferi antigens and DNA in the tissues of a patient with lyme disease. Antibiotics. 2019; 8: 183.
32. Bransfield RC, Goud Gadila SK, Kursawe LJ, Dwork AJ, Rosoklija G, Horn EJ, Cook MJ, Embers ME. Late-stage borreliosis and substance abuse. Heliyon. 2024; 10: e31159.