Lyme disease (LD) and similar Lyme-like Borrelia
infections are caused by members of the Borrelia burgdorferi
(Bb) sensu lato complex or by members of the Borrelia
relapsing fever complex such as B. miyamotoi
, respectively [1
]. Following initial infection, Borrelia
spirochetes can evade host defenses, sequester in immune privileged sites such as joints or the central nervous system, and persist in pleomorphic forms [5
]. Tickborne coinfections including Babesia
may complicate the clinical picture [6
]. If LD is not treated early in the course of infection, chronic illness may result and a variety of symptoms may develop. These symptoms include fatigue, musculoskeletal pain, arthritis, cardiac disease and neurological involvement with peripheral neuropathy, meningitis, encephalitis, cranial neuritis and cognitive dysfunction [6
Although LD was first recognized in 1975, it remains a controversial illness and the topic of polemic debate [6
]. One viewpoint claims that persistent Lyme disease symptoms are related to ongoing spirochetal infection despite antibiotic therapy. This scenario has been demonstrated in animal models including rodents, dogs and horses using various detection methods [16
], and a recent study in non-human primates showing “persistent, intact, metabolically-active B. burgdorferi
after antibiotic treatment of disseminated infection” offers the strongest support for this pathogenesis [37
]. Furthermore, comparable studies have suggested persistent infection after antibiotic therapy as a cause of chronic symptoms in humans [38
]. The opposing viewpoint claims that persistent Lyme disease symptoms may be due to spirochetal “debris” without active infection. While a number of studies from Europe and the USA have demonstrated persistence of Bb DNA or antigens in human bodily tissues or fluids, very few studies have demonstrated culture of live Borrelia
spirochetes, the highest form of evidence for persistent infection in chronic Lyme disease patients [4
In this pilot study, we present detailed evidence of persistent Borrelia infection despite antibiotic therapy in 12 randomly-selected North American patients with ongoing LD symptoms. Spirochetal infection was demonstrated by corroborative microscopic, histopathological and molecular detection of live Borrelia organisms in cultures of body fluids and tissues from these patients.
2.1. Subject Selection
Subjects included in the study were chosen at random from our North American patient population. All of the LD patients in the study were either clinically diagnosed with LD or had positive Bb serological testing prior to study participation. Serological testing for LD was performed by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory (IGeneX Laboratory in Palo Alto, CA, USA), as described in detail elsewhere [60
]. Subjects with Morgellons disease (MD) who were seropositive for LD were included in the study (see below) [61
]. All subjects had been treated with antibiotics prior to the study, and symptomatic patients who remained on antibiotic treatment were included in the study.
2.2. Control Selection
Ten healthy subjects were recruited as controls after informed consent was obtained. These subjects were then tested serologically for LD and those who were negative were accepted as controls. Vaginal or seminal fluids were collected from negative controls and cultured for Borrelia, as described below. Culture pellets underwent PCR testing for Borrelia in a blinded manner at the University of New Haven and Australian Biologics, as described below.
2.3. Informed Consent
All subjects were adults who gave informed consent to participate in the study. Signed informed consent to collect specimens was obtained in accordance with the ethics approval requirements for sample collection of the Western Institutional Review Board, Puyallup, WA, USA (Study # 1148461). Approval for anonymous sample testing was also obtained from the Institutional Review Board of the University of New Haven, West Haven, CT, USA. Additional signed informed consent to publish the results was obtained from each subject.
To avoid contamination, all cultures were performed under strict aseptic conditions in a laboratory that was free of Borrelia
reference strains, and cultures of control and patient samples were processed in an identical manner. Inocula were placed in Barbour-Stoner-Kelly H (BSK) complete medium with 6% rabbit serum (Sigma-Aldrich, #B8291, St. Louis, MO, USA) containing the following antibiotics: phosphomycin (0.02 mg/mL) (Sigma-Aldrich), rifampicin (0.05 mg/mL) (Sigma-Aldrich), and amphotericin B (2.5 μg/mL) (Sigma-Aldrich), as described previously [62
]. Inocula were prepared as follows:
A. Blood—whole blood (10 mL) was collected by venipuncture and left at room temperature to clot, then centrifuged at low speed to separate red blood cells from sera. The serum supernatants with a small amount of blood cells below the serum layer were collected and were inoculated into the BSK medium.
B. Skin—whole calluses or skin from lesions were removed from MD subjects by scraping with a scalpel blade.
C. Vaginal—vaginal secretions were collected by swabbing inside the vagina with sterile cotton-tipped swabs that were then introduced into the BSK medium.
D. Seminal—semen was self-collected into a sterile vial, then was pipetted into the BSK medium.
8 mL tubes of inoculated medium were filled to minimize the airspace present, thus providing a microaerobic environment, and incubated at 32 °C. Culture fluid was examined by darkfield microscopy for visible spirochetes weekly for up to 4 weeks. Cultures were concentrated by centrifuging the fluid at 15,000 g for 20 min, retaining the pellet and discarding the supernatant. For imaging, a small amount of culture pellet was resuspended in 50 μL 0.85% saline solution, washed and centrifuged again. The pellet was mixed with gelatin and then fixed with formalin for further staining.
2.5. Dieterle and Anti-Bb Immunostaining
Dermatological specimens and/or culture pellets from patients were fixed, sectioned and processed for specialized staining at either McClain Laboratories LLC, Smithtown, NY, USA, or the Department of Biology and Environmental Science, University of New Haven, West Haven, CT, USA, as previously described [59
]. Dieterle silver-nitrate staining was performed at McClain Laboratories. Anti-Bb immunostaining was performed at McClain Laboratories or the University of New Haven. In brief, immunostaining was performed using an unconjugated rabbit anti-Bb polyclonal antibody (Abcam ab20950, Cambridge, MA, USA), incubated with an alkaline phosphatase probe (Biocare Medical #UP536L, Pacheco, CA, USA), followed by a chromogen substrate (Biocare Medical #FR805CHC), and counterstained with hematoxylin. Positive and negative controls were prepared for comparison purposes using liver sections from Bb-inoculated and uninfected C3H/HeJ mice followed by Dieterle and immunostaining. Culture pellets from mixed Gram-positive bacteria (Streptococcus
) and Gram-negative bacteria (Escherichia coli
) were also prepared for comparison purposes as negative controls to exclude cross-reactivity with commonly encountered microorganisms.
2.6. Molecular Testing
Patient and negative control samples were submitted in a blinded manner to the laboratories performing polymerase chain reaction (PCR) amplification of DNA, as described below. PCR detection of Borrelia was performed for research purposes only. No data resulting from this study was used diagnostically.
2.7. PCR—University of New Haven
DNA was extracted from culture pellets as previously described [59
]. Reactions of blinded samples were performed in triplicate.
DNA in extracted samples was detected using a published TaqMan assay targeting a 139-bp fragment of the gene encoding the Borrelia
16S rRNA, as described previously [59
]. Amplifications were conducted on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) with cycling of 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s, and fluorescent signals were recorded using CFX96 Real-Time software with the Cq threshold set automatically.
Nested PCR primers for the 16S rRNA, flagellin (Fla), OspC, uvrA and pyrG genes were used as previously described [59
], with a final volume of 50 μL using 10 μL template DNA and final concentrations of 20 mM Tris-HCl (pH 8.4), 50 mM KCl (1 × Buffer B, Promega, Fitchburg, WI, USA), 2 mM MgCl2
, 0.4 mM dNTP mix, 2 μM of each primer, and 2.5 U Taq polymerase (Invitrogen, Carlsbad, CA, USA). The first reaction used “outer” primers and the second reaction used “inner” primers, and 1 μL of PCR product from the first reaction was used as template for the second. Cycling was programmed as follows: 94 °C for 5 min followed by 40 cycles of denaturation at 94 °C for 1 min, annealing for 1 min, and extension at 72 °C for 1 min, with a final extension step at 72 °C for 5 min. DNA products were visualized in 1–2% agarose gels.
PCR amplification was followed by Sanger sequencing. PCR products were extracted using the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Eluates were sequenced in both directions, then were compared by BLAST analysis using the GenBank database (National Center for Biotechnology Information).
2.8. PCR—Australian Biologics
DNA was extracted from culture pellets using the DNeasy Blood and Tissue kit®
(Qiagen) in accordance with the manufacturer’s instructions. Samples were forwarded to Australian Biologics for Borrelia
DNA and Treponema denticola
DNA testing. Blinded samples were run in duplicate with positive and negative controls using primers for the Borrelia
16S rRNA and rpoC gene targets, as previously described [59
DNA was detected by real-time PCR targeting the 16S rRNA gene and/or by endpoint PCR targeting the rpoC gene, as previously described [59
], using the Eco™ Real-Time PCR system with software version 184.108.40.206. Thermal profiles were performed with incubation for 2 min at 50 °C, polymerase activation for 10 min at 95 °C then PCR cycling for 40 cycles of 10 s at 95 °C dropping to 60 °C sustained for 45 s. The PCR signal magnitude generated (∆R) was interpreted as either positive or negative as compared to positive and negative controls.
For endpoint PCR, amplicons were visualized on 1–2% agarose gels and extracted from the gels using the QIAquick Gel Extraction kit (Qiagen) in accordance with the manufacturer’s instructions. Sanger sequencing was used for gene analysis, as described previously [59
2.9. PCR—University California Irvine
The presence of Bb sensu stricto DNA in a set of blinded samples was confirmed by the laboratory of Dr. Alan Barbour (University of California Irvine) by first quantitative PCR [69
], and then by sequence of the PCR-amplified 16S-23S intergenic spacer [70
]. The samples studied included specimens from two of the subjects in this paper, Case 2 and Case 10, as described below.
In this pilot study, we cultured live Borrelia
organisms from 12 antibiotic-treated subjects with persistent Lyme disease symptoms, thus showing that viable spirochetes can be found in LD patients despite antibiotic therapy. Half of these subjects were taking antibiotics at the time of sampling. Patient cultures showed Borrelia
spiral forms and spherical bodies, as described in other publications (Figure 1
]. We demonstrated the presence of Borrelia
infection in cultures from these patients using corroborative microscopy, histopathology and PCR techniques, and we obtained sequences for amplicons from 10/12 patients. Repeat cultures of blood, semen and vaginal secretions were positive for Bb by microscopy, histopathology and PCR in six patients tested by four different laboratories. Cultures from healthy Borrelia
-seronegative controls were consistently negative using microscopy, histopathology and PCR techniques, making the possibility of Borrelia
contamination in LD patient samples extremely unlikely.
infection may result in part from the wide variety of tissues and fluids that support spirochetal growth [16
]. The tissues susceptible to Borrelia
infection include fibroblasts, skin, synovial tissue, ligaments, cardiac tissue, glial cells, neurons, endothelial cells, lymphoid tissue and hepatic tissue [5
]. The pleotropic nature of Borrelia
infection may allow the spirochete to evade the host immune system and antibiotic therapy, as outlined below.
The role of round body cysts and biofilms in persistent Borrelia
infection is controversial [10
]. Ongoing Lyme disease symptoms may arise from spirochetes hidden in biofilms or surviving as round body cysts or cell wall-deficient L-forms, by intracellular Borrelia
sequestration or by sequestration within privileged sites where antibiotics do not attain therapeutic levels [13
]. Regardless of the mechanism by which Borrelia
spirochetes persist in tissues, persistent Borrelia
infection requires treatment, and options at present are limited and controversial [10
]. The controversy is fueled by disagreement over viability of the spirochetes, as described below.
Although there is evidence of post-treatment Borrelia
infection in animals and humans, some researchers speculate that Borrelia
antigens and DNA detected in studies are merely spirochetal “debris” [36
]. Wormser et al. offered an “amber” hypothesis as a possible explanation for persistent symptoms, namely that persistent Lyme arthritis is caused by non-viable spirochetes enmeshed in joints within host-derived fibrinous or collagenous matrices [88
]. Bockenstedt et al. proposed that the inflammation seen in mice described in their study following antibiotic treatment was caused by Borrelia
DNA and proteins representing non-infectious spirochetal “debris” deposited in tissues [36
In contrast, those who support the idea that active infection is responsible for persisting Lyme disease symptoms propose that there are various protective mechanisms providing spirochetal resistance or tolerance to antibiotics, including intracellular invasion and formation of cell-wall deficient L-forms, round body cysts, biofilms and persister cells [78
]. Furthermore the “amber” and “debris” hypotheses of symptom persistence are difficult to support because Borrelia
DNA is rapidly cleared from murine tissues after prompt antibiotic treatment [21
], and the DNA of non-viable spirochetes is cleared from mouse tissue within several hours [90
]. The present study confirms the presence of live Borrelia
spirochetes in patients who had been treated with antibiotics for persistent Lyme disease symptoms.
Recent studies have focused on “persister cells” and “sleeper cells” as spirochetal agents of persistence in Lyme disease [91
]. The concept involves organisms that are tolerant to antibiotics and can downregulate their metabolic needs via a “stringent response” to survive in a hostile environment, only to reemerge when the environment becomes more favorable. A similar mechanism of persistent infection has been described in E. coli
]. The survival of metabolically tolerant spirochetes in privileged sites would explain our findings of viable Borrelia
in antibiotic-treated patients once the antibiotics are withdrawn and culture conditions are optimized. The factors that influence viability of “persister cells” and “sleeper cells” in patients with persistent Lyme disease symptoms merit further study.
Three of our study subjects had a controversial skin condition commonly called Morgellons disease (MD) [61
]. The distinguishing feature of this skin condition is the presence of white, black, or brightly colored filaments that lie under, are embedded in, or project from skin lesions (see Figure 1
D). While some medical practitioners erroneously consider MD to be a purely delusional disorder, MD appears to be a Borrelia
-associated filamentous dermatitis [94
]. MD patients exhibit symptoms that resemble those of Lyme disease such as fatigue, joint pain, and neuropathy, and the skin condition has been shown to be associated with Borrelia
]. Spirochetes from different Borrelia
species have been detected in MD patient specimens [61
]. We obtained positive Borrelia
cultures from all three of our MD subjects.
The mechanism of MD filament evolution has not been resolved, but as collagen and keratin filaments arise from proliferative keratinocytes and fibroblasts in human epithelial tissue, we speculate that Borrelia
infection alters keratin and collagen gene regulation [99
bacteria can invade fibroblasts and keratinocytes where they survive and replicate intracellularly [74
]. As shown by in vitro studies, Borrelia
spirochetes can be isolated from keratinocyte and fibroblast monolayers despite treatment with antibiotics [74
]. Persistent refractory infection in MD patients may therefore result in part from sequestration of live Borrelia
spirochetes within keratinocytes and fibroblasts.
spirochetes have been detected in vaginal and seminal secretions [13
]. We cultured Borrelia
spirochetes in genital secretions from ten of our study subjects who had taken or were currently taking antibiotic therapy. Bb is a complex organism that is related to the spirochetal agent of syphilis, and therefore may have similar infectious capabilities [13
]. As outlined above, Borrelia
spirochetes penetrate tissues, can form cystic structures and L-forms, hide in biofilms, become intracellular, and sequester in privileged sites (brain, eye and synovium) [9
]. These specialized abilities of the Borrelia
spirochete suggest that the genital tract could harbor infection. The vagina and the seminal vesicles are privileged sites, and that may explain why the organism can persist in the genital tract despite antimicrobial therapy in a manner similar to syphilis, chlamydia, human immunodeficiency virus (HIV), Ebola and Zika virus [102