Borrelia burgdorferi and Borrelia miyamotoi seroprevalence in California blood donors

Sharon I Brummitt; Anne M Kjemtrup; Danielle J Harvey; Jeannine M Petersen; Christopher Sexton; Adam Replogle; Andrea E Packham; Evan M Bloch; Alan G Barbour; Peter J Krause; Valerie Green; Woutrina A Smith

doi:10.1371/journal.pone.0243950

. 2020 Dec 28;15(12):e0243950. doi: 10.1371/journal.pone.0243950

Borrelia burgdorferi and Borrelia miyamotoi seroprevalence in California blood donors

Sharon I Brummitt ^1,^*, Anne M Kjemtrup ², Danielle J Harvey ³, Jeannine M Petersen ⁴, Christopher Sexton ⁴, Adam Replogle ⁴, Andrea E Packham ⁵, Evan M Bloch ⁶, Alan G Barbour ⁷, Peter J Krause ⁸, Valerie Green ⁹, Woutrina A Smith ¹

Editor: R Mark Wooten¹⁰

¹Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, California, United States of America

²California Department of Public Health, Sacramento, California, United States of America

³Department of Public Health Sciences, School of Medicine, University of California, Davis, California, United States of America

⁴Division of Vector-Borne Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado, United States of America

⁵Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, California, United States of America

⁶Department of Pathology and Transfusion Medicine, John Hopkins University, Baltimore, Maryland, United States of America

⁷Department of Medicine and Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, California, United States of America

⁸Department of Epidemiology of Microbial Diseases, Yale School of Public Health, and Yale School of Medicine, New Haven, CT, United States of America

⁹Creative Testing Solutions, Tempe, Arizona, United States of America

¹⁰University of Toledo College of Medicine and Life Sciences, UNITED STATES

Competing Interests: The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Dr. Bloch is a member of the U.S. Food and Drug Administration (FDA) Blood Products Advisory Committee. Any views or opinions expressed in this manuscript are Dr. Bloch's and are based on his own scientific expertise and professional judgment; they do not necessarily represent the views of the Blood Products Advisory Committee or the formal position of the FDA and also do not bind or otherwise obligate or commit either the Advisory Committee or the FDA to the views expressed. The views and opinions expressed herein are those of the authors alone and do not represent the official position of the Centers for Disease Control and Prevention.

^✉

* E-mail: Sibrummitt@ucdavis.edu

Roles

Sharon I Brummitt: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing

Anne M Kjemtrup: Conceptualization, Data curation, Methodology, Supervision, Writing – original draft, Writing – review & editing

Danielle J Harvey: Formal analysis, Methodology, Supervision, Writing – review & editing

Jeannine M Petersen: Conceptualization, Methodology, Supervision, Writing – review & editing

Christopher Sexton: Methodology, Writing – review & editing

Adam Replogle: Methodology, Writing – review & editing

Andrea E Packham: Data curation, Investigation, Methodology, Writing – review & editing

Evan M Bloch: Conceptualization, Resources, Writing – review & editing

Alan G Barbour: Conceptualization, Methodology, Resources, Writing – review & editing

Peter J Krause: Conceptualization, Methodology, Writing – review & editing

Valerie Green: Investigation, Resources, Writing – review & editing

Woutrina A Smith: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing

R Mark Wooten: Editor

PMCID: PMC7769429 PMID: 33370341

Abstract

The western blacklegged tick, Ixodes pacificus, an important vector in the western United States of two zoonotic spirochetes: Borrelia burgdorferi (also called Borreliella burgdorferi), causing Lyme disease, and Borrelia miyamotoi, causing a relapsing fever-type illness. Human cases of Lyme disease are well-documented in California, with increased risk in the north coastal areas and western slopes of the Sierra Nevada range. Despite the established presence of B. miyamotoi in the human-biting I. pacificus tick in California, clinical cases with this spirochete have not been well studied. To assess exposure to B. burgdorferi and B. miyamotoi in California, and to address the hypothesis that B. miyamotoi exposure in humans is similar in geographic range to B. burgdorferi, 1,700 blood donor sera from California were tested for antibodies to both pathogens. Sampling was from high endemic and low endemic counties for Lyme disease in California. All sera were screened using the C6 ELISA. All C6 positive and equivocal samples and nine randomly chosen C6 negative samples were further analyzed for B. burgdorferi antibody using IgG western blot and a modified two ELISA test system and for B. miyamotoi antibody using the GlpQ ELISA and B. miyamotoi whole cell sonicate western blot. Of the 1,700 samples tested in series, eight tested positive for antibodies to B. burgdorferi (0.47%, Exact 95% CI: 0.20, 0.93) and two tested positive for antibodies to B. miyamotoi (0.12%, Exact 95% CI: 0.01, 0.42). There was no statistically significant difference in seroprevalence for either pathogen between high and low Lyme disease endemic counties. Our results confirm a low frequency of Lyme disease and an even lower frequency of B. miyamotoi exposure among adult blood donors in California; however, our findings reinforce public health messaging that there is risk of infection by these emerging diseases in the state.

Introduction

California is considered a low incidence state for Lyme disease, defined as a state with a disease incidence of <10 confirmed cases/100,000 annually [1]. The varied ecology results in some counties having higher endemnicity for Lyme disease than others [2–4]. The western blacklegged tick (Ixodes pacificus) is a common human-biting tick in California and is the principle vector for Borrelia burgdorferi sensu stricto, also called Borreliella burgdorferi and hereinafter referred to as B. burgdorferi [5]. B. burgdorferi is the causative agent of Lyme disease in humans and animals in North America. Other potentially zoonotic spirochetes have been documented in the western blacklegged tick [6–8], most notably Borrelia miyamotoi, an emerging tick-borne pathogen that is in same genus as the agents of relapsing fever. It causes a febrile illness that occasionally may relapse [9, 10].

In California, the prevalence of B. burgdorferi is typically higher in nymphal I. pacificus ticks (~3–5%) than adult ticks (~1% or less) [11]. The prevalence for B. miyamotoi is about the same (~1%) in both of these tick stages [12–16]. B. miyamotoi is transmitted both transstadially and transovarially, which means that while the risk of exposure to B. burgdorferi is greater than B. miyamotoi after exposure to nymphal ticks, B. miyamotoi infection may occur after larval, nymphal, or adult tick exposure and thus extends the season of risk [11]. The distribution of B. miyamotoi in I. pacificus ticks appears to be similar to that of B. burgdorferi and is most prevalent in coastal and foothill regions of northern California [11, 17]. Despite ample evidence of B. miyamotoi in California ticks, including ticks that were recovered from humans [18], epidemiological information and case descriptions of B. miyamotoi infections in humans are lacking in California.

There are a handful of tick-borne relapsing fever cases caused by Borrelia hermsii reported each year in California [16]. Although this spirochete is phylogenetically related to B. miyamotoi and shares common antigens [19], its ecology is quite distinct. It is vectored by the soft tick, Ornithodoros hermsi, which can be found in rodent nests built in cabins and houses in the mountains and foothill regions of the western United States [20, 21]. The possibility of human infections with B. miyamotoi in California comes from a recent study that identified seroreactivity to B. miyamotoi in an area highly endemic for Lyme disease in northern California. Seroprevalence was 12%– 14% in the study participants who were at high risk for tick-borne disease, although the GlpQ-based serologic assay used to test for B. miyamotoi antibody could not differentiate B. miyamotoi infection from B. hermsii [22]. The authors surmised that ecology and known behaviors of study participants suggested that they were most likely exposed to B. miyamotoi rather than B. hermsii [22].

Even with a low prevalence of B. burgdorferi and B. miyamotoi, California residents are still at risk for tick borne relapsing fever. The purpose of the present study was to determine whether B. miyamotoi has a broader geographical range in California than previously demonstrated and to compare the seroprevalence of B. burgdorferi and B. miyamotoi over this larger range. We therefore assessed human exposure to B. burgdorferi and B. miyamotoi by testing 1,700 blood bank serum samples from both high and low Lyme disease endemic areas in California. As a broad California-based serosurvey, findings from this study should inform public-health messaging as well as future B. burgdorferi and B. miyamotoi research.

Material and methods

Study population

We obtained 1,700 de-identified human sera samples from Creative Testing Solutions (www.mycts.org) consisting of human sera samples from blood banks. These included 941 samples from high endemic Lyme disease counties in California, defined as > 1 case per 100,000 annually and 759 samples from low endemic Lyme disease counties, defined as < 1 case per 100,000 annually [16] (Fig 1). Sample size calculations were performed using Ausvet Epi tools Epidemiological calculators, with sample size calculated based on the following assumptions: (i) an estimated human prevalence of 2% for Lyme disease in high endemic California counties and an estimated human prevalence of 1% for low endemic Lyme disease counties [11, 16]; (ii) a C6 ELISA screening test with a sensitivity of 97% and a specificity ranging from 93% to 99% for antibodies to B. burgdorferi; and (iii) a desired precision of 0.012 with an α = .05 and 80% power. Inclusion criteria for blood donor serum samples included the zip code of the blood donor, sera that was non-reactive on screening assay for other infectious diseases including Hepatitis B or C, HIV 1 or 2, HTLV I/II, West Nile virus, Zika virus, and syphilis; and a minimum volume of at least 1ml. Samples were collected from April 2017 through June 2017, corresponding with typical seasonality for Lyme disease cases reported in California [11]. Information associated with the samples included general demographic information such as sex, age, ethnicity, county and zip code of the donor.

Fig 1 — The California counties in dark green represent high endemic counties for Lyme disease and those in light green represent low endemic counties for Lyme disease. Number and percentages of sera samples by county are provided. High endemic Lyme disease counties are defined as those with ≥ 1 case per 100,000 annually and low endemic Lyme disease counties are defined as < 1 case per 100,000 annually [16].

Ethics approval

De-identified serum samples with associated demographic variables for each serum sample were submitted for this study. The study was determined to have exempt status by the Institutional Review Board, University of California Davis (Protocol Number 1090480–1, FWA No: 00004557).

Sample processing and storage

Creative Testing Solutions shipped frozen one-ml aliquot sera samples on dry ice to the testing facility at the University of California, Davis (UCD). UCD stored all sera samples at -80°C until thawed in the refrigerator for one to five days until testing. By using the commercial C6 ELISA test as a screening tool, the full set of samples could be initially evaluated for reactivity to B. burgdorferi and B. miyamotoi since both of these agents react to this antigen [23, 24]. The IgG western blot for B. burgdorferi and total Ig GlpQ ELISA and IgG western blot for B. miyamotoi were subsequently used to test all C6 positive/equivocal samples, as well as nine randomly selected C6 negative samples, to serve as controls [23, 25–27]. Additionally, a recently FDA approved test system for detection of B. burgdorferi antibodies, referred to as the Modified Two Tier Test (MTTT) (Zeus Scientific, New Jersey), was performed in accordance with the manufacturer’s guidelines on the C6 positive/equivocal and C6 negative samples [28]. After C6 screening (described below), all C6-positive/equivocal and the randomly chosen negative samples were separated into 250μl aliquots in coded tubes, re-coded to mask county of location, and shipped frozen to the Centers for Disease Control and Prevention (CDC). CDC stored all sera samples in a -80°C freezer until testing (Fig 2).

Fig 2 — Testing strategy followed for the 1,700 blood donors from California. Each step of the diagram details the testing strategy and the number of samples tested.

Standard two-tiered testing

All sera samples were screened using a C6 Lyme ELISA kit (Immunetics, Boston Massachusetts) performed in accordance with the manufacturer’s recommendation. Testing was performed in duplicate for all specimens. A Lyme index value for each sample was calculated by dividing the average of the sample’s OD values by the calibrator cutoff value. Positive and negative controls were provided by the manufacturer. Samples positive or equivocal by C6 Lyme ELISA were tested using the second-tier (IgG) Marblot western blot (MarDx Diagnosis, Trinity Biotech, Carlsbad, California) which was performed in accordance with the manufacturer’s recommendation. The criteria used in the interpretation of the western blot as either negative or positive was based on the manufacturer’s recommendation using established criteria [29].

Modified two-tiered testing

The Borrelia VlsE1/pepC10 IgM/IgG test system (ZEUS Scientific, Branchburg, NJ) was performed on 91 C6 ELISA positive/equivocal samples as well as nine randomly chosen negative samples. All positive and equivocal samples were then tested by the B. burgdorferi ELISA whole cell antigen IgG test system (ZEUS Scientific) [30]. Both ELISAs were performed in accordance with the manufacturer’s recommendation [31].

GlpQ ELISA

B. miyamotoi recombinant his-tagged GlpQ antigen (1 μg/well) was bound to 96 well plates as described [24] with the following modifications. Peroxidase conjugated goat anti-human IgA+IgG+IgM (H&L) (1:2500) and SureBlue TMB peroxidase substrate (SeraCare, Milford, Massachusetts) were utilized to detect bound antibody. The positive cutoff was set at three standard deviations above the mean absorbance of sera from four negative controls (healthy persons). A positive control from a B. miyamotoi PCR positive patient was included in each run. Absorbance was read at 450 nm.

B. miyamotoi western blot

The B. miyamotoi western blot strips were produced using B. miyamotoi strain CT13-2396, which was originally isolated from I. scapularis collected in Connecticut; NCBI accession number: PRJNA310783. Strain CT13-2396 was grown in BSK-R medium and harvested by centrifuging at 10,000(g) for 10 minutes at 4°C. The resulting cell pellet was frozen, thawed, and re-suspended in TE buffer (Fisher Scientific, Pittsburgh, PA), sonicated, and diluted to a final protein concentration of 2.0 mg/ml. The sonicate was mixed with sample buffer with DTT (Bio-Rad, Inverness, CA), heated at 95°C heat block for 10 min, resolved on 12.5% SDS-PAGE gels for 180 minutes at 70mAmps, and soaked in tris/glycine buffer (Bio-Rad) with 20% methanol for 30 minutes at 4°C. Separated proteins were transferred for 30 minutes at 25 volts to a 0.2 μm nitrocellulose membrane using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad). Membranes were soaked overnight in 1% milk (Bio-Rad) and tris-buffered saline and Tween™ 20 (TBST) (Fisher Scientific), dried and then cut into 3 mm strips and stored at 4°C. To perform western blotting, strips were re-hydrated in 1% milk and TBST (blocking buffer), then incubated with sera at a final concentration of 1:200 in blocking buffer for 30 min. Strips were then washed and incubated for 15 min in blocking buffer and phosphatase-labeled goat anti human IgG (H+ L) conjugate (KPL, Gaithersburg, MD) added at a concentration of 1:10,000, followed by a final wash series. Strips were developed using BCIP/NBT phosphatase substrate (KPL, Gaithersburg, MD). A control strip using a monoclonal GlpQ antibody was included as a locator for the GlpQ antigen; it was processed identically, with the exception of using anti mouse IgG (H+ L) conjugate. B. miyamotoi and B. burgdorferi human sera were included as positive and negative controls, respectively.

Statistical analysis

Statistical analysis of B. burgdorferi and B. miyamotoi results were performed separately. The outcome variable for each analysis was the serum sample as positive, equivocal, or negative for the pathogen. The exposure variable was blood donor’s residence in high or low Lyme disease endemic counties, as defined above. Point prevalence (seroprevalence) was calculated as the number of people who tested positive for the pathogen over total number of people tested. Exact 95% confidence intervals were constructed for prevalence estimates. Sample t-tests and chi-square tests were used to compare characteristics of those providing samples between the high and low-endemic counties in the overall sample and those samples carried forward to confirmatory testing. A two-tailed Fisher’s exact test was performed to compare the percentage of Borrelia species–seropositive study participants in the high and low endemic counties. A P value less than 0.05 was considered statistically significant. All analyses were performed using SAS software version 9.4, SAS Institute Inc., Cary, NC, USA.

Results

Sample characteristics

There was no significant difference in the distribution of males to females by risk level for the study population. Participants in low Lyme disease endemic counties were on average slightly younger than those in high Lyme disease endemic counties (41 years and 49 years, respectively P<0.001). The majority of participants listed ethnicity as White and non-Hispanic, with a greater percentage of Hispanics in the low endemic counties (20.5%) than in the high endemic counties (6.7%; P<0.001) (Table 1). In the high endemic counties, over two-thirds of the samples were from San Mateo (38.0%) or Sonoma (20.6%) counties, while most of the samples from the low endemic counties were from Ventura (56.1%) and San Luis Obispo (31.6%) counties (Table 2).

Table 1. Demographic characteristics of sera samples and positive Bb STTT and Bm GlpQ.

	All Sera Samples		P-Value	Positive B. burgdorferi Standard Two Tiered Testing^**		Positive Borrelia miyamotoi GlpQ^**
	All Sera Samples		P-Value	(C6 ELISA + Marblot WB)		(GlpQ ELISA + Bm WB)
	High Endemic	Low Endemic		High Endemic	Low Endemic	High Endemic	Low Endemic
	(N = 941)	(N = 759)
Sex
Male	521 (55.4%)	420 (55.3%)	0.7	7 (0.74%)	0 (0.00%)	0 (0.00%)	0 (0.00%)
Female	413 (43.9%)	346 (45.6%)	0.7	0 (0.00%)	1 (0.13%)	1 (0.11%)	1 (0.13%)
Age
Mean Age (SD^*)	49.0 (16.9)	41.1 (19.7)	< 0.001	65.3 yrs (6.5)	63 yrs (N/A)	17 yrs (N/A)	32 yrs (N/A)
Range	16yrs to 84yrs	16yrs to 84yrs	< 0.001	56 yrs—75 yrs	N/A	N/A	N/A
Ethnicity
Non-Hispanic	878 (93.3%)	603 (79.4%)	< 0.001	7 (0.74%)	1 (0.13%)	1 (0.11%)	0 (0.00%)
Hispanic	63 (6.7%)	156 (20.5%)	< 0.001	0 (0.00%)	0 (0.00%)	0 (0.00%)	1 (0.13%)

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* Standard deviation not calculated for (n = 1)

** Percentages calculated out of all sera samples in high or low endemic areas

Table 2. Testing results of sera samples for B. burgdorferi and B. miyamotoi by county.

Counties where Sera was Sampled	C6 Lyme ELISA/Screening Test^**	Bb Standard Two Tier Testing^***	Borrelia miyamotoi GlpQ^***
N	n (%^*)	n (%^*)	n (%^*)
High Endemic Counties
Marin (n = 157)	12 (7.64%)	2 (1.27%)	0 (0.00%)
Mendocino (n = 38)	3 (7.89%)	0 (0.00%)	1 (2.63%)
Napa (n = 108)	3 (2.78%)	1 (0.93%)	0 (0.00%)
San Mateo (n = 358)	19 (5.31%)	3 (0.84%)	0 (0.00%)
Santa Clara (n = 86)	3 (3.49%)	0 (0.00%)	0 (0.00%)
Sonoma (n = 194)	9 (4.64%)	1 (0.51%)	0 (0.00%)
Low Endemic Counties
Orange (n = 34)	0 (0.00%)	0 (0.00%)	0 (0.00%)
San Luis Obispo (n = 240)	16 (6.67%)	1 (0.42%)	0 (0.00%)
Solano (n = 59)	3 (5.08%)	0 (0.00%)	0 (0.00%)
Ventura (n = 426)	23 (5.40%)	0 (0.00%)	1 (0.23%)

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## Positive test

* County percent is based upon the number of samples tested from each county.

** County percent include both positive and equivocal results

***County percent include only positive test results

C6 ELISA screen

Of the 1,700 serum samples screened with C6 ELISA, 64 (3.76%) were positive and 27 (1.59%) were equivocal. Of the 941 samples from high endemic counties, 49 (5.2%) total samples were positive (n = 38) or equivocal (n = 11) for B. burgdorferi antibody compared to 42 (5.5%; 26 positive and 16 equivocal) of the 759 samples from low endemic counties.

Ninety-one samples that were C6 ELISA positive/equivocal and nine randomly selected negative samples had additional testing. A little more than half (58%) of the 91 samples that were positive or equivocal were from male individuals. The mean age for males was 42.6 years (range 16 years to 80 years) while the mean age of females was 49.6 years (range 16 years to 77 years). Most individuals with positive or equivocal samples were non-Hispanic (87%). Ethnicity (high endemic: Non-Hispanic (93.9%); low endemic: Non-Hispanic (78.6%); P-value = 0.058), sex (high endemic: male 55.1%; low endemic: male 61.9%; P-value = 0.5) and age (high endemic: mean = 49 years, SD = 16.6; low endemic: mean = 42 years, SD = 20.9; P-value = 0.07), of those with positive or equivocal samples did not differ between high and low endemic counties.

Of the nine randomly selected negative samples, two thirds were from female subjects. The mean age was 40.6 years for males (range 16 years to 72 years) and 35.7 years (range 25 years to 60 years) for females. Seven of the nine (78%) were non-Hispanic. Four of the nine (44%) selected negative of the samples were from high endemic counties while 5 of the 9 (56%) of samples were from low endemic counties.

B. miyamotoi GlpQ seroreactivity

Two of the 1,700 samples had detectable antibodies against B. miyamotoi (0.12%, Exact 95% CI: 0.01%, 0.42%). Both samples tested positive by C6 ELISA, GlpQ ELISA and B. miyamotoi whole cell western blot. Both samples were negative on the IgG western blot for B. burgdorferi (Fig 3, Table 3). None of the nine randomly selected C6 seronegative samples were seropositive for antibodies against B. miyamotoi by the GlpQ ELISA. Seroprevalence was (2.63%, Exact 95% CI: 0.7, 13.81) among Mendocino County residents and (0.23%, Exact 95% CI: 0.01, 1.30) among Ventura County residents. Both seropositive samples were from females with respective ages of 17 years and 32 years (Table 1).

Fig 3 — For B. *burgdorferi*, each run included a serum band locator (SBL) control, which shows reactivity to Lyme diagnostically significant bands at position 93, 66, 58, 45, 41, 39, 30, 28, 23 and 18 kDa and weakly reactive (WR) control, which shows reactivity to the 41 kDa band. Additionally, control serum from persons confirmed positive for B. *burgdorferi* or B. *miyamotoi* were included. Serum samples seropositive for 5 or more bands are shown. For B. *miyamotoi* western blots, control sera from patients confirmed positive for B. *burgdorferi* or B. *miyamotoi* were included along with a negative control serum. A monoclonal antibody to GlpQ was also included as a locator for the GlpQ antigen. The two samples with seroreactivity to GlpQ are shown.

Table 3. Antibody testing results of human samples.

Study Subject	B. burgdorferi (Standard Two Tier Testing)					B. burgdorferi (Modified Two Tier Testing)					B. miyamotoi (GlpQ Testing)
Sample Number	C6 ELISA		Marblot WB (IgG)^*		Interpretation	Zeus vslE1/pepC10		Zeus whole cell IgG		Interpretation	GlpQ ELISA		B. miyamotoi Western Blot
	Index Value	Results	Pos Band	Results		Index Value	Results	Index Value	Results		OD value	> pos cutoff	GlpQ Band	Result
High Endemic Counties
Sample 3	6.660	Positive	7	Positive	Positive	4.468	Positive	3.362	Positive	Positive	1.210	2.799	No	Negative
Sample 17	1.926	Positive	5	Positive	Positive	0.269	Negative	N/A	N/A	Negative	1.471	2.799	No	Negative
Sample 21	4.510	Positive	6	Positive	Positive	2.337	Positive	1.428	Positive	Positive	1.278	2.799	No	Negative
Sample 29	1.468	Positive	5	Positive	Positive	1.448	Positive	2.920	Positive	Positive	0.953	2.145	No	Negative
Sample 56	3.783	Positive	6	Positive	Positive	1.295	Positive	2.799	Positive	Positive	0.589	2.234	No	Negative
Sample 58	1.156	Positive	5	Positive	Positive	0.991	Equivocal	2.083	Positive	Positive	0.622	2.234	No	Negative
Sample 87	1.190	Positive	4	Negative	Negative	0.254	Negative	N/A	N/A	Negative	2.493	2.246	Yes	Positive
Sample 100	1.082	Equivocal	5	Positive	Positive	1.366	Positive	2.365	Positive	Positive	1.145	2.246	No	Negative
Low Endemic Counties
Sample 14	0.990	Equivocal	2	Negative	Negative	0.675	Negative	N/A	N/A	Negative	3.094	2.799	Yes	Positive
Sample 23	0.957	Equivocal	6	Positive	Positive	0.327	Negative	N/A	N/A	Negative	2.015	2.799	No	Negative
Sample 59	3.540	Positive	2	Negative	Negative	1.604	Positive	0.965	Equivocal	Positive	1.591	2.234	No	Negative
Sample 79	>7.00	Positive	3	Negative	Negative	6.437	Positive	2.518	Positive	Positive	0.360	1.666	No	Negative
Sample 85	0.220	Negative	5	Positive	Negative	0.225	Negative	N/A	N/A	Negative	0.865	1.666	No	Negative

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* Marblot Western Blot—A positive sample is ≥ 5 bands out of 10 bands (All results were visually read).

B. burgdorferi IgG western blot

Eight of 1,700 samples had detectable antibodies against B. burgdorferi (0.47%, Exact 95% CI: 0.20, 0.93). Of the eight sera samples that were positive by the C6 ELISA and B. burgdorferi IgG western blot (STTT), seven (0.74%, Exact 95% CI: 0.30, 1.53) were from high Lyme disease endemic counties and one positive sample (0.13%, Exact 95% CI: 0.00, 0.73) was from a low Lyme disease endemic county (P = 0.08) (Table 1). Marin County had an overall seroprevalence of 1.3% (Exact 95% CI: 0.15, 4.53), Napa county had an overall seroprevalence of 0.93%(Exact 95% CI: 0.02, 5.05), San Mateo County had an overall seroprevalence of 0.84% (Exact 95% CI: 0.17, 2.43), and Sonoma County had an overall seroprevalence of 0.52% (Exact 95% CI: 0.01, 2.84). San Luis Obispo had an overall seroprevalence (0.4%) (Exact 95% CI: 0.01, 2.30) (Table 2).

Modified two-tiered testing

Eight of the 91 C6 ELISA positive/equivocal sera were positive by modified two-tiered testing (MTTT) (Table 3). Six of the 91 C6 ELISA positive/equivocal sera positive by both STTT and MTTT were from a high endemic county and two of 91 C6 ELISA positive/equivocal sera positive by MTTT only were from a low endemic county. There was little agreement in STTT and MTTT positivity among C6 ELISA positive/equivocal sera from a low endemic county. Seven of the 91 C6 ELISA positive/equivocal sera positive by MTTT were male with mean age of 65 years (range 56 years to 75 years) and the age of the female was 16 years.

Discussion

This study represents the largest serosurvey across a broad geographic area in California estimating human exposure to B. miyamotoi and B. burgdorferi. We found higher B. burgdorferi antibody estimates from higher risk Lyme disease endemic areas in northern California but similar B. miyamotoi antibody estimates from high and low risk Lyme disease endemic areas in the state. Our large sample size of 1,700 and broad geographic expanse increases the confidence of our seroprevalence estimate. Although the overall risk of human acquisition of either pathogen is lower in California compared with those in high risk Lyme disease endemic areas in the Northeast and northern Midwest, endemic areas are shifting with climate and human habitat changes that alter the epidemiology of these infections [32, 33]. Our data align with previous research that demonstrated the C6 Lyme ELISA test can detect seroreactivity to both B. burgdorferi and B. miyamotoi because the C6 peptide sequence in the C6 ELISA test kit found in B. burgdorferi is very similar to the relapsing fever Borrelia variable large protein (Vlp) sequence, including Vlp 15/16 of B. miyamotoi [23, 34]. The cross-reactive antibodies against the C6 peptide occurs in 90% of patients with B. miyamotoi disease [34]. The C6 ELISA test can also detect antibodies to all of the major pathogenic European Borrelia species: B. afzelii, B. garinii, and B. burgdorferi [35].

Only two of the 1,700 serum samples were positive for both C6 and GlpQ antibodies (0.12%, Exact 95% CI: 0.01, 0.42). This result suggests that these two persons had prior B. miyamotoi infection, given that the glpQ gene is not present in Lyme spirochetes [23, 24]. With so few B. miyamotoi seropositive samples, we had insufficient power to detect a difference between high and low Lyme disease endemic counties. Overall in California, the risk of exposure to B. miyamotoi is relatively low. However, there may be ecologic foci of exposure risk, similar to what is seen for Lyme disease in California [2]. For example, the B. miyamotoi seroprevalence in Mendocino County (2.6%) is similar to that noted in a previous study [22]. which documented seroprevalence values of 1.98% and 6.93% over several years in a Mendocino community. The population studied was at high risk of tick-borne disease because of well-documented I. pacificus tick exposure [22].

There is a potential for cross reactivity to the B. miyamotoi GlpQ antigen in persons with prior exposure to B. hermsii [36, 37]. Borrelia hermsii is the primary etiologic agent of tick-borne relapsing fever (TBRF) in the western United States and transmitted by the argasid (soft) tick Ornithodoros hermsi [38, 39] with about 2 to 20 cases per year in California [40]. The primary hosts for these ticks are rodents [38, 39]. The range of the vector that carries B. hermsii are typically found at higher elevations (914 meters to 2743 meters) from the southern Cascades to the Eastern Sierra Nevada mountain range down to the Southern California Mountains [38]. Ventura County’s proximity to B. hermsii endemic areas (within 120 km), and the fact that no I. pacificus ticks have tested positive for B. miyamotoi from Ventura County [16, 20, 40, 41], suggest that we cannot rule out that the B. miyamotoi seropositive sample from Ventura County represents seropositivity to B. hermsii. Mendocino County, by contrast, has had I. pacificus ticks with documented B. miyamotoi infection and is geographically distant from O. hermsii distribution [22], supporting the glpQ -positive sample more likely to represent B. miyamotoi exposure. Both GlpQ positive samples for B. miyamotoi, not only can represent cross reactivity with B. hermsii, but the infections may have been acquired outside the county or state. Although B. miyamotoi has a low seroprevalence in California, our study findings are consistent with a previous study that humans in California are exposed to B. miyamotoi [22].

The overall seroprevalence for B. burgdorferi from both high and low Lyme disease endemic counties in California for Lyme disease was 0.47% (Exact 95% CI: 0.20, 0.93). The STTT, utilizing the C6 ELISA and IgG western blot, is more stringent and specific then the IgM western blot [42]. Since blood bank donors are generally healthier than the general population [43, 44] and the incidence of Lyme disease in California is about 0.2 cases per 100,000 population [16], a B. burgdorferi seroprevalence of less than 0.5% in California appears to be a reasonable estimate.

For California, a local approach to estimating risk is important for public health communication, given the well-documented non-uniform exposure due to local ecological influences [2, 11, 17, 45]. Few studies are available from California measuring B. burgdorferi seroprevalence in specific communities. In one study in Sonoma County, 1.4% of a small community were found to be seropositive for B. burgdorferi, an estimate within our confidence level estimates for that county, while in the same study, no samples from a blood bank collection from Sacramento County tested positive [46]. Although we did not find a statistically significant difference in seroprevalence between high and low Lyme disease endemic counties, all but one of our B. burgdorferi positive samples came from high endemic counties. Our study provides an updated estimate of B. burgdorferi exposure in a broad geographic area of California and helps demonstrate that risk for Lyme disease is geographically diverse. A more recent study found that 3.2% of residents in high endemic counties for Lyme disease in northern California may have been exposed to B. burgdorferi [47] as measured by the C6 ELISA alone. This estimate is within our confidence level estimates for C6 positive/equivocal samples. However, it is important that our C6 positive or equivocal samples were followed by testing with a more specific IgG Lyme western blot assay to avoid over estimation of B. burgdorferi exposure and to differentiate B. miyamotoi exposure. The increased sensitivity and decreased specificity of the C6 ELISA could over-estimate true exposure if used as a stand-alone test for estimating seropositivity, particularly in low incident states [48, 49] and should be used in conjunction with second tier assays in the recommended STTT format for Lyme disease diagnosis [50, 51].

The MTTT is intended for the qualitative detection of antibodies to B. burgdorferi in human serum [30]. With the advent of both the VslE1 and peptide C10 in the first tier ELISA assay, the MTTT assay we utilized is designed to be both sensitive and specific for early and late infection in patients with Lyme disease [30, 52]. The C6 peptide derived from VlsE1 does not bind to IgM well, and therefore the C6 ELISA is not ideal for detecting early cases of Lyme disease [48, 53]. On the other hand, an IgM response is generated early in disease in response to the pepC10 protein [52]. Among the 91 samples seropositive or equivocal by the C6 ELISA, the overall seropositivity was the same for MTTT and STTT. However, minor discrepancies were noted: Samples 59 and 79 were negative by STTT but positive by MTTT and Samples 17 and 23 were positive by STTT but negative by MTTT. Samples 59 and 79 had high Lyme index values by C6 ELISA but were negative on the Lyme disease western blot (IgG), possibly indicating that both samples were taken early after infection or the person had been treated early after illness onset and did not produce an expanded IgG response detectable by the Lyme disease western blot criteria. In contrast, samples 17 and 23 had lower index values by C6 ELISA and were positive by western blot, possibly indicating an old infection, with waning pepC10 antibody response that could not be picked up by the MTTT.

There are some limitations to this study. The necessary deidentified nature of human serum from blood bank samples precludes analysis of potential risk factors such as travel to B. burgdorferi, B. miyamotoi or B. hermsii endemic areas or degree of tick exposure. Human antibodies to the C6 peptide for B. burgdorferi wane after two years following untreated B. burgdorferi infection and probably more rapidly if treated [54]. The sensitivity of C6 for detecting B. miyamotoi infection and the longevity of the C6 antibody response in B. miyamotoi infection is also not known. Human IgG antibody response dynamics to B. miyamotoi are still unknown [55]. Lack of exposure information decreases the overall prior probability of having the disease in relation to diagnostic testing. Only nine negative samples were chosen due to resource limitations of second tier testing. Though the samples could have been matched by gender, given the small number of negative samples available, the authors chose to balance by endemnicity status. Since there was no difference in seropositivity between males and females, we felt that the 2/3 bias towards females in the negative samples did not compromise the study. Sampling sera from blood donors may decrease the generalizability of the results to the general population [43].

Conclusions

Our study demonstrates that California residents are at risk for infection by the emerging I. pacificus-transmitted pathogens B. miyamotoi and B. burgdorferi, although they are at relatively low risk of these infections even in the most highly endemic counties for Lyme disease. Standard two-tier B. burgdorferi testing and GlpQ serologic testing for B. miyamotoi on C6 positive or equivocal samples determined specific reactivities to these agents. Among samples testing C6 positive/equivocal, the STTT and MTTT performed fairly consistently even in this population with no known exposure history and in a state with low endemicity for Lyme disease. Further investigation of risk mapping related to geography and habitat type are needed for B. miyamotoi.

Supporting information

S1 Raw images

(PDF)

Click here for additional data file.^{(1.2MB, pdf)}

Acknowledgments

We would like to acknowledge Dr. Christopher Barker from the University of California, Davis for his helpful input and review on earlier drafts of this manuscript as well as his support as a member of my dissertation committee. We would like to also acknowledge the California Lyme Disease Association for their matching funds with the experiment.com crowdfunding platform. We would also like to thank Ruwini K. Rupasinghe, Phd Student in Epidemiology at UC Davis for her help in C6 Testing of all 1700 blood samples.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This research was supported in part by a crowdfunding platform through experiment.com set up by S.I.B.: https://experiment.com/projects/ticks-carry-more-than-lyme-disease-in-california-are-californians-at-risk. We would like to acknowledge the California Lyme Disease Association for providing matching funds to the experiment.com crowdfunding platform. This research was also supported in part by National Institutes of Health grant AI-100236 to A.G.B, The National Heart, Lung, and Blood Institute of the National Institutes of Health under award number K23HL151826 to E.M.B, and the Gordon and Llura Gund Foundation to P.J.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study.

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PLoS One. doi: 10.1371/journal.pone.0243950.r001

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10 Sep 2020

PONE-D-20-23819

Borrelia burgdorferi and Borrelia miyamotoi Seroprevalence in California Blood Donors

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"The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Dr. Bloch is a member of the U.S. Food and Drug Administration (FDA) Blood Products Advisory Committee. Any views or opinions expressed in this manuscript are Dr. Bloch's and are based on his own scientific expertise and professional judgment; they do not necessarily represent the views of the Blood Products Advisory Committee or the formal position of the FDA and also do not bind or otherwise obligate or commit either the Advisory Committee or the FDA to the views expressed. The views and opinions expressed herein are those of the authors alone and do not represent the official position of the Centers for Disease Control and Prevention."

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: I Don't Know

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: Review: Borrelia burgdorferi and Borrelia miyamotoi Seroprevalence in California Blood Donors.

This study assessed the seroprevalence of B. burgdorferi and B. miyamotoi in 1700 blood donors in California. Donors were selected from counties with reported high prevalence of B. burgdorferi infection as well as those with low prevalence. Of the 1700 donors, 91 were positive (or equivocal) by a standalone C6 ELISA assay for B. burgdorferi. When two tier (STTT or MTTT) assay was applied only 8 of the 91 donors were positive for B. burgdorferi antibodies. Two donors were positive for B. miyamotoi antibodies. No major differences in seroprevalence were observed between high and low prevalence areas. In total 0.47% of patients had serological evidence of B. burgdorferi infection and 0.12% had serological evidence of B. miyamotoi infection. The findings demonstrate low prevalence of B. burgdorferi and B. miyamotoi infection in California.

A key strength of this study is the assessment of seroprevalence for both agents at two independent testing sites (UCD and CDC) and the use of 2 testing methodologies (STTT and MTTT) for B. burgdorferi. The results are largely consistent with previous findings. The major comment is regarding the data presentation in the Results, which could be improved.

1) Figure 1 should include the number of samples that came from each county as well as the percentage. Figure Legends should be provided.

2) The 2nd paragraph of the Results (C6 ELISA Screen) provides information on how the 91 patients were selected for downstream analyses, but this information is not presented anywhere. My suggestion is to present this in a form of a Table or a flow chart since all subsequent analyses in the manuscript stem from this selection/screen.

3) In the second paragraph of the C6 ELISA Screen section, the authors discuss the distribution of Hispanics and non-Hispanics among low and high prevalence areas in California. They make a point that individuals with positive / equivocal result were usually non-Hispanic however one would assume this based on the lower prevalence of Hispanics in this cohort. The seroprevalence to C6 peptide should be assessed directly in Hispanics vs non-Hispanics taking into account the sample size for each.

4) The authors selected randomly 9 negative samples by standalone C6 ELISA, two thirds of which were female. What is the rationale for selecting 9 samples (vs more) and why were they not gender matched with the C6 seropositive populations?

5) Table 2 should include a row that lists the C6 findings for all 1700 patients. It should also include data for all 941 samples from high endemic area, as well as for 759 patients from low enedemic area; followed by the data for each county in that area. The data need to be clearly labeled (what are the data in parenthesis? Do the data represent positive results?). It would help to present the number of positives in addition to percentages.

6) The paragraph on B. miyamotoi GlpQ seroreactivity discusses seroprevalence in high vs low endemic regions for B. burgdorferi. This discussion is based on only two positive samples, and it is difficult to conclude much in the difference in prevalence between the two regions, except that the seroprevalence is relatively low. One could also postulate that the infection could have been acquired elsewhere and not in the counties/state where the samples were obtained.

7) A similar comment holds true for the paragraph on B. burgdorferi IgG western blot. The authors provide percentages and CI for seroprevalence in Marin county, but only one donor was seropositive there. With such small numbers it is difficult to assess the significance of seroprevalence and one should be careful to not overinterpret the data. For example, is it fair to state that San Luis Obispo county had the highest prevalence in the low endemic area since it had the only positive case (out of 759) in this region?

8) Line 369 in the Discussion: is it low specificity or high sensitivity of C6 ELISA that would over-estimate true exposure?

Reviewer #2: This is a very well written paper from several experienced investigators. They report a comprehensive sero-survey of B. burgdorferi and B. miyamotoi in counties of high and low endemicity for LD in California. The study design is straightforward and the results confirm previous observations of low prevalence of LD in CA (<0.5%). They found an even lower prevalence of antibodies to B. miyamotoi (0.1%). It is interesting that they found that the two positive miyamotoi cases were positive in C6 and Qlp. The following paper should be discussed: Borrelia miyamotoi infection leads to cross-reactive antibodies to the C6 peptide in mice and men. Koetsveld J, Platonov AE, Kuleshov K, Wagemakers A, Hoornstra D, Ang W, Szekeres S, van Duijvendijk GLA, Fikrig E, Embers ME, Sprong H, Hovius JW. Clin Microbiol Infect. 2020 Apr;26(4):513.e1-513.e6. doi: 10.1016/j.cmi.2019.07.026. Epub 2019 Aug 9. PMID: 31404672

The results are well discussed and limitations of the study have be identified. I have no further major or minor suggestions to improve this paper.

**********

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Reviewer #2: No

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PLoS One. 2020 Dec 28;15(12):e0243950. doi: 10.1371/journal.pone.0243950.r002

Author response to Decision Letter 0

19 Nov 2020

Manuscript Name: Borrelia burgdorferi and Borrelia miyamotoi Seroprevalence in California Blood Donors

Response to Reviewers

Reviewer 1:

Comment 1) Figure 1 should include the number of samples that came from each county as well as the percentage. Figure Legends should be provided.

Response to Comment #1: The California county map (figure 1) now includes both the number and percent of the sera samples from each county sampled. The figure legends have been provided in line with the text per instructions to authors. Figure 1 and legend to figure 1 starts at line 117. Figure 2 and legend to figure 2 starts at line 157 and figure 3 and legend to figure 3 starts at line 311.

Comment 2) The 2nd paragraph of the Results (C6 ELISA Screen) provides information on how the 91 patients were selected for downstream analyses, but this information is not presented anywhere. My suggestion is to present this in a form of a Table or a flow chart since all subsequent analyses in the manuscript stem from this selection/screen.

Response to Comment #2: The authors appreciate this comment. A flow chart has been added (figure 2) displaying the process of testing strategies along with the number of samples included for each test. Figure 2 and legend start at line 157. See separate figure 2 uploaded in Plos One submission website

Comment 3) In the second paragraph of the C6 ELISA Screen section, the authors discuss the distribution of Hispanics and non-Hispanics among low and high prevalence areas in California. They make a point that individuals with positive / equivocal result were usually non-Hispanic however one would assume this based on the lower prevalence of Hispanics in this cohort. The seroprevalence to C6 peptide should be assessed directly in Hispanics vs non-Hispanics taking into account the sample size for each.

Response to Comment #3: The authors appreciate this comment. We have re-worded the results of the C6 ELISA in terms of ethnicity. The paragraph starts at line 278. To address the small cell counts in ethnicity, a Fisher’s exact test was performed, which does take into account the sample sizes for each group.

Comment 4) The authors selected randomly 9 negative samples by standalone C6 ELISA, two thirds of which were female. What is the rationale for selecting 9 samples (vs more) and why were they not gender matched with the C6 seropositive populations?

Response to Comment #4: The authors thank the reviewer for this insight. We selected only 9 negative samples and not more due to resource limitations of second tier testing. Though the samples could have been matched on gender, given the small number of negative samples we could afford, the authors chose to balance by endemnicity status. Ultimately there was no difference in seropositivity between males and females, so the authors do not feel that the 2/3 bias towards females in the negative samples compromises the study. Nonetheless we noted this bias in the limitations paragraph starting at line number 491.

Comment 5) Table 2 should include a row that lists the C6 findings for all 1700 patients. It should also include data for all 941 samples from high endemic area, as well as for 759 patients from low endemic area; followed by the data for each county in that area. The data need to be clearly labeled (what are the data in parenthesis? Do the data represent positive results?). It would help to present the number of positives in addition to percentages.

Response to Comment #5: We have revised table 2 and added the findings from the C6 ELISA, which was used as a screening test. The data are clearly labeled and are presented as the number positive (% positive). Table 2 is on line 252.

Comment 6) The paragraph on B. miyamotoi GlpQ seroreactivity discusses seroprevalence in high vs low endemic regions for B. burgdorferi. This discussion is based on only two positive samples, and it is difficult to conclude much in the difference in prevalence between the two regions, except that the seroprevalence is relatively low. One could also postulate that the infection could have been acquired elsewhere and not in the counties/state where the samples were obtained.

Response to Comment #6: The authors agree that two positive samples limit detailed discussion on regional differences. We deleted line the sentence starting on line 288 that states that there was significant difference between high and low endemic counties. Beginning on line 417 we mentioned that seropositivity could have been acquired outside county of residence

Comment 7) A similar comment holds true for the paragraph on B. burgdorferi IgG western blot. The authors provide percentages and CI for seroprevalence in Marin County, but only one donor was seropositive there. With such small numbers it is difficult to assess the significance of seroprevalence and one should be careful to not over interpret the data. For example, is it fair to state that San Luis Obispo county had the highest prevalence in the low endemic area since it had the only positive case (out of 759) in this region?

Response to Comment #7: The sentence comparing Marin County seroprevalence to San Luis Obispo County seroprevalence was deleted. We substituted text on the overall seroprevalence along with its exact 95% CI were presented on all IgG western blot for all B. burgdorferi seropositive samples from each of the high endemic counties as well as the one positive B. burgdorferi from the low endemic county for LD, starting on line 308.

Comment 8) Line 369 in the Discussion: is it low specificity or high sensitivity of C6 ELISA that would over-estimate true exposure?

Response to Comment #8: The authors agree that high sensitivity and low specificity can over- estimate true exposure of LD, especially in a low prevalence state. Wormser et al, 2013 addressed this issue in his paper (reference 49): Single-tier testing with the C6 peptide ELISA kit was compared with two-tier testing for Lyme disease and the authors concluded that the C6 ELISA as a “single-step serodiagnostic test provided increased sensitivity in early Lyme disease with comparable sensitivity in later manifestations of Lyme disease. The C6 ELISA had slightly decreased specificity”. See revisions on manuscript starting on line 456 citing increased sensitivity and decreased specificity as potential cause of over-estimation.

Reviewer #2:

Comment 1) This is a very well written paper from several experienced investigators. They report a comprehensive sero-survey of B. burgdorferi and B. miyamotoi in counties of high and low endemicity for LD in California. The study design is straightforward and the results confirm previous observations of low prevalence of LD in CA (<0.5%). They found an even lower prevalence of antibodies to B. miyamotoi (0.1%). It is interesting that they found that the two positive miyamotoi cases were positive in C6 and Qlp. The following paper should be discussed: Borrelia miyamotoi infection leads to cross-reactive antibodies to the C6 peptide in mice and men. Koetsveld J, Platonov AE, Kuleshov K, Wagemakers A, Hoornstra D, Ang W, Szekeres S, van Duijvendijk GLA, Fikrig E, Embers ME, Sprong H, Hovius JW. Clin Microbiol Infect. 2020 Apr;26(4):513.e1-513.e6. doi: 10.1016/j.cmi.2019.07.026. Epub 2019 Aug 9. PMID: 31404672

The results are well discussed and limitations of the study have be identified. I have no further major or minor suggestions to improve this paper.

Response to Comment #1: Thank you for your suggestion on an article that should be discussed in this manuscript. The article has been discussed starting at line 386.

Attachment

Submitted filename: Brummitt et al Repsonse to Reviewers comments.docx

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PLoS One. doi: 10.1371/journal.pone.0243950.r003

Decision Letter 1

R Mark Wooten

1 Dec 2020

Borrelia burgdorferi and Borrelia miyamotoi Seroprevalence in California Blood Donors

PONE-D-20-23819R1

Dear Mrs. Brummitt,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The revised manuscript addresses the reviewer’s concerns. It brings to light important issue regarding seroprevalence of B. burgdorferi and B. miyamotoi in California which caries public health implications.

Reviewer #2: (No Response)

**********

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Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0243950.r004

Acceptance letter

R Mark Wooten

7 Dec 2020

PONE-D-20-23819R1

Borrelia burgdorferi and Borrelia miyamotoi Seroprevalence in California Blood Donors

Dear Dr. Brummitt:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Associated Data

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Borrelia burgdorferi and Borrelia miyamotoi seroprevalence in California blood donors

Sharon I Brummitt

Anne M Kjemtrup

Danielle J Harvey

Jeannine M Petersen

Christopher Sexton

Adam Replogle

Andrea E Packham

Evan M Bloch

Alan G Barbour

Peter J Krause

Valerie Green

Woutrina A Smith

Roles

Abstract

Introduction

Material and methods

Study population

Fig 1. Counties in California in which samples were collected.

Ethics approval

Sample processing and storage

Fig 2. Flowchart of testing strategies.

Standard two-tiered testing

Modified two-tiered testing

GlpQ ELISA

B. miyamotoi western blot

Statistical analysis

Results

Sample characteristics

Table 1. Demographic characteristics of sera samples and positive Bb STTT and Bm GlpQ.

Table 2. Testing results of sera samples for B. burgdorferi and B. miyamotoi by county.

C6 ELISA screen

B. miyamotoi GlpQ seroreactivity

Fig 3. B. burgdorferi and B. miyamotoi western blot seroreactivity in blood donors from higher and lower risk counties for Lyme disease in California.

Table 3. Antibody testing results of human samples.

B. burgdorferi IgG western blot

Modified two-tiered testing

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

R Mark Wooten

Roles

Author response to Decision Letter 0

Decision Letter 1

R Mark Wooten

Roles

Acceptance letter

R Mark Wooten

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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