用于抗体检测的可定制多重蛋白微阵列及其在蜱传和其他传染病中的应用-动脉网

A customizable multiplex protein microarray for antibody testing and its application for tick-borne and other infectious diseases

Nature 等信源发布 2025-01-20 02:08

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可切换为仅中文

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Abstract

摘要

Tick-borne infections are the most common vector-borne diseases in the USA. Ticks harbor and transmit several infections with Lyme disease being the most common tickborne infection in the US and Europe. Lack of awareness about tick populations, specific diagnostic tests, and overlapping signs and symptoms of tick-borne infections can often lead to misdiagnosis affecting treatment and the prevalence data reported especially for non-Lyme tick-borne infections.

蜱传感染是美国最常见的媒介传播疾病。蜱携带并传播几种莱姆病感染，莱姆病是美国和欧洲最常见的蜱传感染。缺乏对蜱虫种群，特定诊断测试以及蜱传感染的重叠体征和症状的认识，通常会导致误诊，影响治疗和报告的流行率数据，尤其是非莱姆病蜱传感染。

The diagnostic tests currently available for tick-borne diseases are severely limited in their ability to provide accurate results and cannot detect multiple pathogens in a single run. The multiplex protein microarray developed at Vibrant was designed to detect multiple serological antibodies thereby detecting exposure to multiple pathogens simultaneously.

目前可用于蜱传疾病的诊断测试在提供准确结果的能力方面受到严重限制，并且不能在一次运行中检测多种病原体。Vibrant开发的多重蛋白质微阵列旨在检测多种血清学抗体，从而同时检测多种病原体的暴露。

Our microarray in its present form can accommodate 400 antigens (molecules that can bind to specific antibodies) and can multiplex across antigen types, whole cell lysates, recombinant proteins, and peptides. A designed array containing multiple antigens of several microbes including .

我们目前形式的微阵列可以容纳400种抗原（可以与特定抗体结合的分子），并且可以跨抗原类型，全细胞裂解物，重组蛋白和肽进行多重化。一种设计的阵列，包含多种微生物的多种抗原，包括。

Borrelia burgdorferi

博雷利亚 Burgdorferi

, the Lyme disease spirochete, was manufactured and evaluated. The immunoglobulin M (IgM) and G (IgG) responses against several tick-borne microbes and other infectious agents were analyzed for analytical and clinical performance. The microarray improved IgM and IgG sensitivities and specificities of individual microbes when compared with the respective gold standards.

莱姆病螺旋体的制造和评估。分析了针对几种蜱传微生物和其他感染因子的免疫球蛋白M（IgM）和G（IgG）反应的分析和临床表现。与相应的金标准相比，微阵列改善了单个微生物的IgM和IgG敏感性和特异性。

The testing was also performed in a single run in comparison to multiple runs needed for comparable testing standards. In summary, our study presents a flexible multiplex microarray platform that can provide quick results with high sensitivity and specificity for evaluating exposure to varied infectious agents especially tick-borne pathogens..

与可比测试标准所需的多次运行相比，测试也在一次运行中进行。总之，我们的研究提供了一个灵活的多重微阵列平台，可以提供快速的结果，具有高灵敏度和特异性，用于评估暴露于各种传染源，尤其是蜱传病原体。。

Introduction

导言

Most vector-borne infections in the USA can be attributed to pathogens transmitted via tick bites. Of all tick-borne infections identified to date, Lyme disease is the most prevalent infection

美国大多数媒介传播的感染可归因于通过蜱叮咬传播的病原体。在迄今为止确定的所有蜱传感染中，莱姆病是最普遍的感染

. Lyme disease is a potentially serious bacterial infection transmitted by ticks and was first reported in the mid-1970s in the USA. The etiological agent was identified later as

莱姆病是一种由蜱传播的潜在严重细菌感染，于20世纪70年代中期在美国首次报道。病原体后来被确定为

Borrelia burgdorferi

博雷利亚 Burgdorferi

. Several studies have reported the presence of co-infections along with

几项研究报告了合并感染的存在

B. burgdorferi

including

包括

Babesia spp.

巴贝斯虫属。

Bartonella spp.

巴尔通体属。

Ehrlichia spp.

埃立克体属。

Anaplasma phagocytophilum

Anaplasma 细胞

, Powassan virus (POWV)

波瓦桑病毒

Toxoplasma gondii

弓形虫

Rickettsia spp.

立克次体属。

, tick-borne encephalitis virus (TBEV)

，蜱传脑炎病毒（TBEV）

, and West Nile virus (WNV)

和西尼罗河病毒（WNV）

. Additionally, prolonged exposure to

此外，长期暴露于

B. burgdorferi

and other tick-borne pathogens could potentially weaken the patient’s immune system increasing the risk of infections by Epstein Barr virus (EBV)

其他蜱传病原体可能会削弱患者的免疫系统，增加感染爱泼斯坦-巴尔病毒（EBV）的风险

, cytomegalovirus (CMV)

，巨细胞病毒（CMV）

, parvovirus B19 (B19V)

, coxsackie B virus

，柯萨奇B病毒

, herpes simplex virus 1 (HSV-1)

，单纯疱疹病毒1（HSV-1）

, herpes simplex virus 2 (HSV-2)

，单纯疱疹病毒2型（HSV-2）

, and human herpes virus 6 (HHV-6)

和人类疱疹病毒6（HHV-6）

Ticks have been shown to transmit more than one infectious agent in a single bite. For instance, a study by Wormser et al. showed that there was a risk of getting infected with

蜱已被证明在一次叮咬中传播不止一种传染源。例如，Wormser等人的一项研究表明，有感染的风险

(A) phagocytophilum

（A）吞噬细胞

(30%) and

（30%）和

(B) microti

（B） microti

(24%) along with

（24%）以及

B. burgdorferi

. Currently, multi-tiered testing is carried out for diagnosing tick-borne infections

.目前，正在进行多层测试以诊断蜱传感染

(

https://tinyurl.com/33564b8z

). In this method, the infectious agents are tested sequentially, starting with

)。在这种方法中，传染源按顺序进行测试，从

B. burgdorferi

. This method is time-consuming and can often lead to delayed diagnosis, accompanied with high cost to the patient

。这种方法耗时，通常会导致诊断延迟，并给患者带来高昂的成本

(

https://tinyurl.com/33564b8z

). Testing for multiple infections in a single run can help physicians arrive at an accurate diagnosis especially since Lyme disease shares symptoms with other vector-borne co-infections

)。

. The existing diagnostic assays possess various limitations that restrict their applicability in the diagnosis of these infections. The diagnosis of Lyme disease and other infectious diseases using several blot-based and single-plex ELISA tests remain rudimentary in terms of arriving at a diagnostic conclusion.

现有的诊断分析方法具有各种局限性，限制了它们在诊断这些感染中的适用性。在得出诊断结论方面，使用几种基于印迹和单重ELISA测试来诊断莱姆病和其他传染病仍然是初步的。

. Additionally, blot-based assays may have overlapping proteins with similar mass requiring additional testing to tease out the specific antigen to which the antibody is bound. A multiplex system can detect the biomarkers of Lyme disease, potential co-infections, and other infections in a single run.

此外，基于印迹的测定可能具有相似质量的重叠蛋白质，需要额外的测试来梳理抗体结合的特定抗原。多重系统可以在一次运行中检测莱姆病的生物标志物，潜在的合并感染和其他感染。

A serology-based multiplexing system may be preferred to a PCR multiplex system mainly due to its accessibility, for instance using dried blood spots (DBS).

基于血清学的多路复用系统可能优于PCR多路复用系统，主要是由于其可访问性，例如使用干血斑（DBS）。

. Due to the transient nature of the organisms, the timeline in which the patients are bacteremic/viremic/parasitemic is short making it difficult to obtain genetic material of the pathogens for nucleic acid-based diagnosis. However, this limitation is overcome by using serology

。然而，通过使用血清学可以克服这一局限性

. A serology-based system is also ideal for population screening and surveillance since it can indicate past exposure to a pathogen.

基于血清学的系统也是人群筛查和监测的理想选择，因为它可以表明过去接触过病原体。

Our customisable protein microarray design includes antigens physically separated by design unlike blot assays and can multiplex across species. Multiplexing can also be done across antigen types such as recombinant proteins, peptides, and lysates simultaneously. This method can lower test costs since all the manufacturing is automated using bio customised semiconductor processes similar to how electronic chips are made.

我们可定制的蛋白质微阵列设计包括通过设计物理分离的抗原，这与印迹分析不同，并且可以跨物种进行多重化。也可以同时跨抗原类型（例如重组蛋白，肽和裂解物）进行多路复用。这种方法可以降低测试成本，因为所有的制造都是使用类似于电子芯片制造的生物定制半导体工艺自动化的。

The multiplex microarray has three main advantages over the existing technologies. It has an ultra-high-density array surface with high reproducibility and better throughput. It can detect a large number of antibodies against varied infectious agents at the same time. Detection of antibodies can be performed using low sample volumes with low cost and a fast turnaround time.

与现有技术相比，多重微阵列具有三个主要优势。它具有超高密度阵列表面，具有高重现性和更好的通量。它可以同时检测大量针对各种传染源的抗体。抗体的检测可以使用低成本和快速周转时间的低样本量进行。

. Given the flexible nature of the multiplex platform, we aimed to provide a multiplexed testing solution for Lyme disease, its co-infections, and other possible infections of interest.

鉴于多重平台的灵活性，我们旨在为莱姆病及其合并感染和其他可能感兴趣的感染提供多重测试解决方案。

Materials and methods

材料和方法

Patients sera

患者血清

The sera were collected from 2990 (843 samples and 2147 controls) individuals after seeking appropriate Institutional Review Board (IRB) approvals under respective collaborators (Supplementary Table 1). The study was conducted under the ethical principles that have their origins in the Declaration of Helsinki.

在寻求各自合作者的适当机构审查委员会（IRB）批准后，从2990名（843个样本和2147个对照）个体中收集了血清（补充表1）。这项研究是根据源自《赫尔辛基宣言》的道德原则进行的。

The Western Institutional Review Board, WIRB (work order #1-1574995-1) waived the need for informed consent requirement. Table .

西方机构审查委员会WIRB（工作指令#1-1574995-1）放弃了知情同意的要求。表。

lists the provided samples for Lyme disease, co-infections, and other infections along with the counts, respective collaborators and methods used to ascertain the clinical diagnosis by the physician. Supplementary Table 2 provides information regarding the vendors and strains of the pathogens used in the study.

列出提供的莱姆病、合并感染和其他感染的样本，以及计数、各自的合作者和用于确定医生临床诊断的方法。补充表2提供了有关研究中使用的病原体的供应商和菌株的信息。

These reference sera were tested at Vibrant America Clinical Labs (Clinical Laboratory Improvement (CLIA) and College of American Pathologists (CAP) accredited facility) by laboratory personnel in a blinded manner. The sera from healthy patients were considered negative and were used to set the cut-off values and were investigated under IRB (work order #1-1574995-1) determined by WIRB to employ de-linked and de-identified human specimens and medical data for research findings.

这些参考血清由实验室人员在Vibrant America Clinical Labs（临床实验室改进（CLIA）和美国病理学家学院（CAP）认可的设施）进行盲法测试。。

The negative sera were collected from across the US including endemic and nonendemic regions for these infections..

这些阴性血清是从美国各地收集的，包括这些感染的地方性和非流行性地区。。

Table 1 Sample cohort for each pathogen. The table provides an overview of the pathogens used in the study, along with total number of samples, sample source, and basis of diagnosis.

表1每种病原体的样本队列。该表概述了研究中使用的病原体，以及样本总数，样本来源和诊断依据。

Full size table

全尺寸表

Processing of wafers

晶圆的加工

Wafers were functionalized as described previously

晶片如前所述进行了功能化

(

https://tinyurl.com/mr9ctppy

). Briefly, silicon wafers were exposed to an environment of pure oxygen for 2 h followed by washing (deionized water) and coating (1% (vol/vol) with 3-aminopropyltriethoxysilane (APTES) in N-methylpyrrolidone (NMP). Curing was carried out at 120 °C for 60 min under an N

)。简而言之，将硅片暴露于纯氧环境中2小时，然后洗涤（去离子水）并用N-甲基吡咯烷酮（NMP）中的3-氨丙基三乙氧基硅烷（APTES）涂覆（1%（体积/体积））。固化在120°C下进行60分钟

atmosphere and humidity-controlled environment. Coating and incubation of the wafer with a co-polymer solution of poly (L-lysine) and poly (lactic acid) for 24 h were carried out to increase the binding efficiency of the surface on to which the antigens were immobilized via passive adsorption/hydrophobic interactions with the copolymers [Fig. .

大气和湿度受控环境。用聚（L-赖氨酸）和聚（乳酸）的共聚物溶液对晶片进行涂覆和孵育24小时，以提高通过被动吸附/疏水相互作用将抗原固定在其上的表面的结合效率。与共聚物[图。

Fig. 1

图1

Wafer Processing, Antigen Immobilisation, Pillar Plate Assembly. A poly (lactic acid) and poly (L-lysine) copolymer solution is coated onto the silicon wafers and further immobilized with protein probes [steps 1–3]. The wafers are then diced into microchips using a stealth dicing process [step 4]. A standard die sorting system is used to pick and place the microchips onto carrier plates [step 5].

晶圆加工，抗原固定，柱板组装。将聚（乳酸）和聚（L-赖氨酸）共聚物溶液涂覆在硅片上，并进一步用蛋白质探针固定[步骤1-3]。然后使用隐形切割工艺将晶片切成微芯片[步骤4]。使用标准模具分选系统将微芯片拾取并放置在载体板上[步骤5]。

The carrier tapes are loaded onto a high throughput surface mount technology (SMT) component placement system and individual microchips are placed onto 24-pillar plates. Each pillar consists of 87 microchips [step 6]. Created using Figma, Version 116.15 (.

载体胶带被装载到高通量表面贴装技术（SMT）组件放置系统上，单个微芯片被放置在24柱板上。每个支柱由87个微芯片组成[步骤6]。使用Figma版本116.15创建（。

https://www.figma.com/

Full size image

全尺寸图像

Immobilization of antigens

抗原的固定化

The antigens included in the assay are listed in Table

表中列出了测定中包括的抗原

. Pathogens transmitted by ticks and their respective antigens for potential future additions are listed in Fig.

图1列出了蜱传播的病原体及其各自的抗原，以备将来添加。

(

https://tinyurl.com/mwz2u6kf

). The recombinant antigens were expressed in

)。重组抗原在

E. coli

E、大肠杆菌

bacteria using full-length cDNA coding for the respective antigens fused with a hexa histidine purification tag. The whole cell lysate was obtained from organisms cultured according to ATCC protocols prior to lysing them which yielded a cocktail of the cell membrane, cell wall, and cytosolic proteins.

使用全长cDNA编码与六组氨酸纯化标签融合的各个抗原的细菌。在裂解它们之前，从根据ATCC方案培养的生物体中获得全细胞裂解物，其产生细胞膜，细胞壁和胞质蛋白的混合物。

Peptide antigens were synthesized by photolithography as shown in our previous publications.

如我们之前的出版物所示，通过光刻合成肽抗原。

. The capture antigens including the recombinant antigens that mimic the natural pathogen and the whole-cell lysate were incubated on the wafer at a concentration of 1.0 µg/ml and reacted for 24 h at 4 °C. The unbound antigens were removed by washing with aqueous phosphate buffer and the unreacted substrate was quenched with a blocking solution containing bovine serum albumin (BSA) and glycine.

。将捕获抗原（包括模拟天然病原体的重组抗原和全细胞裂解物）在晶片上以1.0µg/ml的浓度孵育，并在4°C下反应24小时。通过用磷酸盐缓冲液洗涤除去未结合的抗原，并用含有牛血清白蛋白（BSA）和甘氨酸的封闭溶液淬灭未反应的底物。

The immobilized antigens were classified with unique identifiers assigned to each wafer. In this study, we employed the microarray to detect Lyme disease, co-infections, and other agents of interest including, .

固定的抗原用分配给每个晶片的唯一标识符进行分类。在这项研究中，我们使用微阵列检测莱姆病，合并感染和其他感兴趣的药物，包括。

B. microti

B. 微生物

B. henselae

B. 建议

A. phagocytophilum

A、吞噬细胞

E. chaffeensis

R. typhi

R.typi

R. rickettsii

立克次氏体

, POWV, TBEV, WNV, coxsackie B virus, CMV, EBV, B19V,

，POWV，TBEV，WNV，柯萨奇B病毒，CMV，EBV，B19V，

T. gondii

T、弓形虫

, HSV-1, HSV-2, and HHV-6 [Fig.

，HSV-1，HSV-2和HHV-6[图。

Table 2 Overview of pathogens and antigens.

表2病原体和抗原概述。

Full size table

全尺寸表

Fig. 2

图2

Overview of all the pathogens and their respective antigens used in the study.

研究中使用的所有病原体及其各自抗原的概述。

Full size image

全尺寸图像

Pillar plate assembly

支柱板总成

Individual wafers were stealth diced into 0.70 × 0.70mm

将单个晶片隐形切成0.70×0.70mm

microchips for each antigen. A standard die-sorting system was used to pick and place these wafers onto individual carrier tapes. The carrier tapes were then placed onto a high-throughput surface mount technology (SMT) component placement system. Finally, microchips were mounted onto 24 pillar plates and each pillar contains 87 microchips with each chip designated for one antigen – recombinant protein, peptide or whole cell lysates [Fig. .

每种抗原的微芯片。使用标准的模具分选系统将这些晶片拾取并放置到各个载体胶带上。然后将载带放置在高通量表面贴装技术（SMT）组件放置系统上。最后，将微芯片安装在24柱板上，每个柱包含87个微芯片，每个芯片指定用于一种抗原重组蛋白，肽或全细胞裂解物[图。

Immunochip assay and antibody detection

免疫芯片检测和抗体检测

Serum samples were probed using 1:20 dilution on the pillar plate and incubated for 1 h at room temperature followed by alternate washing and incubation as described previously

血清样品在柱板上用1:20稀释液进行探测，并在室温下孵育1小时，然后如前所述交替洗涤和孵育

. The plate was then incubated for an hour with the secondary antibody (1:2000 dilution of Goat Anti-Human IgG HRP and Goat Anti-Human IgM HRP individually) and washed with TBST buffer followed by DI Water. The plates were left for drying preceding the addition of chemiluminescent substrate and the performance of chemiluminescent imaging.

然后将平板与二抗（分别以1:2000稀释的山羊抗人IgG-HRP和山羊抗人IgM-HRP）孵育1小时，并用TBST缓冲液和去离子水洗涤。在加入化学发光底物和化学发光成像的性能之前，将板放置干燥。

An enhanced IgM sensitivity was achieved by pre-reacting the sera with goat anti-human IgG leading to IgG stripping prior to IgM testing..

通过在IgM测试之前将血清与山羊抗人IgG预反应导致IgG剥离，可以提高IgM敏感性。。

The detection of multiplex antibodies is based on the chemiluminescent immunoassay and can be performed using < 200 µL of serum. Sample dilution, multi-step incubation, and multi-solution washing are programmed into liquid handlers. The immunochip has the capacity to assay 192 individual specimens in 2 h.

多重抗体的检测基于化学发光免疫测定法，可以使用200μL血清进行。样品稀释，多步孵育和多溶液洗涤被编程到液体处理器中。免疫芯片能够在2小时内检测192个个体标本。

Raw chemiluminescent signals for each probe are extracted and converted into intensity plots by an in-house reporter software. This method of automatic antigen detection can dramatically shorten the turnaround time, reduce the cost of labor and instrument, and eliminate the need for manual handling and subjective interpretation of the WB or IB test results when compared to the traditional two-tiered testing recommended by the CDC.

提取每个探针的原始化学发光信号，并通过内部报告软件将其转换为强度图。与CDC推荐的传统双层检测相比，这种自动抗原检测方法可以大大缩短周转时间，降低人力和仪器成本，并且不需要手动处理和主观解释WB或IB检测结果。

All the antibodies are detected in a single run..

所有抗体均在一次运行中检测到。。

Data Analysis

数据分析

An in-house software extracts the chemiluminescent signals from the generated images which were converted to intensity plots. The average intensity of each antibody was compared with the cut-off values assigned for each antigen to track seropositivity.

内部软件从生成的图像中提取化学发光信号，并将其转换为强度图。。

Results

结果

Custom protein microarray platform

定制蛋白质微阵列平台

The main components of the Immunochip platform include multiple silicon-based 0.70 × 0.70 mm

免疫芯片平台的主要组件包括多个硅基0.70×0.70 mm

microchips that are laser diced from antigen-immobilized wafers, a customized 24 well compatible plate containing 24 pillars, each containing 87 microchips that are picked and placed into a multiplex microarray assembly, and a high-resolution imager capable of simultaneously detecting chemiluminescent signals from labelled antigen–antibody reactions at each microchip throughout the multiplex microarray (Fig. .

从抗原固定的晶片上激光切割的微芯片，一个定制的24孔兼容板，其中包含24个柱，每个柱包含87个微芯片，这些微芯片被挑选并放置在多重微阵列组件中，以及一个高分辨率成像仪，能够同时检测整个多重微阵列中每个微芯片上标记的抗原-抗体反应的化学发光信号（图）。

). Each chip can be considered analogous to an individual band in a western blot; however, the proteins are physically separated eliminating cross-reactive issues usually seen in blot-based assays for proteins with similar mass. Figure

)。每个芯片可以被认为类似于蛋白质印迹中的单个条带；然而，这些蛋白质是物理分离的，消除了在基于印迹的蛋白质质量相似的分析中常见的交叉反应问题。图

provides an overview of the microarray manufacturing process. Figure

提供了微阵列制造过程的概述。图

shows the individual chips that are placed in each pillar, a single serum sample will be applied to each pillar thereby assaying the antibodies in serum against all antigens at the same time.

显示了放置在每个柱中的单个芯片，将单个血清样品应用于每个柱，从而同时测定血清中针对所有抗原的抗体。

Analysis of serological response

血清学反应分析

The Vibrant tick-borne disease panel tests for IgG and IgM antibodies for Lyme disease and other infectious agents as mentioned in Table

如表所示，活力蜱传疾病小组测试莱姆病和其他传染源的IgG和IgM抗体

; Fig.

；图。

. The IgM and IgG immune responses of serum samples obtained from various laboratories (Table

) were analysed, and the clinical sensitivities and specificities were tabulated in Table

)进行了分析，并将临床敏感性和特异性列于表中

. The samples reacted with specific immunoreactive epitopes on the 87 different antigens that were being tested.

.样品与正在测试的87种不同抗原上的特异性免疫反应表位反应。

Table 3 Antigen sensitivities and specificities. Table shows the IgG, IgM, and IgG + IgM sensitivities and specificities obtained using the vibrant microarray, and a comparison with the sensitivities and specificities of the current gold standard for respective pathogens.

表3抗原敏感性和特异性。表显示了使用vibrant微阵列获得的IgG，IgM和IgG+ IgM敏感性和特异性，以及与当前金标准对各自病原体的敏感性和特异性的比较。

Full size table

全尺寸表

Enhanced IgM assay

增强的IgM测定

IgM antibodies are markers of an acute primary infection and are produced during the first two weeks of infection, whereas IgG antibodies are produced few days later and may remain for life. Total IgM antibodies make up only 5–10% of all the circulating antibodies.

IgM抗体是急性原发感染的标志物，在感染的前两周产生，而IgG抗体在几天后产生，并可能终生存在。总IgM抗体仅占所有循环抗体的5-10%。

B. burgdorferi

B.伯氏疏螺旋体

IgM antibodies may persist in Lyme disease patients’ years after the initial infection

莱姆病患者初次感染后数年内，IgM抗体可能持续存在

. An in-house IgM assay was developed with removal of most IgG antibodies and other non-specific proteins from the serum prior to the IgM immunoassay. This helped to increase the sensitivity and specificity of the assay. Human IgG was removed by incubating the serum with a purified goat anti-human (GAH) IgG Fc fragment..

。在进行IgM免疫测定之前，开发了一种内部IgM测定法，从血清中去除了大多数IgG抗体和其他非特异性蛋白。这有助于提高测定的灵敏度和特异性。通过将血清与纯化的山羊抗人（GAH）IgG Fc片段孵育来除去人IgG。。

Analytical performance

分析性能

The analytical performance of the immunochip was evaluated for precision (repeatability/reproducibility), analytical sensitivity, reportable range, linearity, and matrix equivalency studies. Samples for negatives, low or moderate positives, and high positives were run with duplication to determine the analytical performance metrics.

。阴性，低或中等阳性和高阳性的样本重复运行，以确定分析性能指标。

The precision study used a panel of 11 samples and was run over a period of 20 days with 2 duplicates per run and 4 runs per day. The results are tabulated as shown in Supplementary Table 3. Lot to Lot reproducibility was also tested to check for variation in the manufacturing of the pillar plates by running a panel of 11 samples with 5 replicates per run, 3 runs per day over a period of 5 days using 3 manufactured lots.

精密度研究使用了一组11个样本，运行了20天，每次运行2次，每天运行4次。结果如补充表3所示。还测试了批次间的再现性，以检查柱板制造的变化，方法是运行一组11个样品，每次运行5个重复，每天运行3次，使用3个制造批次，持续5天。

The results are tabulated as shown in Supplementary Table 4. Testing of protein-free serum matrix samples and low antibody concentration samples with 2 replicates per run, 2 runs per day over a period of three days was used to determine analytical sensitivity. The limit of blank (LoB) and limit of quantitation (LoQ) was calculated using the mean and standard deviation of the blank and the low antibody concentration samples as shown in Supplementary Table 5.

结果如补充表4所示。测试无蛋白血清基质样品和低抗体浓度样品，每次重复2次，每天2次，持续三天，以确定分析灵敏度。如补充表5所示，使用空白和低抗体浓度样品的平均值和标准偏差计算空白限（LoB）和定量限（LoQ）。

The linearity and reportable range were verified by running samples with varying levels of antibodies and checking assay recovery, the results are tabulated in Supplementary Table 6. Matrix equivalence studies are shown in Supplementary Table 7. The potential interference of specific endogenous and exogenous substances with the immunochip was evaluated by performing an interfering substance study.

通过运行具有不同抗体水平的样品并检查测定回收率来验证线性和可报告范围，结果列于补充表6中。矩阵等效性研究见补充表7。通过进行干扰物质研究，评估了特定内源性和外源性物质对免疫芯片的潜在干扰。

The interfering substances tested were 60 mg/dl bilirubin, 100 mg/ml cholesterol, 1000 mg/ml triglycerides, 1000 mg/ml hemoglobin, and 6 g/dl albumin. There was no interference .

测试的干扰物质为60 mg/dl胆红素，100 mg/ml胆固醇，1000 mg/ml甘油三酯，1000 mg/ml血红蛋白和6 g/dl白蛋白。没有干扰。

Clinical sensitivity and specificity

临床敏感性和特异性

Table

表

provides an overview of the IgG and IgM sensitivities and specificities measured by the Vibrant microarray. This is compared with the pperformance of the current gold standard tests for the particular pathogen. The Vibrant microarray was able to achieve high sensitivities and specificities when compared with the gold standards for each pathogen.

概述了Vibrant微阵列测量的IgG和IgM敏感性和特异性。这与当前针对特定病原体的金标准测试的性能进行了比较。与每种病原体的金标准相比，充满活力的微阵列能够实现高灵敏度和特异性。

Supplementary Table 8 provides more details on the gold standard diagnostic tests for the pathogens along with the modes of transmission and their endemic regions..

补充表8提供了有关病原体的金标准诊断测试以及传播方式及其流行地区的更多详细信息。。

Evaluating the antigens of

评估抗原

Borrelia burgdorferi

博雷利亚 Burgdorferi

In this study, individual antigens of

在这项研究中，个体抗原

B. burgdorferi

were tested for reactivity with IgG and IgM antibodies (Table

测试了与IgG和IgM抗体的反应性（表

). The heat map (Fig.

)。热图（图）。

) shows the performance metrics of the different antigens. Testing for Lyme disease since 1994 has been based on conventional two-tiered testing (CTTT) where an enzyme immunoassay (EIA) is followed by a specific immunoblot for a definitive diagnosis. Recently, CTTT has been replaced with a modified two-tier testing (MTTT) in which an EIA using whole cell lysate is followed by an EIA using C6 peptide.

)显示了不同抗原的性能指标。自1994年以来，莱姆病的检测一直基于传统的两层检测（CTTT），其中酶免疫测定（EIA）之后是特异性免疫印迹以进行明确诊断。最近，CTTT已被改进的双层测试（MTTT）所取代，其中使用全细胞裂解物的EIA之后是使用C6肽的EIA。

This shows the increasing shift away from blot-based testing to conventional ELISA. MTTT removes the burden of immunoblots which are tedious to run, more expensive and could have subjective interpretation of bands.

这表明越来越多地从基于印迹的测试转向常规ELISA。MTTT消除了免疫印迹的负担，免疫印迹运行繁琐，价格昂贵，并且可能对条带进行主观解释。

. Complete replacement of immunoblots can be done using a microarray platform, such as the one described here. The full data set would be available to the physicians to make a nuanced diagnosis instead of a narrow subset of antigens run on ELISAs.

。医生可以使用完整的数据集进行细微的诊断，而不是在ELISA上运行的一小部分抗原。

Table 4 Individual antigen sensitivities (

表4个体抗原敏感性(

Borrelia burgdorferi)

Borrelia burgdorferi）

. The table shows the IgG and IgM values for the individual antigens of

该表显示了个体抗原的IgG和IgM值

B. Burgdorferi.

B.伯氏疏螺旋体。

Full size table

全尺寸表

Fig. 3

图3

Heat map showing Lyme disease antigen reactivity. The positivity cutoff for each antigen was set at greater than 10 chemiluminescent units (CU) (shown as yellow or red). The color key is as follows: Red - High positive (CU > 20); Yellow, orange - Moderate positive (CU = 10.1–20); White - Negative (CU ≤ 10)..

热图显示莱姆病抗原反应性。每种抗原的阳性截止值设置为大于10个化学发光单位（CU）（显示为黄色或红色）。颜色键如下：红色-高正（CU > 20）；黄色，橙色-中度阳性（CU=10.1-20）；白色-阴性（CU≤10）。。

Full size image

全尺寸图像

Discussion

讨论

Ticks are among the most important sources of vector-borne infections in the US

蜱是美国媒介传播感染的最重要来源之一

. The spread of ticks across the US has been steadily increasing over the past decades. In parallel, the discovery of novel pathogens that are spread by ticks has also seen dramatic increases

。过去几十年来，蜱在美国的传播一直在稳步增加。与此同时，由蜱传播的新型病原体的发现也急剧增加

. Currently, there are 14 major tickborne diseases according to the CDC namely, Lyme disease, babesiosis, ehrlichiosis, Rocky Mountain spotted fever, Southern tick-associated rash illness (STARI), hard tick relapsing fever, soft tick relapsing fever, tularemia, anaplasmosis, Colorado tick fever, bourbon virus, heartland virus, .

根据疾病预防控制中心的数据，目前有14种主要的蜱传疾病，即莱姆病，巴贝斯虫病，埃立克体病，落基山斑点热，南部蜱相关皮疹病（STARI），硬蜱复发热，软蜱复发热，土拉菌病，无浆体病，科罗拉多蜱热病，波旁病毒，心脏地带病毒。

Rickettsia parkeri

Rickettsia 公园

rickettsiosis, 364D rickettsiosis, and Powassan virus

rickettsiosis，364d rickettsiosis，powassan病毒

(

https://www.cdc.gov/ticks/diseases/index.html

). Patients are rarely tested for all possible infections that could be transmitted via a tick bite

)。很少对患者进行可能通过蜱叮咬传播的所有可能感染的检测

. The current diagnostic tests are severely limited in distinguishing various tick-borne infections and several studies have revealed that non-Lyme disease tick-borne infections are heavily underdiagnosed

目前的诊断测试在区分各种蜱传感染方面受到严重限制，一些研究表明，非莱姆病蜱传感染的诊断严重不足

Among varied testing options, PCR and serology-based assays are reliable and most widely used. PCR has several advantages as it detects pathogenic DNA/RNA which conclusively proves the organism’s presence. It has high specificity and has a high throughput with assay run times of about 2 h. It can also detect the infection during its early stages.

在各种测试选项中，基于PCR和血清学的检测方法是可靠且使用最广泛的。PCR有几个优点，因为它可以检测致病性DNA/RNA，从而最终证明生物体的存在。它具有高特异性和高通量，测定运行时间约为2小时。。

. There are however certain drawbacks to testing using PCR, especially with tickborne infections. Pathogens transmitted by vectors may be transient in the blood resulting in clinically false negative PCR in tick-borne diseases and other infections, namely,

。由载体传播的病原体可能在血液中短暂存在，导致蜱传疾病和其他感染的临床假阴性PCR，即，

B. burgdorferi

R. typhi

R.typi

T. gondii

T、弓形虫

, HSV-1, EBV, TBEV, and WNV

，HSV-1，EBV，TBEV和WNV

. PCR testing requires specialised laboratories and equipment for testing. PCR may not detect all strains and variants and is limited to detecting known pathogens

PCR检测需要专门的实验室和设备进行检测。PCR可能无法检测所有菌株和变异，仅限于检测已知病原体

. Multiplexing with PCR is limited due to fixed number of analytes that can be parallelly read using PCR instrumentation.

由于使用PCR仪器可以并行读取固定数量的分析物，因此与PCR的多路复用受到限制。

Serology-based testing has several advantages when it comes to tick-borne infection testing. It has the ability to comprehensively assess immune responses and simultaneously detect exposure to multiple pathogens including previous and unresolved infections. Testing two times with a time interval in between can also help diagnose active infections based on altered serum antibody profiles.

当涉及蜱传感染检测时，基于血清学的检测有几个优点。它能够全面评估免疫反应，同时检测多种病原体的暴露，包括以前和未解决的感染。每隔一段时间进行两次检测，也可以根据血清抗体谱的改变来诊断活动性感染。

Simultaneous detection of antibodies against multiple tick-borne pathogens and other related pathogens using a single sample and providing a comprehensive view of the patient’s immune response is a key advantage of serology-based multiplex testing.

使用单个样品同时检测针对多种蜱传病原体和其他相关病原体的抗体，并提供患者免疫反应的全面视图，是基于血清学的多重检测的关键优势。

. The testing can also be done in resource poor settings with collection using DBS as described for Covid-19

。测试也可以在资源匮乏的环境中进行，并使用针对Covid-19所述的DBS进行收集

. Serological testing can diagnose tick-borne diseases even in the later stages when pathogen detection through molecular methods becomes more challenging

。即使在通过分子方法检测病原体变得更具挑战性的后期阶段，血清学检测也可以诊断蜱传疾病

. It also reduces the risk of false negatives

。它还降低了假阴性的风险

. Serological multiplex testing being cost-effective can also contribute to surveillance and epidemiological studies by providing valuable data on the prevalence and distribution of tick-borne diseases, enhancing our understanding of disease dynamics

。血清学多重检测具有成本效益，还可以通过提供有关蜱传疾病流行和分布的有价值数据，增强我们对疾病动态的理解，为监测和流行病学研究做出贡献

. Patients can be asymptomatic during the initial stages or may not remember the tick-bite. Serology is useful to detect the infection in such cases. However, serological studies have their own limitations. Serological testing may not be able to detect early/recent infections. It relies heavily on the timing of sample collection and the host’s immune responses.

。患者在初始阶段可能无症状，也可能不记得蜱叮咬。血清学有助于检测这种情况下的感染。然而，血清学研究有其自身的局限性。血清学检测可能无法检测早期/近期感染。它严重依赖于样本采集的时间和宿主的免疫反应。

In certain cases, molecular testing may be needed to confirm serological testing.

在某些情况下，可能需要进行分子检测以确认血清学检测。

. Despite all this, the benefits of serology testing outweigh its limitations which is why it is recommended by the CDC as the standard of testing for Lyme disease.

尽管如此，血清学检测的好处超过了它的局限性，这就是为什么CDC推荐它作为莱姆病检测的标准。

Apart from PCR and ELISA serology tests, IFA and culture methods have also been suggested for diagnosing tick-borne infections. Testing using IFA is limited due to a lack of standardized antigenic targets, the subjective establishment of positive thresholds, and cross reactivity. These factors can result in varying accuracy of IFA results across laboratories.

除了PCR和ELISA血清学检测外，IFA和培养方法也被建议用于诊断蜱传感染。由于缺乏标准化的抗原靶标，主观建立阳性阈值和交叉反应性，使用IFA的测试受到限制。这些因素可能导致实验室间IFA结果的准确性不同。

. Furthermore, bacterial or viral cultures are not recommended for the diagnosis of tick-borne infections. This is due to the time-consuming nature of the test, the need for special media, and procedures that are only performed at specific laboratories

此外，不建议将细菌或病毒培养物用于蜱传感染的诊断。这是由于测试的耗时性，对特殊介质的需求以及仅在特定实验室进行的程序

This study employed a serology-based microarray developed at Vibrant to multiplex Lyme disease and other tick-borne infections along with a few other infections of interest. The uniqueness of the microarray lies in the application of the immunodominant antigens that eliminate nonspecific binding with high sensitivity needed for accurate diagnosis.

这项研究采用了Vibrant开发的基于血清学的微阵列，以多重莱姆病和其他蜱传感染以及其他一些感兴趣的感染。微阵列的独特性在于应用免疫显性抗原，消除非特异性结合，具有准确诊断所需的高灵敏度。

Antigens could be evaluated in a multiplex setting to gauge their performance with clinical samples to pick the ideal set of antigens for any infection. To the best of our knowledge, this is the first report on a broad panel of antigens for Lyme disease, co-infections, and other related infections in such a flexible format.

抗原可以在多重环境中进行评估，以评估其在临床样本中的表现，从而为任何感染选择理想的抗原集。据我们所知，这是第一份关于莱姆病，合并感染和其他相关感染的广泛抗原的报告。

The structure of the Vibrant pillar plate is designed to encompass 400 probe chips at each pillar facilitating the detection of an array of co-infections in a single run, saving cost, labor, and time. Further compaction of the chip allows improved performance by enhancing multiplexing and widening its clinical applications.

振动柱板的结构设计为在每个柱上包含400个探针芯片，便于在一次运行中检测一系列共感染，节省了成本，人力和时间。芯片的进一步压缩可以通过增强多路复用和扩大其临床应用来提高性能。

The microarray platform is advantageous over other existing gold standards for tick-borne diseases and was able to overcome several of their limitations. Average time for multitier testing for several tickborne pathogens could take several months whereas the microarray technology takes only about a day to perform.

微阵列平台优于蜱传疾病的其他现有黄金标准，并且能够克服其一些局限性。对几种蜱传病原体进行多层检测的平均时间可能需要几个月，而微阵列技术只需要大约一天的时间。

. The microarray detected 17 tick-borne and other infections along with Lyme disease with sensitivities and specificities listed in Table

微阵列检测到17种蜱传和其他感染以及莱姆病，其敏感性和特异性列于表中

In conclusion, the protein microarray with a multiplex of antigens was validated for Lyme disease, co-infections, and other related infections. Simultaneous testing for Lyme disease, co-infections, and other related infections makes the diagnosis and treatment easier and quicker. This approach caters to the diagnostic needs of patients owing to its high sensitivity and specificity, affordable cost, quick availability of results, and low sample volume requirement.

总之，具有多种抗原的蛋白质微阵列已被验证用于莱姆病，合并感染和其他相关感染。同时检测莱姆病、合并感染和其他相关感染可以使诊断和治疗变得更容易、更快。由于其高灵敏度和特异性，可承受的成本，结果的快速可用性以及低样本量要求，这种方法满足了患者的诊断需求。

Measures for syndromic surveillance, diagnostic preparedness in disease outbreak investigations, personal protection, and education of clinical health professionals and patients could play a role in controlling tick-borne infections. As the known repertoire of antigens increases, this flexible microarray format can be customised to include the new antigens.

症状监测、疾病爆发调查中的诊断准备、个人保护以及临床卫生专业人员和患者的教育措施可能在控制蜱传感染方面发挥作用。随着已知抗原库的增加，可以定制这种灵活的微阵列格式以包括新抗原。

Future editions could also include other infections/agents namely Colorado tick fever, heartland virus, .

未来的版本还可能包括其他感染/病原体，即科罗拉多蜱热病，心脏病病毒。

R. parkeri

R.帕克里

, tick-borne relapsing fever, and tularemia which can be tested in parallel. Novel antigens for pathogens which may include whole cell lysates, recombinant proteins or peptide epitopes can be added as the science progresses leading to continuous improvement in diagnostic technology for detecting tick-borne infections..

，蜱传复发热和土拉菌病，可以并行测试。随着科学的进步，可以添加新的病原体抗原，包括全细胞裂解物，重组蛋白或肽表位，从而不断改进用于检测蜱传感染的诊断技术。。

Data availability

数据可用性

The data used to support the findings of this study can be acquired from Karthik Krishna (karthik@vibrantsci.com).

用于支持这项研究结果的数据可以从Karthik Krishna那里获得(karthik@vibrantsci.com)。

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Acknowledgements

致谢

Vibrant Sciences LLC developed the microarray technology showcased in the publication. All IP associated with the microarray manufacture and diagnostics belongs to Vibrant Sciences. The specific roles of authors are stated in the author contribution section.

Vibrant Sciences LLC开发了出版物中展示的微阵列技术。与微阵列制造和诊断相关的所有IP都属于充满活力的科学。作者的具体角色在作者贡献部分中进行了说明。

Funding

资金

Vibrant America provided funding for this study in the form of salaries for authors [CS, SM, KK, VJ, TW, KB, HKK, JJR]. The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript.

Vibrant America以作者工资的形式为这项研究提供了资金[CS，SM，KK，VJ，TW，KB，HKK，JJR]。资助者在研究设计，数据收集，分析，决定发表或准备稿件方面没有任何作用。

Author information

作者信息

Authors and Affiliations

作者和隶属关系

Vibrant Sciences LLC., San Carlos, CA, USA

Vibrant Sciences LLC.，美国加利福尼亚州圣卡洛斯

Hari Krishnan Krishnamurthy, Vasanth Jayaraman, Karthik Krishna, Tianhao Wang, Kang Bei & John J. Rajasekaran

Krishnan Krishnamurthy Day，[UNK]VasanthJayaraman，[UNK]Karthik Krishna，[UNK.]TianhaoWang，[UNK-]KangBei[UNK]&[UNK][UNK]John J.Rajasekaran

Vibrant America LLC., San Carlos, CA, USA

Vibrant America LLC.，美国加利福尼亚州圣卡洛斯

Chithra Changalath & Shiny Matilda

Chithra Changalath和Shiny Matilda

Irving Medical Center, Department of Pathology & Cell Biology, Columbia University, New York, USA

美国纽约哥伦比亚大学病理与细胞生物学系欧文医学中心

Alex J. Rai

亚历克斯·J·雷

Department of Parasitology, Faculty of Biology, Diagnostic Laboratory of Parasitic Diseases and Zoonotic Infections, Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland

波兰华沙华沙大学生物与化学研究中心寄生虫病与人畜共患感染诊断实验室生物学系

Renata Welc-Falęciak

Renata Welc-Falęciak 的

Department of Immunopathology of Infectious and Parasitic Diseases, Medical University of Warsaw, 3C Pawińskiego Street, 02-106, Warsaw, Poland

华沙医科大学传染病和寄生虫病免疫病理学系，波兰华沙Pawińskiego街3C号，02-106

Agnieszka Pawełczyk

Agnieszka Pawełczyk 的

Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, 77555, USA

德克萨斯大学医学院内科传染病科，加尔维斯顿，德克萨斯州，77555，美国

Lucas S. Blanton

卢卡斯·S·布兰顿

Department of Infectious Diseases, Hospital Ceske Budejovice, Ceske Budejovice, Czech Republic

捷克共和国塞斯克·布德约维奇塞斯克·布德约维奇医院传染病科

Aleš Chrdle

AlešChrdle

Veterinary Research Institute, Brno, Czech Republic

兽医研究所,布尔诺,捷克共和国

Andrea Fořtová

安德烈福尔特

Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, Ceske Budejovice, Czech Republic

捷克共和国Ceske Budejovice捷克科学院生物学中心寄生虫学研究所

Daniel Růžek

丹尼尔罗泽克

Biomedical Sciences Department, College of Health Sciences, Qatar University, P.O. Box 2713, Doha, Qatar

卡塔尔大学健康科学学院生物医学系，卡塔尔多哈邮政信箱2713

Gheyath K. Nasrallah, Laith J. Abu-Raddadi, Duaa W. Al-Sadeq & Marah Abed Alhakim Abdallah

Gheyath K.Nasrallah，Laith J.Abu Raddadi，Duaa W.Al Sadeq和Marah Abed Alhakim Abdallah

Microbiologia E Virologia, Fondazione IRCCS Policlinico San Matteo, 27100, Pavia, Italy

微生物学和病毒学，IRCCS基金会Policlinico San Matteo，27100，帕维亚，意大利

Daniele Lilleri, Chiara Fornara, Piera D’Angelo & Milena Furione

Daniele Lilleri，Chiara Fornara，Piera D'Angelo和Milena Furione

University of Helsinki, Haartmaninkatu 3, 00290, Helsinki, Finland

赫尔辛基大学，Haartmaninkatu 3，00290，赫尔辛基，芬兰

Maria Söderlund-Venermo & Klaus Hedman

Maria Söderlund Venermo&Klaus Hedman

Laboratory of Clinical Microbiology and Microbial Pathogenesis, School of Medicine, University of Crete, 70013, Heraklion, Crete, Greece

克里特大学医学院临床微生物学和微生物发病机制实验室，70013，希腊克里特岛赫拉克利翁

Dimosthenis Chochlakis, Eirini Makridaki, Artemis Ntoula & Anna Psaroulaki

Dimosthenis Chochlakis、Eirini Makridaki、Artemis Ntoula和Anna Psaroulaki

Laboratory of Biotechnology at, Institute of Biomedical Science, University of Ciudad Juárez, Ciudad Juárez, Mexico

墨西哥华雷斯城华雷斯城大学生物医学研究所生物技术实验室

Angélica Escárcega-Ávila

安吉丽卡·斯卡尔切加-阿维拉

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Hari Krishnan Krishnamurthy

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Vasanth Jayaraman

瓦桑特贾亚拉曼

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Kang Bei

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Renata Welc-Falęciak

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Lucas S. Blanton

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Aleš Chrdle

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Marah Abed Alhakim Abdallah

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Contributions

捐款

Conception and study design: HKK, JJR, VJ. Performing experiments: KK, TW. Analysis and interpretation: KB, KK. Writing-original draft: HKK, CC, SM. Review and editing: HKK, CC, AJR, RWF, AP, LSB, AC, AF, DR, GKN, LJA, DWA, MAA, DL, CF, PD, MF, MSV, KH, DC, AP, EM, and AN. Sample resources: RWF, AP, LSB, AC, AF, DR, GKN, LJA, DWA, MAA, DL, CF, PD, MF, MSV, KH, DC, EM, AN, AP, and AE.

概念和研究设计：HKK，JJR，VJ。执行实验：KK，TW。分析和解释：KB，KK。撰写原稿：HKK，CC，SM。审阅和编辑：HKK，CC，AJR，RWF，AP，LSB，AC，AF，DR，GKN，LJA，DWA，MAA，DL，CF，PD，MF，MSV，KH，DC，AP，EM和AN。示例资源：RWF，AP，LSB，AC，AF，DR，GKN，LJA，DWA，DL，CF，PD，MF，MSV，KH，DC，EM，AN，AP和AE。

All authors reviewed and approved the final manuscript..

所有作者都审查并批准了最终稿件。。

Corresponding author

通讯作者

Correspondence to

通信对象

Hari Krishnan Krishnamurthy

克里希那穆提

Ethics declarations

道德宣言

Competing interests

相互竞争的利益

The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: CC and SM are paid employees of Vibrant America LLC. KK, VJ, TW, KB, HKK, and JJR are paid employees of Vibrant Sciences LLC. RWF, AP, LSB, AC, AF, DR, GKN, LJA, DWA, MAA, DL, CF, PD, MF, MSV, KH, DC, EM, AN, AP, and AE are academic collaborators who provided samples and assisted in the review and editing of the manuscript.

作者阅读了该期刊的政策，本手稿的作者有以下相互竞争的利益：CC和SM是Vibrant America LLC的付费员工。KK，VJ，TW，KB，HKK和JJR是Vibrant Sciences LLC的付费员工。RWF，AP，LSB，AC，AF，DR，GKN，LJA，DWA，MAA，DL，CF，PD，MF，MSV，KH，DC，EM，AN，AP和AE是提供样本并协助稿件审查和编辑的学术合作者。

AJR is a paid consultant of Vibrant America LLC. Vibrant America offers commercial testing for Lyme disease and other infectious diseases and could benefit from increased testing..

AJR是Vibrant America LLC的付费顾问。Vibrant America提供莱姆病和其他传染病的商业检测，并可能从增加检测中受益。。

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Springer Nature在已发布的地图和机构隶属关系中的管辖权主张方面保持中立。

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以下是电子补充材料的链接。

Supplementary Material 1

补充材料1

Supplementary Material 2

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Supplementary Material 3

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Supplementary Material 4

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Supplementary Material 5

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Supplementary Material 6

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Supplementary Material 7

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Krishnamurthy, H.K., Jayaraman, V., Krishna, K.

克里希那穆西，H.K.，贾亚拉曼，V.，克里希纳，K。

et al.