METAGENOMIC NEXT-GENERATION SEQUENCING OF MICROBIAL CELL-FREE NUCLEIC ACIDS IN SUBJECTS WITH LYME DISEASE
What is disclosed herein is a method of detecting and treating Lyme disease, particularly Lyme arthritis. The method includes metagenomic next-generation sequencing (mNGS) of plasma microbial cell-free DNA (mcfDNA) to detect and quantify Borrelia spp. in the circulation. Also disclosed is a method of treating Lyme arthritis by treating the patient with an anti-microbial treatment until Borrelia mcfDNA is lowered to below detectable levels, such as less than 1 molecule per microliter of plasma.
1 . A method of treating a Borrelia spp. infection in a subject that has received a diagnosis of the Borrelia spp. infection, wherein the diagnosis of the Borrelia spp. infection was based at least in part on [[the]]a method comprising:
a. collecting one or more blood samples from the subject at a time when the subject does not have an erythema migrans (EM) rash and wherein the one or more blood samples comprise microbial cell-free nucleic acids (mcfNA);
b. detecting mcfNA from Borrelia spp. in the one or more blood samples; and
c. diagnosing the subject with the Borrelia spp. infection based at least in part on the detecting the mcfNA from the Borrelia spp.
2 . The method of claim 1 , further comprising quantifying the mcfNA from Borrelia spp.
3 . (canceled)
4 . The method of claim 1 , wherein the one or more blood samples are one or more plasma samples.
5 . The method of claim 1 , further comprising attaching nucleic acid adapters to cell-free nucleic acids in the one or more blood samples to prepare a sequencing library comprising the mcfNA.
6 . The method of claim 5 , further comprising generating sequence reads from the sequencing library comprising the mcfNA, aligning the sequence reads to Borrelia spp. genomic sequences in a reference data set to obtain aligned sequence reads, and identifying the Borrelia spp. based on the aligned sequence reads.
7 . The method of claim 1 , the method further comprising wherein the treating comprises administering a therapeutic treatment to the subject to treat a Borrelia spp. infection.
8 . The method of claim 7 , wherein the therapeutic treatment is a Borrelia -directed therapy.
9 . The method of claim 8 , wherein the Borrelia -directed therapy is at least one therapy selected from the group consisting of: doxycycline, amoxicillin, cefuroxime axetil, ceftriaxone, and cefotaxime.
10 . The method of claim 1 , further comprising spiking the one or more blood samples with a known concentration of synthetic DNA.
11 . The method of claim 4 , further comprising spiking the one or more plasma samples with a known concentration of synthetic DNA.
12 . (canceled)
13 . (canceled)
14 . (canceled)
15 . The method of claim 1 , wherein the subject has arthritis of a joint.
16 . The method of claim 15 , wherein the joint comprises at least one joint selected from the group consisting of knee, elbow, temporomandibular joint, and hip.
17 . The method of claim 1 , wherein the subject is blood culture negative for Borrelia at the time of the collecting of the one or more blood samples.
18 . The method of claim 1 , wherein the subject is negative for Borrelia when measured by a polymerase chain reaction (PCR) test of a blood sample or synovial fluid sample of blood from the subject.
19 . (canceled)
20 . The method of claim 1 , wherein the subject was bitten by a tick carrying Borrelia bacteria at least 6 months prior to the collecting of the one or more blood samples.
21 . (canceled)
22 . (canceled)
23 . The method of claim 1 , wherein the subject is serologically positive for Borrelia antibodies.
24 . (canceled)
25 . The method of claim 1 , wherein the concentration of Borrelia mcfDNA is 1-1,000 molecules per microliter (MPM) of plasma.
26 . The method of claim 1 , wherein the subject has disseminated late-stage Lyme disease.
27 . The method of claim 1 , wherein a sensitivity of detecting the Borrelia mcfDNA is at least 60%.
28 . The method of claim 1 , wherein the Borrelia mcfNA comprises mcfNA derived from B. burgdorferi or B. maynoii bacteria.
29 - 36 . (canceled)