SEQUENCING MICROBIAL CELL-FREE DNA FROM ASYMPTOMATIC INDIVIDUALS
Disclosed herein are methods of detecting and treating subjects for bloodstream infections (BSI). Disclosed herein are methods of predicting a bloodstream infection prior to an onset of a symptom. Disclosed herein are method of detecting bloodstream infections using high-throughput sequencing of microbial cell free nucleic acids.
1 . A method of treating a subject at risk for a bloodstream infection comprising:
a. collecting one or more blood samples from the subject, wherein the one or more blood samples comprise microbial cell-free nucleic acids (mcfNA) and the subject is afebrile or blood-culture negative;
b. detecting an amount of a pathogen associated with the bloodstream infection based on the microbial cell-free nucleic acids (mcfNA) in the one or more blood samples;
c. predicting that the subject at risk for the bloodstream infection will experience a sign or symptom of a bloodstream infection based on the amount of the pathogen associated with the bloodstream infection, wherein the predicting has a predictive sensitivity of at least 75% for samples collected at least three days prior to onset of the sign or symptom of the bloodstream infection; and
d. administering a therapeutic treatment to the subject prior to onset of the symptom of the bloodstream infection.
2 . The method of claim 1 , wherein the pathogen associated with the bloodstream infection is a bacterium, and the bloodstream infection is a bacterial bloodstream infection.
3 - 4 . (canceled)
5 . The method of claim 1 , wherein the pathogen associated with the bloodstream infection is a fungus and the bloodstream infection is a fungal infection.
6 . The method of claim 5 , wherein the fungus is a Candida spp.
7 . The method of claim 5 , wherein the fungus is a Candida krusei.
8 . The method of claim 1 , wherein the mcfNA are microbial cell-free DNA.
9 . The method of claim 1 , wherein the one or more blood samples are one or more plasma samples.
10 - 11 . (canceled)
12 . The method of claim 1 , wherein the predictive sensitivity of at least 75% is a predictive sensitivity of at least 85% for a bacterial bloodstream infection.
13 - 15 . (canceled)
16 . The method of claim 1 , wherein the therapeutic treatment is a pathogen-directed therapy.
17 . The method of claim 16 , wherein the pathogen-directed therapy is at least one therapy selected from the group consisting of: vancomycin, ampicillin, cefepime, penicillin, meropenem, ceftriaxone, levofloxacin, and linezolid.
18 . The method of claim 1 , wherein the subject has no infection within seven days prior to collecting the one or more blood samples.
19 - 23 . (canceled)
24 . The method of claim 1 , wherein the subject is blood culture negative during the collecting of the one or more blood samples.
25 - 30 . (canceled)
31 . A method of processing and analyzing a blood sample from a blood cancer patient who is asymptomatic for an infection by a bacterial or fungal microbe or blood-culture negative for the bacterial or fungal microbe comprising:
a. preparing at least one plasma sample comprising microbial cell-free nucleic acids (mcfNA) from the blood cancer patient asymptomatic for the infection by the bacterial or fungal microbe or blood-culture negative for the bacterial or fungal microbe;
b. preparing a first library comprising the mcfNA attached to adapters;
c. subjecting the first library comprising the mcfNA attached to the adapters to next generation sequencing to produce sequence reads;
d. aligning the sequence reads to bacterial or fungal DNA sequences in a reference data set to obtain aligned sequence reads; and
e. detecting a presence of and quantifying the bacterial or fungal organism at a species or strain level based on the aligned sequence reads.
32 . The method of claim 0 , wherein the microbial cell-free nucleic acids comprise microbial cell-free DNA.
33 - 38 . (canceled)
39 . The method of claim 31 , further comprising spiking the at least one plasma sample with a known concentration of synthetic DNA molecules.
40 . The method of claim 31 , wherein the blood cancer is a leukemia.
41 . (canceled)
42 . The method of claim 31 , wherein the patient is receiving chemotherapy.
43 . The method of claim 31 , wherein the patient is the recipient of a hematopoietic stem cell transplant.
44 - 48 . (canceled)
49 . The method of claim 31 , wherein the infection by a bacterial or fungal microbe is a bacterial bloodstream infection.
50 . (canceled)
51 . The method of claim 31 , wherein the infection by a bacterial or fungal microbe is an invasive fungal infection.
52 . (canceled)
53 . A method of processing and analyzing a blood sample from a cancer patient at risk of a bacterial or fungal infection comprising:
a. preparing plasma samples comprising microbial cell-free DNA (mcfDNA) from at least two longitudinal blood samples collected from the cancer patient within seven days prior to the onset of a bloodstream infection;
b. isolating the mcfDNA from the plasma samples;
c. attaching adapters to the mcfDNA to produce a DNA library comprising mcfDNA attached to the adapters;
d. obtaining sequence reads from the DNA library;
e. aligning the sequence reads to bacterial or fungal DNA sequences in a reference data set to obtain aligned sequence reads;
f. identifying the infection by a bacterial or fungal organism based on the aligned sequence reads; and
g. quantifying the mcfDNA at the species or strain level prior to onset of the infection by a bacterial or fungal organism.
54 - 70 . (canceled)
71 . The method of claim 1 , wherein the onset of infection is the onset of an infection that is blood-culture positive.