WO2018045359A1

WO2018045359A1 - Detection and treatment of infection during pregnancy

Info

Publication number: WO2018045359A1
Application number: PCT/US2017/049980
Authority: WO
Inventors: Sivan BERCOVICI; Lily BLAIR; Timothy A. Blauwkamp; Jason BLUE-SMITH; Daid K. HONG; Michael Kertesz; Simona ZOMPI
Original assignee: Karius, Inc.
Priority date: 2016-09-02
Filing date: 2017-09-01
Publication date: 2018-03-08

Abstract

This disclosure provides methods for detecting and/or treating a pathogenic infection in a pregnant subject, a subject contemplating pregnancy or a neonate.

Description

DETECTION AND TREATMENT OF INFECTION DURING PREGNANCY

CROSS-REFERENCE

[0001] The application claims the benefit of U.S. Provisional Patent Application No. 62/383,353 (Attorney Docket No. 47697-708.101) filed on September 2, 2016 and U.S. Provisional Patent Application No. 62/483,921 (Attorney Docket No. 47697-708.102) filed on April 10, 2017; the entire contents of which are incorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Chorioamnionitis is inflammation within the uterus caused by infection of the fetal membranes and placenta during pregnancy and can have severe consequences such as preterm birth, neonatal infection and neonatal death. These infections may be due to a variety of pathogens and can be clinically silent. In some instances, analysis of amniotic fluid using amniocentesis may detect chorioamnionitis; however, amniocentesis is an invasive procedure with its own risks of infection and preterm birth. Even when these tests are available, in up to 30% of the cases of preterm births with inflammation on histology, a pathogen cannot be identified.

[0003] Chorioamnionitis is particularly frequent in cases of preterm, premature rupture of the membranes (PPROM) and in pregnant women at high risk of preterm birth. Antibiotic treatment can increase the latency time in PPROM, e.g., time from rupture of the membranes to delivery, increasing the gestational age at birth for the neonate and thus decreasing risk of neonatal complications as well as complications later in life associated with preterm birth. There is thus an urgent need in the art for non-invasive approaches to detecting

chorioamnionitis at early stages of the disease to target antimicrobial treatment and prevent severe neonatal consequences.

[0004] Additional pregnancy-related conditions may also be currently difficult to detect in a non-invasive manner. For example, analysis of maternal blood by polymerase chain reaction (PCR) may often provide a negative result during congenital infections. Currently, diagnosis of congenital infections relies on serological tests in the mother, which cannot confirm whether an infection is current or past, and on invasive tests such as PCR performed on amniotic fluid samples obtained by amniocentesis. There is thus a need for accurate, noninvasive approaches for detecting congenital infections caused by pathogens.

[0005] Lastly, newborns are susceptible to infections arising in utero or during delivery. Most neonatal infections are caused by bacteria, which normally live in the birth canal and can expose the baby to infection during birth. For example, the baby may swallow or breathe in the fluid in the birth canal and then the bacteria may get into the baby's lungs and bloodstream causing early onset neonatal sepsis (EONS). Current diagnostic methods may require testing of blood by blood culture, of which very little can be obtained from a neonate, or other invasive procedure such as spinal tap, and the results may be inconclusive if mother has been treated for the infecting pathogen during or before delivery. If the baby is not treated until later, the baby may get very sick and need intensive care to recover. Delayed treatment can cause high mortality; thus, EONS is often treated preemptively without microbiological confirmation. There is an additional need for accurate, non-invasive approaches for testing neonates for infections present at delivery to avoid unnecessary antibiotic treatment which may prolong the hospital stay and can have its own side effects, and/or to make sure that infected neonates are treated with the correct antibiotics.

SUMMARY OF THE INVENTION

[0006] Provided herein are methods of detecting pregnancy-associated infections, thereby reducing adverse effects that may negatively impact a developing fetus or neonate. The methods may include sequencing and analyzing cell-free nucleic acids in biological fluid (e.g., blood, plasma) from a pregnant subject (e.g., pregnant woman) or subject

contemplating pregnancy (e.g., woman contemplating pregnancy) or from a neonate (e.g., cord blood, peripheral blood, plasma). In some cases, the method may be used to detect pathogens that may have infected fetal membranes, the placenta, the pregnant subject or subject contemplating pregnancy, or the fetus or neonate itself. The pregnant subject may be clinically suspected of having chorioamnionitis, being at risk of preterm birth, being at risk of or experiencing preterm premature rupture of fetal membranes (PPROM), or may have another condition.

[0007] Using the methods provided herein, the causal pathogen may be detected and a subject may be treated accordingly to increase the time to delivery and reduce the likelihood of preterm birth, to monitor the pregnancy accordingly, and/or to treat a neonatal infection. Similarly, by performing cell-free nucleic acid sequencing of plasma or other biological fluids from a pregnant woman with a fetus suspected of a congenital infection, the causal pathogen may be detected. In such cases, the pregnant subject may be clinically suspected of having an acute infection which may infect the fetus or neonate. In some cases, the pregnant subject may be carrying a fetus with fetal anomalies detected by ultrasound. In some cases, such anomalies may be evocative of congenital infection, such as, but not limited to, microcephaly, intra-cerebral calcifications, multiple organ anomalies, fetal growth restrictions, and/or placental enlargement. In some cases, the methods provided herein make it possible to determine whether an infection is an active infection affecting the fetus, as opposed to a latent or past infection detected in the pregnant subject. Using the methods provided herein, the causal pathogen may be detected and a subject may be treated and monitored accordingly and/or earlier termination of pregnancy may be discussed.

[0008] In an aspect, the present disclosure provides a method of detecting pathogen nucleic acids in a pregnant subject comprises (a) obtaining a sample comprising cell-free nucleic acids from the pregnant subject, wherein the pregnant subject is at risk of having an infection; and (b) conducting a sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids from a collection of pathogen nucleic acids, wherein the pathogen nucleic acids are specifically selected for the collection primarily based on a known association between the pathogen nucleic acids and chorioamnionitis.

[0009] In another aspect, the present disclosure provides a method of detecting pathogen nucleic acids in a pregnant subject comprises (a) obtaining a sample comprising cell-free nucleic acids from the pregnant subject, wherein the pregnant subject is at risk of having an infection; and (b) conducting a high-throughput sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids from a collection of pathogen nucleic acids, wherein the pathogen nucleic acids are specifically selected for the collection primarily based on a known association between the pathogen nucleic acids and at least one infection selected from the group consisting of fetal membrane infection, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection, umbilical cord infection, and neonatal infection.

[0010] In some embodiments, the method comprises quantifying each detected pathogen nucleic acid in order to obtain initial values for the detected pathogen nucleic acids and comparing the initial values for the detected pathogen nucleic acids with at least one reference value in order to obtain relative values for the detected pathogen nucleic acids. In some embodiments, the method comprises identifying detected pathogen nucleic acids with relative values above a threshold value, thereby obtaining a selected set of one or more pathogen nucleic acids.

[0011] In some embodiments, the method comprises designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids exceeds a threshold value of different pathogen nucleic acids. In some embodiments, the threshold value is any value within the range of 2 to 20. In some embodiments, the threshold value is 2. In some embodiments, the method comprises designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids comprises 20 different pathogen nucleic acids, or pathogen nucleic acids from 20 different pathogens. In some embodiments, the method comprises designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids are pathogen nucleic acids associated with number of different pathogens wherein the number is between 2 and 20. In some embodiments, the different pathogen nucleic acids are from different pathogen taxa, different pathogen genera, different pathogen species, different pathogen strains, or different pathogen variants. In some embodiments, the at least one reference value is based on levels of the pathogen nucleic acids detected in one or more samples selected from the group consisting of water sample, blood sample, plasma sample, serum sample, urine sample, body fluid sample, reagent sample, sample from a healthy subject, sample from a healthy pregnant subject, and any combination thereof.

[0012] In some embodiments, the method comprises electronically transmitting data reflecting the selected set of one or more pathogen nucleic acids to a recipient. In some embodiments, the electronically-transmitted data is used by the recipient to determine a treatment plan for the pregnant subject. In some embodiments, the electronically-transmitted data is used by the recipient to detect, monitor or diagnose a condition of the pregnant subject. In some embodiments, the obtained sample is associated with a patient identifier and the method further comprises separating the patient identifier from the obtained sample to obtain de-identified samples; obtaining de-identified sample sequence data from the high- throughput sequencing assay; uploading the de-identified sample sequence data to a server; detecting the pathogen nucleic acids within the de-identified sample sequence data in order to obtain de-identified result data, such that the de-identified result data is on the server; downloading the de-identified result data from the server. In some embodiments, the method comprises associating the patient identifier with the de-identified result data.

[0013] In some embodiments, the method comprises administering a therapeutic regimen to the pregnant subject based on the selected set of one or more pathogen nucleic acids. In some embodiments, the administering a therapeutic regimen comprises treating the pregnant subject with a specific drug to reduce or eliminate the source of the detected pathogen nucleic acids. In some embodiments, the drug is selected from the group consisting of antibiotic, antiviral, ampicillin, sulbactam, penicillin, vancomycin, gentamycin, aminoglycoside, clindamycin, cephalosporin, metronidazole, timentin, ticarcillin, clavulanic acid, cefoxitin, antiretroviral, immunoglobulins, and any combination thereof. In some embodiments, the therapeutic regimen comprises administering a therapy to the pregnant subject and then repeating steps a and b to monitor effects of the therapy on the selected set of one or more pathogen nucleic acids. In some embodiments, the method comprises terminating the pregnancy of the pregnant subject based on the selected set of one or more pathogen nucleic acids. In some embodiments, the method comprises determining a risk of congenital defects in a developing fetus of the pregnant subject based on the detection results.

[0014] In some embodiments, the method comprises using the high-throughput sequencing assay to determine relative amounts of organ cell-free RNA of the fetus or pregnant subject compared to a control value. In some embodiments, the one or more pathogen nucleic acids and increased amounts of organ cell-free RNA indicate the presence of an infection in the organ. In some embodiments, the one or more pathogen nucleic acids are Zika nucleic acids and the organ is fetal brain. In some embodiments, the one or more pathogen nucleic acids are pathogen nucleic acids associated with chorioamnionitis and the organ is uterus. In some embodiments, the high-throughput sequencing assay detects cell-free pathogen DNA or cell-free pathogen RNA of the pregnant subject in order to prognose a risk of preterm labor or preterm delivery. In some embodiments, the high-throughput sequencing assay detects cell-free pathogen DNA or cell-free pathogen RNA in order to prognose a risk of congenital defects in the fetus.

[0015] In some embodiments, the pregnant subject has one or more clinical symptoms of chorioamnionitis or funisitis. In some embodiments, the one or more clinical symptoms of chorioamnionitis or funisitis are selected from the group consisting of fever, rapid heartbeat or tachycardia of the pregnant woman, rapid fetal heartbeat or fetal tachycardia, uterine tenderness, vaginal discharge with an unusual or foul odor or discoloration, amniotic fluid with a foul smell, maternal leukocytosis, and any combination thereof.

[0016] In some embodiments, the pregnant subject has one or more risk factors for chorioamnionitis or funisitis. In some embodiments, the one or more risk factors for chorioamnionitis or funisitis are selected from the group consisting of longer duration of membrane rupture, prolonged labor, internal monitoring of labor, multiple vaginal examinations, meconium-stained amniotic fluid, smoking, alcohol abuse, drug abuse, compromised immune system, epidural anesthesia, colonization with group B streptococcus, sexually transmissible genital infections, vaginal colonization with ureaplasma, and any combination thereof.

[0017] In some embodiments, the pregnant subject is a pregnant woman with one or more risk factors for premature labor and delivery. In some embodiments, the one or more risk factors for premature labor and delivery are selected from the group consisting of having a premature rupture of the membranes; having a personal or family history of premature birth, miscarriage, or stillbirth; being underweight or overweight before pregnancy; not gaining enough weight during pregnancy; having certain health conditions such as diabetes, high blood pressure, preeclampsia, or blood clot disorders; becoming pregnant after in vitro fertilization (IVF); becoming pregnant up to 18 months after a previous birth; having a social risk associated with preterm birth; having a positive test result from an assay giving a prognosis of preterm birth, and any combination thereof. In some embodiments, the pregnant subject is a pregnant woman who previously had an amniocentesis test or chorionic villus sampling test. In some embodiments, the amniocentesis test or chorionic villus sampling test indicated inflammation or infection but did not identify a specific pathogen.

[0018] In some embodiments, the one or more pathogen nucleic acids are derived from one or more pathogens present in the sample. In some embodiments, the one or more pathogens comprise one or more bacteria or one or more viruses. In some embodiments, the one or more pathogens comprise Escherichia coli, group B streptococcus (Streptococcus agalactiae), anaerobic bacterium, Staphylococcus aureus, cytomegalovirus (CMV), Mycoplasma spp., Mycoplasma hominis, Ureaplasma spp., Ureaplasma urealyticum, Ureaplasma parvum, human immunodeficiency virus (HIV), lentivirus, herpes simplex, human herpes viruses, varicella, B19 erythrovirus, Toxoplasma gondii, Treponema pallidum, Listeria monocytogenes, Plasmodium falciparum, rubella, Chlamydia trachomatis, hepatitis B virus, hepatitis E virus, Parvovirus, Enterovirus, hepatitis C virus, syphilis, gonorrhea, Fusobacterium nucleatum, Enterococcus faecalis, Corynebacterium aurimucosum, Corynebacterium urealyticum, Gardnerella vaginalis, Bacteroides fragilis, Haemophilus influenzae, Methylobacterium mesophilicum, Prevotella bivia, Rothia mucilaginosa, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopenumoniae, Streptococcus pasteurianus, Sneathia sanguinegens, Candida albicans, Neisseria gonorrhoeae, Citrobacter koseri, Peptoniphilus harei, Klebsiella pneumoniae, Micrococcus luteus, and Flaviviruses. In some embodiments, the one or more pathogen nucleic acids comprise cell-free pathogen DNA, cell-free pathogen RNA, or a mixture of cell-free pathogen DNA and cell-free pathogen RNA.

[0019] In some embodiments, the sample is selected from the group consisting of blood, cord blood, peripheral blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage, urine, stool, saliva, nasal swab, cord blood, amniotic fluid, cell-free plasma, and any combination thereof. In some embodiments, the sample has been processed to remove at least one component from the group consisting of cells, human cells, bacterial cells, viral particles, and exosomes. In some embodiments, the sample is a plasma sample that has been further processed to remove at least one component from the group consisting of cells, human cells, bacterial cells, viral particles, and exosomes.

[0020] In some embodiments, the method reduces the risk of preterm labor by at least 50%. In some embodiments, the detecting the pathogen nucleic acids comprises monitoring the pathogen nucleic acids over time. In some embodiments, the pathogen detected is associated with an intrauterine infection. In some embodiments, the pathogen detected is associated with a congenital fetal infection. In some embodiments, the pathogen detected is associated with a neonatal infection. In some embodiments, the intrauterine infection is acute chorioamnionitis or funisitis. In some embodiments, the intrauterine infection is chronic chorioamnionitis or funisitis. In some embodiments, the intrauterine infection is sub-clinical chorioamnionitis or funisitis. In some embodiments, the detection of one or more pathogen nucleic acids from the collection of pathogen nucleic acids is used to detect, diagnose or prognose an increased risk for infection of intrauterine tissue, chorion, amnion, umbilical cord or placenta.

[0021] In some embodiments, the method comprises diagnosing the pregnant subject with at least one infection at least in part based on the detected pathogen nucleic acids, wherein the at least one infection is selected from the group consisting of fetal membrane infection, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection, umbilical cord infection, and neonatal infection. In some embodiments, the method comprises diagnosing the pregnant subject or subject contemplating pregnancy with at least one infection at least in part based on the detected pathogen nucleic acids, wherein the at least one infection is selected from the group consisting of chorioamnionitis, funisitis, chronic chorioamnionitis, chronic funisitis, acute chorioamnionitis, acute funisitis, neonatal sepsis, and placental infection. In some embodiments, the method comprises diagnosing the pregnant subject or subject contemplating pregnancy with at least one infection at least in part based on the detected pathogen nucleic acids, wherein the at least one infection is selected from the group consisting of uterine, vaginal and reproductive system infection or colonization. In some embodiments, the method comprises detecting in a subject contemplating pregnancy pathogens a presence of one or more pathogens associated with chorioamnionitis or other pregnancy-related infection. In some embodiments, the method comprises detecting, diagnosing, monitoring, or prognosing the pregnant subject with a bacterial infection or viral infection.

[0022] In some embodiments, the sample is not cord blood, amniotic fluid or chorionic villus. In some embodiments, the sample is not cord blood, amniotic fluid or chorionic villus and the sample is used to detect, diagnose or prognose chorioamnionitis, fetal infection, or neonatal infection. In some embodiments, the method comprises conducting an RNA sequencing assay on a sample comprising cell-free RNA from the pregnant subject in order to distinguish between extra-uterine and intra-uterine infection. In some embodiments, the method comprises detecting an infection in the cord blood in utero or after delivery of a neonate. In some embodiments, the method comprises detecting an infection from the cell- free fraction of a blood sample from the human pregnant subject. In some embodiments, one or more of the steps in the method are implemented using a computer.

[0023] In another aspect, a method of predicting preterm labor in a pregnant subject comprises (a) obtaining a sample comprising cell-free nucleic acids from a pregnant subject or subject contemplating pregnancy suspected of having an infection; and (b) conducting a high-throughput sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids associated with infection of the fetal membranes, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection or neonatal infection.

[0024] In some embodiments, the one or more pathogens nucleic acids derived from one or more pathogens comprises Escherichia coli, group B streptococcus (Streptococcus agalactiae), anaerobic bacterium, Staphylococcus aureus, cytomegalovirus (CMV),

Mycoplasma spp., Mycoplasma hominis, Ureaplasma spp., Ureaplasma urealyticum,

Ureaplasma parvum, human immunodeficiency virus (HIV), lentivirus, herpes simplex, varicella, B 19 erythrovirus, Toxoplasma gondii, Treponema pallidum, Listeria monocytogenes, Plasmodium falciparum, rubella, Chlamydia trachomatis, hepatitis B virus, hepatitis E virus, Parvovirus, Enterovirus, hepatitis C virus, syphilis, gonorrhea,

Fusobacterium nucleatum, Enterococcus faecalis, Coryne bacterium aurimucosum,

Corynebacterium urealyticum, Gardnerella vaginalis, Bacteroides fragilis, Haemophilus influenzae, Methylobacterium mesophilicum, Prevotella bivia, Rothia mucilaginosa,

Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopenumoniae,

Streptococcus pasteurianus, Sneathia sanguinegens, Candida albicans, Neisseria gonorrhoeae, Citrobacter koseri, Peptoniphilus harei, Klebsiella pneumoniae, Micrococcus luteus, and Flaviviruses. In some embodiments, the detecting the one or more pathogen nucleic acids comprise monitoring the one or more pathogen nucleic acids over time. [0025] In another aspect, the method comprises a method of treating an infection in a pregnant subject comprises administering to the pregnant subject a therapeutic regimen to treat the infection wherein the pregnant subject has been determined to have an increased level of cell-free pathogen nucleic acids and wherein the cell-free pathogen nucleic acids are associated with an infection selected from the group consisting of fetal membrane infection, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection and neonatal infection. In some embodiments, the determination is conducted by high- throughput sequencing or massively parallel sequencing.

[0026] In another aspect, a method of detecting pathogen nucleic acids in a pregnant subject comprises (a) providing a sample comprising cell-free nucleic acids from a pregnant subject; and (b) conducting a high-throughput sequencing assay on the cell-free nucleic acids from the pregnant subject, thereby obtaining sequence reads from nucleic acids from pathogens present in a tissue or organ of the pregnant subject, wherein the tissue or organ is selected from the group consisting of fetal membrane, chorionic tissue, intra-amniotic tissue or fluid, placenta, uterus, fetal tissue, umbilical cord, fetoplacental circulatory system, and any combination thereof.

[0027] In some embodiments, the tissue or organ is chorionic or amniotic membrane tissue. In some embodiments, the tissue or organ is chorionic or amniotic membrane tissue and the sample is plasma, cord blood, amniotic fluid, serum, or urine. In some embodiments, the method comprises applying a filter to the sequence reads in order to evenly distribute coverage of the sequence reads across a pathogen genome. In some embodiments, the method comprises applying a filter to the sequence reads in order to reduce signal associated with microbes present in the vaginal canal or gut. In still other cases, sequence reads associated with skin pathogens are filtered out.

[0028] In some embodiments, the method further comprises quantifying the sequence reads from the nucleic acids from pathogens present in the tissue or organ of the pregnant subject to obtain initial pathogen values. In some embodiments, the method comprises comparing the initial pathogen values with at least one reference value in order to obtain relative pathogen values. In some embodiments, the method comprises identifying relative pathogen values above a threshold value, thereby obtaining a selected set of one or more pathogen nucleic acids. In some embodiments, the method comprises designating the sample from the pregnant subject as contaminated or inconclusive when the threshold value is any value within the range of 2 to 20. In some embodiments, the method comprises designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids are associated with a number of different pathogens, wherein the number is between 2 and 20. In some embodiments, the method prognoses pathogen infection of the fetus. In some embodiments, the specificity or sensitivity of the method is greater than 75%. In some embodiments, the specificity is greater than 70%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the sensitivity is greater than 70%, 80%, 85%), 90%), 95%), or 99%. In some embodiments, the specificity and specificity is greater than 80%), 85%), 90%), 95%), or 99%. In some embodiments, the cell-free pathogen nucleic acids are not particle-protected viral nucleic acids or are not pathogen nucleic acids associated with a viral particle.

[0029] In another aspect, provided herein are methods of detecting a pathogenic infection in a pregnant subject or subject contemplating pregnancy comprising: (a) obtaining a sample comprising cell-free nucleic acids from a pregnant subject or subject contemplating pregnancy suspected of having an infection; and (b) conducting a high-throughput sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids associated with infection of fetal membranes, placental infection, intra-amniotic infection, intrauterine infection, or congenital fetal infection. Further provided herein are methods of detecting a pathogenic infection in a neonate comprising: (a) obtaining a sample comprising cell-free nucleic acids from maternal cord blood from a post-partum subject and/or from neonatal peripheral blood; and (b) conducting a high-throughput sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids associated with neonatal infections.

[0030] In some cases, the high-throughput sequencing assay is a next generation sequencing assay. In some cases, the method includes quantifying an amount of the one or more pathogens nucleic acids. In some cases, the method includes medical intervention comprising administering a therapeutic regimen, such as treating the pregnant subject, subject contemplating pregnancy or neonate with a specific drug to reduce or eliminate the detected and quantified one or more pathogen nucleic acids based on the pathogen identified in step b. In some cases of a method described herein, the drug is selected from the group consisting of antibiotic, antiviral, ampicillin, sulbactam, penicillin, vancomycin, gentamycin, aminoglycoside, clindamycin, cephalosporin, metronidazole, timentin, ticarcillin, clavulanic acid, cefoxitin, antiretroviral, immunoglobulins, and any combination thereof. In some cases, step b is repeated over time. In some cases, the therapeutic regimen comprises administering a therapy to the pregnant subject or subject contemplating pregnancy and then repeating step b to monitor effects of the therapy on the amount of the one or more pathogen nucleic acids. In some cases, the medical intervention comprises terminating a pregnancy. In some cases, the method further comprises identifying congenitalgenetic defects in the fetus.

[0031] In some cases, the method further comprises performing an RNA sequencing assay on a cell-free RNA sample obtained from the pregnant subject or subject contemplating pregnancy. In some cases, the RNA sequencing assay determines relative amounts of organ cell-free mRNA compared to a control value. In some cases, increased levels of the amount of the one or more pathogen nucleic acids and increased amounts of organ cell-free mRNA indicate the presence of an infection in the organ. In some cases, the one or more pathogen nucleic acids are Zika nucleic acids and the organ is fetal brain. In some cases, the one or more pathogen nucleic acids are pathogen nucleic acids associated with chorioamnionitis and the organ is the uterus. In some cases of a method described herein, the RNA sequencing assay detects RNA of the pregnant subject to prognose a risk of preterm labor or delivery.

[0032] In preferred embodiments, the subject is a woman. In some cases of a method described herein, the pregnant woman is in a first trimester of pregnancy. In some cases of a method described herein, the pregnant woman is in a second trimester of pregnancy. In some cases of a method described herein, the pregnant woman is in a third trimester of pregnancy.

In some cases of a method described herein, the pregnant woman has chorioamnionitis. In some cases of a method described herein, the pregnant woman has one or more clinical symptoms of chorioamnionitis. In some cases of a method described herein, the one or more clinical symptoms of chorioamnionitis are selected from, but not limited to, the group consisting of fever, rapid heartbeat or tachycardia of the pregnant woman, rapid fetal heartbeat or fetal tachycardia, uterine tenderness, vaginal discharge with an unusual or foul odor or discoloration, amniotic fluid with a foul smell, maternal leukocytosis, and any combination thereof. In some cases of a method described herein, the pregnant subject has one or more risk factors for chorioamnionitis. In some cases of a method described herein, the one or more risk factors for chorioamnionitis are selected from the group consisting of longer duration of membrane rupture, prolonged labor, internal monitoring of labor, multiple vaginal examinations, meconium-stained amniotic fluid, smoking, alcohol abuse, drug abuse, compromised immune system, epidural anesthesia, colonization with group B streptococcus, sexually transmissible genital infections, vaginal colonization with Ureaplasma spp., and any combination thereof. In some cases of a method described herein, the pregnant subject has current or recent symptoms evocative of acute infection at risk of congenital infection. In some cases of a method provided herein, the pregnant woman has one or more risk factors for premature labor and delivery. In some cases of a method provided herein, the one or more risk factors for premature labor and delivery are selected from the group consisting of having a premature rupture of the membranes; having a personal or family history of premature birth, miscarriage, or stillbirth; being underweight or overweight before pregnancy; not gaining enough weight during pregnancy; having certain health conditions such as diabetes, high blood pressure, preeclampsia, or blood clot disorders; becoming pregnant after in vitro fertilization (IVF); becoming pregnant up to 18 months after a previous birth; having a social risk associated with preterm birth (e.g., being single or exposed to domestic violence);

having a positive test result from an assay giving a prognosis of preterm birth (e.g., Sera Prognostics PreTRM™ test); and any combination thereof.

[0033] In some cases of a method provided herein, the pregnant subject is considered to have a high-risk pregnancy. In some cases of a method provided herein, the pregnant subject is not considered to have a high-risk pregnancy. In some cases of a method provided herein, the pregnant subject previously had an amniocentesis test or chorionic villus sampling test. In some cases, the amniocentesis test or chorionic villus sampling test indicated

inflammation or infection but did not identify a specific pathogen.

[0034] In some cases of a method outlined herein, the subject is a neonate considered at a high-risk of infection. In some cases, the neonate is delivered by a pregnant subject with a known or suspected infection, while in other cases the fetus or neonate exhibited signs or symptoms of infection during pregnancy, labor or post-labor.

[0035] In some cases of a method described herein, the one or more pathogen nucleic acids are derived from one or more pathogens. In some cases of a method described herein, the one or more pathogens comprise one or more bacteria, one or more viruses, one or more fungi, one or more yeasts and/or one or more eukaryotes. In some cases of a method described herein, the one or more pathogens comprise two or more, three or more, four or more, five or more, ten or more, fifteen or more, twenty or more, fifty or more, or a hundred or more pathogens. In some cases of a method described herein, the one or more pathogens comprise, but are not limited to, Escherichia coli, group B streptococcus (Streptococcus agalactiae), anaerobic bacterium, Staphylococcus aureus, cytomegalovirus (CMV),

Mycoplasma spp. (e.g., Mycoplasma hominis), Ureaplasma spp. (e.g., Ureaplasma urealyticum, Ureaplasma parvum), human immunodeficiency virus (HIV) or other lentivirus, herpes simplex, varicella, B19 erythrovirus, Toxoplasma gondii, Treponema pallidum,

Listeria monocytogenes, Plasmodium falciparum, rubella, Chlamydia trachomatis, hepatitis

B virus, hepatitis E virus, Parvovirus, Enterovirus, hepatitis C virus, syphilis, gonorrhea,

Fusobacterium nucleatum, Enterococcus faecalis, Corynebacterium aurimucosum, Corynebacterium urealyticum, Gardnerella vaginalis, Bacteroides fragilis, Haemophilus influenzae, Methylobacterium mesophilicum, Prevotella bivia, Rothia mucilaginosa, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopenumoniae ,

Streptococcus pasteurianus, Sneathia sanguinegens, Candida albicans, Neisseria

gonorrhoeae, Citrobacter koseri, Peptoniphilus harei, Klebsiella pneumoniae, Micrococcus luteus, and Flaviviruses, including Zika virus.

[0036] In some cases of a method described herein, the one or more pathogen nucleic acids comprise cell-free pathogen DNA. In some cases of a method described herein, the one or more pathogen nucleic acids comprise cell-free pathogen RNA. In some cases of the method described herein, the one or more pathogen nucleic acids comprise particle protected RNA rather than cell-free RNA. In some cases of a method described herein, the one or more pathogen nucleic acids comprise a mixture of cell-free pathogen DNA and cell-free pathogen RNA. In some cases of a method described herein, the cell-free nucleic acids comprise cell- free DNA. In some cases of a method described herein, the cell-free nucleic acids comprise cell-free RNA. In some cases of a method described herein, the cell-free nucleic acids comprise a mixture of cell-free DNA and cell-free RNA.

[0037] In some cases of a method described herein, the sample is selected from the group consisting of blood, plasma, serum, umbilical cord blood, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage, urine, stool, saliva, nasal swab, cord blood, amniotic fluid and any combination thereof. In some cases of a method described herein, the sample is a cell-free sample.

[0038] In some cases of a method described herein, the method is conducted without conducting an amniocentesis or chorionic villus sampling test on the pregnant subject or subject contemplating pregnancy. In some cases of a method described herein, the method is conducted without performing an invasive test to detect intra-uterine infection or

inflammation. In some cases of a method described herein, the method reduces the risk of preterm labor by at least 10%, at least 20%, at least 30%>, at least 40%, at least 50%, at least 60%), at least 70%, at least 80%, or at least 90%. In some cases of a method described herein, the method reduces the risk of mortality and/or morbidity related to early onset neonatal sepsis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0039] In some cases of a method described herein, the method further comprises adjusting the therapeutic regimen administered to the pregnant subject or subject

contemplating pregnancy or neonate. In some cases of a method described herein, the detecting the one or more pathogen nucleic acids comprises monitoring the one or more pathogen nucleic acids over time. In some cases of a method described herein, the pathogens are associated with an intrauterine infection. In some cases of a method described herein, the pathogens are associated with a congenital fetal infection. In some cases of a method described herein, the pathogens are associated with a neonatal infection. In some cases of a method described herein, the method comprises detecting chorioamnionitis or an increased risk thereof. In some cases, the chorioamnionitis is acute chorioamnionitis, chronic chorioamnionitis, sub-clinical chorioamnionitis, or other type of chorioamnionitis. In some cases of a method described herein, the method comprises detecting an increased risk for infection of intrauterine tissue, chorion, amnion, or placenta. In some cases of a method described herein, the method further comprises discussion of earlier termination of pregnancy (e.g., related to irreversible sequelae of the fetus).

[0040] In some cases of a method described herein, the method further comprises performing one or more tests on the pregnant subject or subject contemplating pregnancy or a fetus or neonate. In some cases of a method described herein, the one or more tests comprise an ultrasound. In some cases of a method described herein, the one or more tests comprise a Magnetic Resonance Imaging (MRI). In some cases of a method described herein, the one or more tests comprise molecular diagnostics, PCR, digital PCR, sequencing, or analyzing one or more genetic defects in the fetus. In some cases of a method described herein, the one or more tests comprise a test that determines fetal heart rate, amniotic fluid level, fetal or maternal biophysical profile, bowel dilation or development of hydrops.

[0041] In some cases of a method described herein, the method comprises detecting an infection in the pregnant subject or subject contemplating pregnancy. In some cases of a method described herein, the method comprises detecting an infection in the pregnant subject or subject contemplating pregnancy and conducting an RNA sequencing assay on a sample comprising cell-free RNA from the pregnant subject or subject contemplating pregnancy to distinguish between extra-uterine and intra-uterine infection. In some cases of a method described herein, the method further comprises repeating the method over time to (a) monitor infection in the pregnant subject or subject contemplating pregnancy or (b) monitor efficacy of a treatment. In some cases of a method described herein, the method comprises detecting an infection in the cord blood in utero or after delivery of a neonate. In some cases of a method described herein, the method further comprises administering a therapeutic regimen to the neonate when the infection is detected.

[0042] In some cases of a method described herein, the high throughput sequencing comprises generating sequence reads at a rate of at least 1,000 reads per hour. In some cases of a method described herein, the nucleic acids are circulating cell-free nucleic acids. The cell-free nucleic acids may, in some cases, comprise highly fragmented nucleic acid sequences that are free from cells, viral particles or virions in the host. In some cases of a method described herein, the nucleic acids are isolated from viral particles.

[0043] Another aspect of the present disclosure provides a non-transitory computer- readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods described herein.

[0044] Another aspect of the present disclosure provides a system comprising one or more computer processors and a non-transitory computer-readable medium coupled thereto. The non-transitory computer readable medium comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods described herein.

INCORPORATION BY REFERENCE

[0045] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entireties to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0047] FIG. 1 shows a schematic of a basic method of this disclosure.

[0048] FIG. 2 shows a computer control system that is programmed or otherwise configured to implement methods provided herein.

[0049] FIG. 3A-3B show heatmaps of the abundance (MPM) of all pathogens in cord blood, overlaid with clinical information about each sample. A. all data, without filtering. B. cleaned heatmap to identify taxa most likely to be causing infection. A black box means the taxa was called as significantly above baseline, and a grey box means the taxa was called as significantly above baseline, but filtered out because the abundance is only slightly above baseline and presence may be due to deep sequencing of this particular sample. V: Vaginal birth, C: Cesarean section; V: Very preterm (less than or equal to 31weeks and 6 days of gestational age), P: preterm (between 32 weeks 0 days and 35 weeks 6 days), M: marginal preterm (between 36 weeks 0 days and 36 weeks 6 days)), and A: at term (more than or equal to 37 weeks), C: clinical chorioamnionitis; H: Presence of chorioamnionitis on histology, - : Absence of chorioamnionitis on histology; S: confirmed early onset neonatal sepsis.

[0050] FIG. 4A-4B show a quantile-quantile plot of the spearman correlation p-values comparing the significance levels of taxa in the maternal plasma to the significance levels in the cord blood in A. individuals with chorioamnionitis confirmed by histology, and B.

individuals without histological chorioamnionitis.

[0051] FIG. 5A-5C show the enrichment of pathogens that have been reported as a cause of chorioamnionitis: A. enrichment of these pathogens in cord blood of individuals who have histological chorioamnionitis, B. enrichment of these pathogens in maternal blood of individuals who have histological chorioamnionitis and C. enrichment of these pathogens in maternal blood in individuals who had preterm birth. Only pathogens that are present in at least one cord blood or maternal plasma sample, respectively, are included.

[0052] FIG. 6A-6D show exemplary performance of the method in cord blood in cases with and without chorioamnionitis, either confirmed by histology (Fig. 6A and 6C) or either clinically diagnosed or diagnosed by histology (Fig. 6B and 6D), in all type of births (Fig 6A-6C) or after Cesarean section births only (Fig. 6D). PTL: PreTerm Labor; Chorio:

Chorioamnionitis; Se: Sensitivity; Sp: Specificity; PPV: Positive Predictive Value; PV: Negative Predictive Value.

[0053] Fig. 7 shows an exemplary detection rate of pathogens in cord blood by mode of delivery.

[0054] FIG. 8A-8C show exemplary performance of the method in maternal blood in cases with and without chorioamnionitis confirmed by histology. A. performance of the method using maternal blood from subjects with preterm labor, B. performance of the method using maternal blood from subjects with at term labor and C. performance of the method using maternal blood from subjects with at term labor but after removing cases of chronic chorioamnionitis. PTL: PreTerm Labor; Chorio: Chorioamnionitis; Se: Sensitivity; Sp: Specificity; PPV: Positive Predictive Value; NPV: Negative Predictive Value.

[0055] FIG. 9 shows the concordance in pathogen identification between cord blood and maternal plasma. DETAILED DESCRIPTION OF THE INVENTION

[0056] Overview

[0057] The methods provided herein include methods of non-invasively detecting and/or methods of treating pregnancy-associated infections, thereby reducing adverse effects that may negatively impact fetal, neonatal or maternal health. The methods may often involve direct sequencing of pathogen-derived cell-free nucleic acids (e.g., DNA, RNA) present in a body fluid of a pregnant subject (e.g., pregnant woman), a woman contemplating pregnancy or in the umbilical cord blood (e.g., fetal and/or maternal blood). FIG. 1 provides a general overview of some of the methods provided herein. The methods may involve obtaining a sample from a pregnant subject or woman contemplating pregnancy 110. In other cases, the subject may be a neonate and the testing is conducted using cord blood post-delivery or peripheral blood. The pregnant subject or subject contemplating pregnancy may be healthy or may have, or be at risk of having, a pregnancy associated infection such as an infection of fetal membranes, of the placenta, or of the fetus itself either prior to or during delivery. In some instances, the pregnant subject may have or be clinically suspected of having chorioamnionitis, which generally is intra-uterine inflammation of the amnion, chorion, or placenta, or funisitis if the infection has spread to the umbilical cord. Chronic

chorioamnionitis can be defined by the infiltration of lymphocytes in the chorioamniotic membranes and the chorionic plate, similar to that of neutrophils in acute chorioamnionitis. Funisitis, also a histopathologic diagnosis, is the extension of infection or inflammation to the umbilical cord.

[0058] Chorioamnionitis is usually caused by a bacterial infection but can also have other causes such as viral infection or other types of infection. In some cases, the pregnant subject may have had an amniocentesis that indicated the presence of intra-uterine inflammation 120. In some cases, no prior amniocentesis, chorionic villus sampling test, or other invasive procedure was performed on the subject 130. Indeed, in some cases, the methods may involve detecting and treating an infection or other condition without performing an amniocentesis test or other invasive procedure at any point during the method. In some cases, the pregnant subject may be carrying a fetus with a congenital infection, or at risk of a congenital infection. In some cases, the pregnant subject or subject contemplating pregnancy may have an active or latent infection. In some cases, the pregnant subject may be at risk of preterm birth, at risk or experiencing preterm premature rupture of the fetal membranes, or may have another condition.

[0059] The sample may be a blood sample 140 or plasma sample 150, as depicted, or cord blood sample or any other type of biological sample, especially a biological sample containing a bodily fluid, tissue, and/or cells. In some preferred examples, the sample may be any bodily fluid containing cell-free nucleic acids including, but not limited to, whole blood, serum, plasma, maternal peripheral blood, or urine. In a preferred embodiment, the cell -free nucleic acids (e.g., DNA, RNA) 160 are extracted from the sample. In some cases, the nucleic acids are enriched for a certain population of nucleic acids such as pathogenic sequences or exon sequences or viral particle sequences. In some cases, a collection of oligonucleotides can be used as primers in primer extension reactions, PCR reactions, or reverse-transcription reactions and/or in hybridization and/or pull-down assays to enrich for certain pathogen-specific nucleic acids. In some cases, the collection of oligonucleotides comprises at least 1,000, at least 5,000, or at least 10,000 oligonucleotides with different nucleotide sequences, wherein the different nucleotide sequences are specifically selected to contain target (e.g., pathogen, organ) nucleic acid sequences at least 10 nucleotides in length. In some cases, the target nucleic acid sequences are at least 10 nucleotides in length, or from 10 to 12 nucleotides in length, or from 12 to 15 nucleotides in length.

[0060] The methods may include sequencing 170 the cell-free nucleic acids, generally by Next Generation Sequencing or by another form of high-throughput sequencing. The sequencing may be directed to specific targets, or may be initially agnostic to a particular target sequence (e.g., whole genome sequencing, whole exome sequencing, whole transcriptome sequencing, metagenomic sequencing). The results from the sequencing may be analyzed (e.g., bioinformatically) to detect pathogen sequences 180. The pathogen sequences may be analyzed further to determine a quantity of pathogen in the sample and to assess whether the pregnant subject is experiencing a pregnancy-related infection such as infection of fetal membranes, placental infection, or a congenital infection. To estimate the abundance of each pathogen, DNA and/or RNA molecules of various lengths, GC content and at known concentrations can be spiked into the sample before conducting an assay such as a sequencing assay. A statistical framework can be used to estimate the relative abundance of the pathogens. In some cases, the sample is analyzed to distinguish a latent or past infection from an active infection. In some cases, the pathogen sequences may be analyzed to determine the causal pathogen for a particular pregnancy-associated condition such as chori oamni oniti s .

[0061] When a causal pathogen is identified by methods provided herein, the methods may include treating the pregnant subject or subject contemplating pregnancy with an appropriate drug such as an antibiotic or antiviral medication 185. In some cases where the subject is a pregnant subject, early treatment may lengthen the period of time to delivery and avert premature labor and/or a preterm birth 190. As a result, the methods may reduce the risk of neonatal complications that may be associated with preterm or premature birth, such as stillbirth, and various injuries to the infant including injury to the nervous system, respiratory system, immune system, cardiovascular system, visual system and/or auditory system 195. The method may also reduce the need for (or risk of) early termination of pregnancy, particularly when an infection is caught at an early stage. In other cases, the pregnant subject may need to undergo early termination. In some cases, the methods described herein may reduce a risk of early labor.

[0062] The pregnant subject or subject contemplating pregnancy may be monitored over time, for example, by repeating a method provided herein one or more times or by conducting one or more additional tests (e.g., ultrasound or MRI) on the pregnant subject or subject contemplating pregnancy or fetus. In some cases, the course of the infection is monitored following drug treatment. In such instances, temporal differences in the amount of one or more nucleic acids from one or more pathogens can be used to monitor

effectiveness of treatment or to modify a treatment. For instance, the amount of one or more nucleic acids from one or more pathogens can be determined before and after a treatment. A decrease in the one or more nucleic acids from one or more pathogens after treatment may indicate that the treatment was successful. Additionally, the amount of one or more nucleic acids from one or more pathogens can be used to choose between treatments, for examples, treatments targeting different pathogens.

[0063] In some cases, where the subject is a neonate subject, the methods may be used where the mother has a confirmed or suspected infection that may have been treated during pregnancy. In other cases, the methods may be used as a preliminary diagnostic to determine if the fetus was exposed to infection in utero or during labor. In some cases, the methods include treating the neonate subject and/or repeating the method provided herein one or more times.

[0064] The methods provided herein may have a multitude of advantages and potential uses. The methods described herein may detect an infection in difficult clinical settings such as when: (a) there is low pathogen titer in the blood or body fluid sample; (b) the infection is clinically silent or the pregnant subject or subject contemplating pregnancy is asymptomatic; (c) the infectious pathogens are particularly difficult to detect, e.g., because they are difficult to culture or fail to propagate in vitro or because the patient received preemptive treatment (e.g., "loss of diagnostic window opportunity"); or (d) the infection is a localized infection (rather than a systemic infection) particularly an infection localized to uterine tissues or fetus and not expected to be detectable by analysis of target nucleic acids in a body fluid such as blood, plasma, serum or urine. The methods described herein are particularly useful in such difficult clinical settings because, in some embodiments, the methods may have high sensitivity, high specificity and/or may be capable of detecting low concentrations of target nucleic acids (e.g., nucleic acids from one or more pathogens, from the fetus, or from the uterus) among much higher concentrations of background nucleic acids (e.g., nucleic acids of the pregnant subject or subject contemplating pregnancy or from other organs of the pregnant subject or subject contemplating pregnancy).

[0065] Some of the methods described herein may be combined with other methods to improve detection of a pathogen as well as to enable a determination of the site of infection. In some cases, the methods may be combined with a cell-free RNA sequencing method to identify organs that may be infected. For example, the methods may detect the presence of a pathogen in a pregnant subject or subject contemplating pregnancy by virtue of the nature of cell-free circulating DNA. In such case, the method may further be able to detect that the infection is within a uterus (such as by detection of increased levels of circulating cell-free RNA derived from uterine tissues or fetal tissues (e.g., fetal brain) (see, Koh W. et al, Noninvasive in vivo monitoring of tissue-specific global gene expression in humans, PNAS 2014: 111 (7361-7366), which publication is hereby incorporated by reference in its entirety for all purposes). Conversely, the presence of increased levels of circulating cell-free RNA from a different organ such as the lungs of the pregnant subject or subject contemplating pregnancy may indicate that the detected pathogen has not infected the uterine tissues (e.g., chorion, amnion, placenta) or, in the case of a pregnant subject, fetal tissues

[0066] The methods provided may involve detection of specific causative agents of inflammation or infection, particularly in the context of pregnancy. As such, the disclosed methods may promote antibiotic stewardship, for example, by indicating a specific drug (e.g., antibiotic) for a detected pathogen, thereby preventing overuse of broad-spectrum antibiotics. In other cases, the methods may eliminate the use of an antibiotic or other drug altogether. For example, the methods provided herein may indicate that the source of an infection or inflammation is not bacterial, which may eliminate the need to administer an antibiotic to the pregnant subject or subject contemplating pregnancy.

[0067] Subjects

[0068] The subjects described herein are preferably human subjects, and more preferably women, particularly pregnant women. In further preferred embodiments, the subject may be a neonate. As used herein, the term "neonate" refers to a newborn baby, generally less than a month old. The pregnant woman or subject may be tested and/or treated during any point of her pregnancy including during her first trimester, her second trimester, or her third trimester. In some cases, a test provided herein may be administered during a perinatal period (e.g., from 24 weeks gestation to seven days following birth). As used herein, the term "woman" includes adult women and teenagers. In some cases, a subject may not be pregnant, but may be contemplating pregnancy.

[0069] In preferred embodiments, a pregnant subject is a pregnant woman infected with a pathogen, at risk of infection by a pathogen, or suspected of having a pathogenic infection. In some cases, the subject is suspected of having a particular infection of known cause, such as a known pathogen. In other cases, the subject is suspected of having an infection of unknown origin. In some examples, the subject has been exposed to a pathogen, or suspected to have been exposed to a pathogen such as by travel to a particular geographical region or by interaction with an infected individual (including sexual interaction).

[0070] A pregnant subject provided herein may have one or more clinical symptoms of chorioamnionitis. Such symptoms may include, but are not limited to, fever (e.g., >100° F), rapid heartbeat or tachycardia of the subject (e.g., greater than 120 beats per minute), rapid fetal heartbeat or fetal tachycardia (e.g., greater than 160-180 beats per minute), uterine tenderness, vaginal discharge with an unusual or foul odor or discoloration, amniotic fluid with a foul smell, and/or maternal leukocytosis (e.g., total blood leukocyte count of >15,000- 18,000 cells/nL).

[0071] A pregnant subject or subject contemplating pregnancy may have certain risk factors for chorioamnionitis. Such risk factors may include, but are not limited to, longer duration of membrane rupture, prolonged labor, internal monitoring of labor, multiple vaginal examinations, meconium-stained amniotic fluid, smoking, alcohol abuse, drug abuse, compromised immune system, epidural anesthesia, colonization with group B streptococcus, sexually transmissible genital infections, and vaginal colonization with Ureaplasma spp.

[0072] The pregnant subject or subject contemplating pregnancy may be healthy or may have, or be at risk of having, a pregnancy associated infection such as an infection of fetal membranes, of the placenta, or of the fetus itself (congenital infection). In some instances, the pregnant subject may be clinically suspected of having chorioamnionitis. As used herein, the term chorioamnionitis generally refers to inflammation of fetal membranes (e.g., amnion, chorion) and/or inflammation of the placenta. The cause of the chorioamnionitis may be an infection of any type, including bacterial, viral, fungal, parasitic, etc. In some cases, the pregnant subject may have a fetus with a congenital infection, or at risk of a congenital infection. In some cases, the pregnant subject or subject contemplating pregnancy may have an active or latent infection. In some cases, the pregnant subject or subject contemplating pregnancy may be at risk of preterm birth, at risk or experiencing preterm premature rupture of the fetal membranes, or may have another condition.

[0073] Generally, the methods provided herein involve methods of detecting the infectious cause of a pregnancy-related condition without performing an invasive procedure such as an amniocentesis or chorionic villus sampling. As such, the subject may have not had an amniocentesis or chorionic villus sampling prior to the performance of methods herein. But in some cases, the subject may have had or may have an amniocentesis test or other invasive procedure. The amniocentesis may be performed prior to the sequencing step or following the sequencing step. The sequencing process may detect a pathogenic infection that was not detected by the subject's amniocentesis or chorionic villus sampling. In some cases, a subject may have an amniocentesis or chorionic villus sampling test that indicates the presence of an infection, e.g., by Gram stain, or an abnormal differential count of white blood cells and then may have a test disclosed herein to identify the causal pathogen of the infection.

[0074] The subject may have been treated or may be treated with an antimicrobial, antibacterial, antiviral, or antiparasitic drug. In some cases, the subject is infected (e.g., with one or more microbes, pathogens, bacteria, viruses, fungi, or parasites). In some cases, the subject is not infected (e.g., with one or more microbes, pathogens, bacteria, viruses, fungi, or parasites). In some cases, the subject is healthy. In some cases, the subject is infected but asymptomatic. In some cases, the subject is susceptible or at risk of an infection. The subject may have or be at risk of having another disease or disorder.

[0075] In preferred embodiments, the subject is human; but the subject may also be a non-human animal. In some cases, the subject fits within a category of subjects, including but not limited to, mammals, non-human mammals, non-human primates, primates, domesticated animals (e.g., laboratory animals, household pets, or livestock), or non- domesticated animals (e.g., wildlife). In some particular embodiments, the subject is a dog, cat, rodent, mouse, hamster, cow, bird, chicken, pig, horse, goat, sheep, rabbit, ape, monkey, or chimpanzee.

[0076] Samples

[0077] The samples provided herein are preferably any type of clinical sample. In some cases, the samples contain cells, tissue, or a bodily fluid. In preferred embodiments, the sample is a liquid or fluid cell-free sample. In some cases, the sample contains a body fluid such as whole blood, blood component, plasma, serum, cord blood, tissue obtained from fetal surgery, urine, saliva, lymph, spinal fluid, bronchoalveolar lavage, nasal swab, respiratory secretions, vaginal fluid, amniotic fluid, semen and/or menses. In some cases, the sample is made up of, in whole or in part, cells or tissue. The sample may be from any part of the body including the central nervous system, the brain, spinal cord, bone marrow, pancreas, thyroid, gall bladder, liver, heart, spleen, colon, rectum, lung, respiratory system, throat, nasal cavity, stomach, esophagus, ears, eyes, skin, limbs, uterus, prostate, reproductive organ, circulatory system, or any other organ or region of the body. In some cases, a sample is derived from a pregnant subject (e.g., a pregnant woman) or a woman contemplating pregnancy. In some cases, a sample is derived from the umbilical cord, placenta, fetal membranes or a fetus.

[0078] The sample may be a nucleic acid sample; in some cases, the sample contains a certain amount of nucleic acids. Nucleic acids within a sample may include double-stranded (ds) nucleic acids, single stranded (ss) nucleic acids, DNA, RNA, cDNA, mRNA, cRNA, tRNA, ribosomal RNA, dsDNA, ssDNA, miRNA, siRNA, circulating nucleic acids, circulating cell-free nucleic acids, circulating DNA, circulating RNA, cell-free nucleic acids, cell-free DNA, cell-free RNA, cell-free pathogen nucleic acids, cell-free viral nucleic acids, cell-free bacterial nucleic acids, cell-free maternal nucleic acids, circulating cell-free DNA, circulating cell-free RNA, genomic DNA, exosomes, mitochondrial nucleic acids, non- mitochondrial nucleic acids, nuclear DNA, nuclear RNA, chromosomal DNA, circulating tumor DNA, circulating tumor RNA, circulating maternal DNA, circulating maternal RNA, circulating fetal DNA, circulating fetal RNA, or any combination thereof. In some cases, sample nucleic acids may include synthetic nucleic acids. In some cases, nucleic acids are maternal nucleic acids. In some cases, nucleic acids are fetal nucleic acids.

[0079] In some cases, different types of nucleic acids may be present in a sample. For example, in a preferred embodiment, the sample may comprise cell-free RNA and cell-free DNA. Likewise, a method provided herein may include a method where both the RNA and the DNA present in a sample are analyzed, singly or in combination; in some cases, the RNA and DNA in a mixed sample are detected singly, or in combination..

[0080] In some cases, the sample may be an unprocessed sample (e.g., whole blood) or a processed sample (e.g., serum, plasma) that contains cell-free or cell-associated nucleic acids. In some cases, the sample has been enriched for a certain type of nucleic acid, e.g.,

DNA, RNA, cell-free DNA, cell-free RNA, cell-free circulating DNA, cell-free circulating RNA, etc. In some cases, a sample has been processed in some way to isolate nucleic acids or to separate nucleic acids from other components within the sample, e.g. bacterial cells, human cells, viral particles or exosomes. In some cases, the sample has been enriched for pathogen-specific nucleic acids. For example, the sample may be enriched for nucleic acids from pathogens associated with chorioamnionitis, e.g., by size selection and/or by using a collection of oligonucleotides. In some cases of the methods provided herein, nucleic acids are extracted from plasma or from plasma that has been subjected to further processing (e.g., centrifugation, filtration, ultrafiltration, etc.) to further remove cells or other components.

[0081] As used herein, the term "cell-free" refers to the condition of the nucleic acid as it appeared in the body immediately or directly before the sample is obtained from the body and generally refers to nucleic acids that, at the time of sample collection, are present in the body in a "free" state, outside of a cell or virion. In some cases, the cell-free state may also include nucleic acids that are free from viral particles. In some examples, circulating cell-free nucleic acids in a sample may have originated as, or be derived from, cell-free nucleic acids circulating in the bloodstream prior to collection from the subject. In contrast, nucleic acids that are extracted post-collection from a solid tissue, such as a biopsy, or removed post- collection from intact virions in a sample such as a plasma sample, are generally not considered to be "cell-free."

[0082] Often, the sample is a fresh sample. In some cases, the sample is a frozen sample. In some cases, the sample is fixed, e.g., with a chemical fixative such as formalin-fixed paraffin-embedded tissue.

[0083] In addition, patient samples can be obtained at any point from the pregnant subject or from a subject contemplating pregnancy. The samples may be obtained prior to treatment, during the treatment process, following exposure to a pathogen, throughout the course of infection, immediately prior to labor or any other point. The samples may be obtained at specific intervals, such as daily, weekly or monthly, or during routine pregnancy examinations.

[0084] Target Nucleic Acids

[0085] The methods provided herein may be used to detect any number of target nucleic acids. The target nucleic acids may include whole or partial genomes, exomes, genetic loci, genes, exons, introns, modified nucleic acids (e.g., methylated nucleic acids), etc. Often, the methods provided herein can be used to detect pathogen target nucleic acids; in some cases, the pathogen target nucleic acids are present in complex clinical sample containing nucleic acids from the subject. The pathogen target nucleic acid may be associated with an infectious disease, such as influenza, tuberculosis, meningitis, sepsis, toxoplasmosis, malaria, zika infection, or any other known infectious disease or disorder, including those described further herein. Often, the methods include methods of enriching a sample for certain target nucleic acids.

[0086] The pathogen target nucleic acid may be present in a tissue sample, such as a tissue sample from a site of infection. In other cases, the pathogen target nucleic acid has migrated from the site of infection; for example, it may be obtained from a sample containing circulating cell-free nucleic acids (e.g., cell-free RNA, cell-free DNA).

[0087] In some cases, the target nucleic acid may make up only a very small portion of the entire sample, e.g., less than 0.1 %, less than 0.01%, less than 0.001%, less than

0.0001%, less than 0.00001%, less than 0.000001%, less than 0.0000001% of the total nucleic acids in a sample. Often, the total nucleic acids in an original sample may vary. For example, total cell-free nucleic acids (e.g., DNA, mRNA, RNA) may be in a range of 1-100 ng/ml, e.g., (about 1, 5, 10, 20, 30, 40, 50, 80, 100 ng/ml). In some cases, the total concentration of cell-free nucleic acids in a sample is outside of this range (e.g., less than 1 ng/ml; in other cases, the total concentration is greater than 100 ng/ml). This may be the case with cell-free nucleic acid (e.g., DNA) samples that are predominantly made up of human DNA and/or RNA. In such samples, pathogen target nucleic acids may have scant presence compared to the human or host nucleic acids.

[0088] The length of target nucleic acids can vary. In some cases, target nucleic acids may be about or at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,

160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500 or 2000 nucleotides

(or base pairs) in length. In some cases, target nucleic acids may be up to about 20, 30, 40,

50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,

450, 500, 750, 1000, 1500 or 2000 nucleotides (or base pairs) in length. In some particular embodiments, the target nucleic acids are relatively short, e.g., less than 50 base pairs, less than 75 base pairs, less than 100 base pairs, less than 500 base pairs (or nucleotides) or less than 1000 base pairs (or nucleotides) in length. In some cases, the target nucleic acids are relatively long, e.g., greater than 1000, greater than 1500, greater than 2000, greater than

2500, greater than 3000, or greater than 5000 base pairs (or nucleotides) in length.

[0089] As is the case with the sample nucleic acids, the target nucleic acids may be any type of nucleic acid including: double-stranded (ds) nucleic acids, single stranded (ss) nucleic acids, DNA, RNA, cDNA, mRNA, cRNA, tRNA, ribosomal RNA, dsDNA, ssDNA, miRNA, siRNA, circulating nucleic acids, circulating DNA, circulating RNA, cell-free nucleic acids, cell-free DNA, cell-free RNA, circulating cell-free DNA, circulating cell-free RNA, genomic DNA, circulating maternal nucleic acids, circulating maternal DNA, circulating maternal RNA, circulating fetal DNA, circulating fetal RNA, cell-free maternal nucleic acids, cell-free maternal RNA, cell-free maternal DNA, cell-free pathogen nucleic acids, circulating pathogen nucleic acids, mitochondrial nucleic acids, non-mitochondrial nucleic acids, nuclear DNA, nuclear RNA, chromosomal DNA, or any combination thereof.

[0090] The target nucleic acids are preferably nucleic acids derived from pathogens including but not limited to viruses, bacteria, fungi, parasites and any other microbe, particularly infectious microbe. Often, the methods provided herein are designed to detect pathogen nucleic acids from one or more different taxa. Taxa can include a taxonomic group of any rank or type, such as a strain, variant, species, genus, family, order, class, phylum, kingdom, or domain. The target nucleic acids (e.g., pathogen nucleic acids) may be derived from pathogens associated with chorioamnionitis, intra-uterine infection, fetal or neonatal infection, pregnancy-related infection or any other infection potentially experienced by a pregnant subject or subject contemplating pregnancy. Examples of pathogen target nucleic acids include, but are not limited to, nucleic acids derived from Escherichia coli, group B streptococcus (Streptococcus agalactiae), anaerobic bacterium, Staphylococcus aureus, cytomegalovirus (CMV), Mycoplasma spp. (e.g., Mycoplasma hominis), Ureaplasma spp. (e.g., Ureaplasma urealyticum, Ureaplasma parvum), human immunodeficiency virus (HIV) or other lentivirus, herpes simplex, varicella, B19 erythrovirus, Toxoplasma gondii,

Treponema pallidum, Listeria monocytogenes, Plasmodium falciparum, rubella, Chlamydia trachomatis, hepatitis B virus, hepatitis E virus, Parvovirus, Enterovirus, hepatitis C virus, syphilis, gonorrhea, Fusobacterium nucleatum, Enterococcus faecalis, Coryne bacterium aurimucosum, Corynebacterium urealyticum, Gardnerella vaginalis, Bacteroides fragilis, Haemophilus influenzae, Methylobacterium mesophilicum, Prevotella bivia, Rothia mucilaginosa, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus

pseudopenumoniae, Streptococcus pasteurianus, Sneathia sanguinegens, Candida albicans, Neisseria gonorrhoeae, Citrobacter koseri, Peptoniphilus harei, Klebsiella pneumoniae, Micrococcus luteus, and Flaviviruses (e.g., West Nile virus, Dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus). In some cases, pathogens detected by the methods provided herein may be present and/or replicating in a particular tissue or organ such as fetal membrane (e.g., amnion, chorion), amnion, chorion, placenta, umbilical cord, fetal tissue, and/or uterus.

[0091] In some cases, one or more pathogen nucleic acids from a collection, panel, set or group of pathogen nucleic acids may be detected using the methods provided herein. The collection of pathogen nucleic acids can be selected based on a known association between the pathogen nucleic acids and an infection. For example, a collection of pathogen nucleic acids can be specifically selected to detect different pathogens associated with

chorioamnionitis. In another example, a collection of pathogen nucleic acids can be specifically selected to detect any fetal membrane infection. In some cases, the collection of pathogen nucleic acids may be selected in order to detect different pathogens or infections that affect a common organ, different pathogens that are associated with the same disease or infection, different pathogens associated with different diseases or infections, or any combination thereof. In some cases, a collection of unique pathogen nucleic acids associated only with one infection can be used to detect only that infection. In some case, the collection, panel, set or group of pathogen nucleic acids may include 1, 2, 3, 4, 5, 6, 10, or more pathogen nucleic acids that are associated with an infection.

[0092] In some cases, the target nucleic acids may be nucleic acids derived from a particular organ or tissue. Examples of organ/tissue target nucleic acids include but are not limited to nucleic acids (e.g., RNA) preferentially expressed or present in organs or tissues such as uterus, cervix, chorion, amniotic tissue, placenta, fetus, fetal brain, blood, heart, lungs, maternal brain, kidneys, vagina, reproductive organs, gastro-intestinal tissue, optic nerve, fetal optic nerve, or organs/tissue associated with the central or peripheral nervous system.

[0093] Nucleic Acid Enrichment and Library Preparation

[0094] In the methods provided herein, nucleic acids can be isolated from a sample using any means known in the art. For example, nucleic acids can be extracted using liquid extraction (e.g., Trizol, DNAzol) techniques. Nucleic acids can also be extracted using commercially available kits (e.g., QIAamp Circulating Nucleic Acid Kit, Qiagen DNeasy kit, QIAamp kit, Qiagen Midi kit, QIAprep spin kit).

[0095] Nucleic acids can be concentrated or precipitated by known methods, including, by way of example only, centrifugation. Nucleic acids can be bound to a selective membrane

(e.g., silica) for the purposes of purification. Nucleic acids can also be enriched for fragments of a desired length, e.g., fragments which are less than 1000, 500, 400, 300, 200 or 100 base pairs in length. Such an enrichment based on size can be performed using, e.g., PEG-induced precipitation, an electrophoretic gel or chromatography material (Huber et al. (1993) Nucleic

Acids Res. 21 : 1061-6), gel filtration chromatography, or TSKgel (Kato et al. (1984) J.

Biochem, 95:83- 86), which publications are hereby incorporated by reference in their entireties for all purposes.

[0096] The nucleic acid sample can be enriched for target polynucleotides, particularly target nucleic acids associated with inflammation or infection. In preferred cases, the target nucleic acids are associated with chorioamnionitis, intra-uterine infection, neonatal infection or congenital fetal infection. In some preferred cases, the target nucleic acids are pathogen nucleic acids (e.g., cell-free pathogen nucleic acids). In some preferred cases, the target nucleic acids are cell-free RNA associated with a particular organ or tissue including but not limited to uterus, lung, fetal brain, liver, or cervical tissue.

[0097] Target enrichment can be by any means known in the art. For example, the nucleic acid sample may be enriched by amplifying target sequences using target-specific primers (e.g., primers specific for pathogen nucleic acids associated with chorioamnionitis, intrauterine infection, fetal congenital infection, or other pregnancy-related infection). The target amplification can occur in a digital PCR format, using any methods or systems known in the art. The nucleic acid sample may be enriched by capture of target sequences onto an array immobilized thereon target-selective oligonucleotides. The nucleic acid sample may be enriched by hybridizing to target-selective oligonucleotides free in solution or on a solid support. The oligonucleotides may comprise a capture moiety which enables capture by a capture reagent. In some embodiments, the nucleic acid sample is not enriched for target polynucleotides, e.g., represents a whole genome.

[0098] In some cases, target (e.g., pathogen, organ) nucleic acids can be enriched relative to background (e.g., pregnant subject or subject contemplating pregnancy, healthy tissue) nucleic acids in the sample, for example, by pull-down (e.g., preferentially pulling down target nucleic acids in a pull-down assay by hybridizing them to complementary

oligonucleotides conjugated to a label such as a biotin tag and using, for example, avidin or streptavidin attached to a solid support), targeted PCR, or other methods. Examples of enrichment techniques include, but are not limited to: (a) self-hybridization techniques in which the major population in a sample of nucleic acids self-hybridizes more rapidly than the minor population in the sample; (b) depletion of nucleosome-associated DNA from free DNA; (c) removing and/or isolating DNA of specific length intervals; (d) exosome depletion or enrichment; and (e) strategic capture of regions of interest.

[0099] In some cases, an enriching step comprises (a) providing a sample of nucleic acids from a host, wherein the sample of nucleic acids from the host is a sample of single- stranded nucleic acids from the host and comprises host nucleic acids and non-host nucleic acids; (b) renaturing at least a portion of the single-stranded nucleic acids from the host, thereby producing a population of double-stranded nucleic acids within the sample; and (c) removing at least a portion of the double-stranded nucleic acids within the sample using a nuclease, thereby enriching non-host sequences in the sample of nucleic acids from the host. In some cases, an enriching step comprises (a) providing a sample of nucleic acids from a host, wherein the sample of nucleic acids from the host comprises host nucleic acids associated with nucleosomes and non-host nucleic acids; and (b) removing at least a portion of the host nucleic acids associated with nucleosomes, thereby enriching the non-host nucleic acids in the sample of nucleic acids from the host. In some cases, an enriching step comprises (a) providing a sample of nucleic acids from a host, wherein the sample of nucleic acids from the host comprises host nucleic acids and non-host nucleic acids; and (b) removing or isolating DNA of one or more length intervals, thereby enriching the non-host nucleic acids in the sample of nucleic acids from the host. In some cases, an enriching step comprises (a) providing a sample of nucleic acids from a host, wherein the sample of nucleic acids from the host comprises host nucleic acids, non-host nucleic acids, and exosomes; and (b) removing or isolating at least a portion of the exosomes, thereby enriching non-host sequences in the sample of nucleic acids from the host. In some cases, an enriching step comprises

preferentially removing nucleic acids with lengths that are above about 300 bases in length from the sample. In some cases, an enriching step comprises preferentially amplifying or capturing non-host nucleic acids from the sample.

[0100] An enriching step can comprise preferentially removing nucleic acids from the sample that are above about 120, about 150, about 200, or about 250 bases in length. In some cases, an enriching step comprises preferentially enriching nucleic acids from the sample that are between about 10 bases and about 60 bases in length, between about 10 bases and about 120 bases in length, between about 10 bases and about 150 bases in length, between about 10 bases and about 300 bases in length between about 30 bases and about 60 bases in length, between about 30 bases and about 120 bases in length, between about 30 bases and about 150 bases in length, between about 30 bases and about 200 bases in length, or between about 30 bases and about 300 bases in length. In some cases, an enriching step comprises

preferentially digesting nucleic acids derived from the host (e.g., pregnant subject or subject contemplating pregnancy). In some cases, an enriching step comprises preferentially replicating the non-host nucleic acids.

[0101] In some cases, an enriching step increases the ratio of non-host nucleic acids relative to host (e.g., pregnant subject or subject contemplating pregnancy) nucleic acids by at least 2X, at least 3X, at least 4X, at least 5X, at least 6X, at least 7X, at least 8X, at least 9X, at least 10X, at least 1 IX, at least 12X, at least 13X, at least 14X, at least 15X, at least 16X, at least 17X, at least 18X, at least 19X, at least 20X, at least 3 OX, at least 40X, at least 50X, at least 60X, at least 70X, at least 80X, at least 90X, at least 100X, at least 1000X, at least 5000X, or at least ΙΟ,ΟΟΟΧ.

[0102] In some cases, a nucleic acid library is prepared. The nucleic acid library can be a single-stranded nucleic acid library or a double-stranded nucleic acid library. In some cases, a single-stranded nucleic acid library can be a single-stranded DNA library (ssDNA library) or an RNA library. In some cases, a double-stranded nucleic acid library is a double-stranded DNA library (dsDNA library). A method of preparing an ssDNA library can comprise denaturing a double stranded DNA fragment into ssDNA fragments, ligating a primer docking sequence onto one end of the ssDNA fragment, and hybridizing a primer to the primer docking sequence. The primer can comprise at least a portion of an adaptor sequence that couples to a next-generation sequencing platform. The method can further comprise extension of the hybridized primer to create a duplex, wherein the duplex comprises the original ssDNA fragment and an extended primer strand. The extended primer strand can be separated from the original ssDNA fragment. The extended primer strand can be collected, wherein the extended primer strand is a member of the ssDNA library. A method of preparing an RNA library can comprise ligating a primer docking sequence onto one end of the RNA fragment and hybridizing a primer to the primer docking sequence. The primer can comprise at least a portion of an adaptor sequence that couples to a next-generation sequencing platform. The method can further comprise extension of the hybridized primer to create a duplex, wherein the duplex comprises the original RNA fragment and an extended primer strand. The extended primer strand can be separated from the original RNA fragment. The extended primer strand can be collected, wherein the extended primer strand is a member of the RNA library. A method of preparing a dsDNA library can comprise ligating an adaptor sequence onto one or both ends of the dsDNA fragment.

[0103] In various aspects, dsDNA can be fragmented by any means known in the art or as described herein. In some cases, dsDNA can be fragmented by physical means (e.g., by mechanical shearing, nebulization, or sonication), by enzymatic means, or by chemical means.

[0104] In some embodiments, cDNA is generated from RNA. For example, cDNA may be generated using random primed reverse transcription (RNaseH+) to generate randomly sized cDNA.

[0105] The lengths of the nucleic acids may vary. The nucleic acids or nucleic acid fragments (e.g., dsDNA fragments, RNA, or randomly sized cDNA) can be less than 1000 bp, less than 800 bp, less than 700 bp, less than 600 bp, less than 500 bp, less than 400 bp, less than 300 bp, less than 200 bp, or less than 100 bp. The DNA fragments can be about 40 to aboutlOO bp, about 50 to about 125 bp, about 100 to about 200 bp, about 150 to about 400 bp, about 300 to about 500 bp, about 100 to about 500, about 400 to about 700 bp, about 500 to about 800 bp, about 700 to about 900 bp, about 800 to about 1000 bp, or about 100 to about 1000 bp.

[0106] The ends of dsDNA fragments can be polished (e.g., blunt-ended). The ends of DNA fragments can be polished by treatment with a polymerase. Polishing can involve removal of 3' overhangs, fill-in of 5' overhangs, or a combination thereof. The polymerase can be a proof-reading polymerase (e.g., comprising 3' to 5' exonuclease activity). The proofreading polymerase can be, e.g., a T4 DNA polymerase, Pol 1 Klenow fragment, or Pfu polymerase. Polishing can comprise removal of damaged nucleotides (e.g., abasic sites), using any means known in the art.

[0107] Ligation of an adaptor to a 3' end of a nucleic acid fragment can comprise formation of a bond between a 3' OH group of the fragment and a 5' phosphate of the adaptor. Therefore, removal of 5' phosphates from nucleic acid fragments can minimize aberrant ligation of two library members. Accordingly, in some embodiments, 5' phosphates are removed from nucleic acid fragments. In some embodiments, 5' phosphates are removed from at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 95% of nucleic acid fragments in a sample. In some embodiments, substantially all phosphate groups are removed from nucleic acid fragments. In some embodiments, substantially all phosphates are removed from at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%), or greater than 95% of nucleic acid fragments in a sample. Removal of phosphate groups from a nucleic acid sample can be by any means known in the art. Removal of phosphate groups can comprise treating the sample with heat-labile phosphatase. In some embodiments, phosphate groups are not removed from the nucleic acid sample. In some embodiments ligation of an adaptor to the 5' end of the nucleic acid fragment is performed.

[0108] Sequencing

[0109] The nucleic acids produced according to the present methods may be analyzed to obtain various types of information including genomic, epigenetic (e.g., methylation), and

RNA expression. Methylation analysis can be performed by, for example, conversion of methylated bases followed by DNA sequencing. RNA expression analysis can be performed, for example, by polynucleotide array hybridization, by RNA sequencing techniques, or by sequencing cDNA produced from RNA.

[0110] In preferred embodiments, the sequencing is performed using a next generation sequencing assay. As used herein, the term "next generation" generally refers to any high- throughput sequencing approach including, but not limited to one or more of the following: massively-parallel signature sequencing, pyrosequencing (e.g., using a Roche 454

sequencing device), Illumina (Solexa) sequencing, sequencing by synthesis (Illumina), Ion torrent sequencing, sequencing by ligation (e.g., SOLiD sequencing), single molecule realtime (SMRT) sequencing (e.g., Pacific Bioscience), polony sequencing, DNA nanoball sequencing, heliscope single molecule sequencing (Helicos Biosciences), and nanopore sequencing (e.g., Oxford Nanopore). In some cases, the sequencing assay uses nanopore sequencing. In some cases, the sequencing assay includes some form of Sanger sequencing. In some cases, the sequencing involves shotgun sequencing. In some cases, the sequencing includes bridge PCR. In some cases, the sequencing is broad spectrum. In some cases, the sequencing is targeted.

[0111] In some cases, the sequencing assay comprises a Gilbert's sequencing method. In such approach, nucleic acids (e.g., DNA) are chemically modified and then cleaved at specific bases. In some cases, a sequencing assay comprises dideoxynucleotide chain termination or Sanger-sequencing.

[0112] A sequencing-by-synthesis approach may be used in the methods provided herein.

In some cases, fluorescently-labeled reversible-terminator nucleotides are introduced to clonally-amplified DNA templates immobilized on the surface of a glass flowcell. During each sequencing cycle, a single labeled deoxynucleoside triphosphate (dNTP) may be added to the nucleic acid chain. The labeled terminator nucleotide may be imaged when added to identify the base and may then be enzymatically cleaved to allow incorporation of the next nucleotide. Since all four reversible terminator-bound dNTPs (A, C, T, G) are generally present as single, separate molecules, natural competition may minimize incorporation bias.

[0113] In some cases, a method called Single-molecule real-time (SMRT) is used. In such approach, nucleic acids (e.g., DNA) are synthesized in zero-mode wave-guides

(ZMWs), which are small well-like containers with capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the

ZMW bottom) and fluorescently labelled nucleotides flowing freely in the solution. The fluorescent label is detached from the nucleotide upon its incorporation into the DNA strand, leaving an unmodified DNA strand. A detector such as a camera may then be used to detect the light emissions; and the data may be analyzed bioinformatically to obtain sequence information.

[0114] In some cases, a sequencing by ligation approach is used to sequence the nucleic acids in a sample. One example is the next generation sequencing method of SOLiD

(Sequencing by Oligonucleotide Ligation and Detection) sequencing (Life Technologies). This next generation technology may generate hundreds of millions to billions of small sequence reads at one time. The sequencing method may comprise preparing a library of DNA fragments from the sample to be sequenced. In some cases, the library is used to prepare clonal bead populations in which only one species of fragment is present on the surface of each bead (e.g., magnetic bead). The fragments attached to the magnetic beads may have a universal PI adapter sequence attached so that the starting sequence of every fragment is both known and identical. In some cases, the method may further involve PCR or emulsion PCR. For example, the emulsion PCR may involve the use of microreactors containing reagents for PCR. The resulting PCR products attached to the beads may then be covalently bound to a glass slide. A sequencing assay such as a SOLiD sequencing assay or other sequencing by ligation assay may include a step involving the use of primers. Primers may hybridize to the PI adapter sequence or other sequence within the library template. The method may further involve introducing four fluorescently labelled di-base probes that compete for ligation to the sequencing primer. Specificity of the di-base probe may be achieved by interrogating every first and second base in each ligation reaction. Multiple cycles of ligation, detection and cleavage may be performed with the number of cycles determining the eventual read length. In some cases, following a series of ligation cycles, the extension product is removed and the template is reset with a primer complementary to the n- 1 position for a second round of ligation cycles. Multiple rounds (e.g., 5 rounds) of primer reset may be completed for each sequence tag. Through the primer reset process, each base may be interrogated in two independent ligation reactions by two different primers. For example, the base at read position 5 is assayed by primer number 2 in ligation cycle 2 and by primer number 3 in ligation cycle 1.

[0115] In any of the embodiments, the detection or quantification analysis of the oligonucleotides can be accomplished by sequencing. The subunits or entire synthesized oligonucleotides can be detected via full sequencing of all oligonucleotides by any suitable methods known in the art, e.g., Ulumina HiSeq 2500, including the sequencing methods described herein.

[0116] Sequencing can be accomplished through classic Sanger sequencing methods which are well known in the art. Sequencing can also be accomplished using high- throughput systems some of which allow detection of a sequenced nucleotide immediately after or upon its incorporation into a growing strand, e.g., detection of sequence in real time or substantially real time. In some cases, high throughput sequencing generates at least 1,000, at least 5,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, or at least 500,000 sequence reads per hour. In some cases, each read is at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, or at least 150 bases per read. In some cases, each read is up to 2000, up to 1000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 400, up to 300, up to 200, or up to 100 bases per read. Long read sequencing can include sequencing that provides a contiguous sequence read of for example, longer than 500 bases, longer than 800 bases, longer than 1000 bases, longer than 1500 bases, longer than 2000 bases, longer than 3000 bases, or longer than 4500 bases.

[0117] In some cases, high-throughput sequencing involves the use of technology available by Illumina's Genome Analyzer IIX, MiSeq personal sequencer, NextSeq series or

HiSeq systems, such as those using HiSeq 4000, HiSeq 3000, HiSeq 2500, HiSeq 1500,

HiSeq 2000, or HiSeq 1000. These machines use reversible terminator-based sequencing by synthesis chemistry. These machines can do 200 billion DNA or more reads in eight days.

Smaller systems may be utilized for runs within 3, 2, or 1 days or less time. Short synthesis cycles may be used to minimize the time it takes to obtain sequencing results.

[0118] In some cases, high-throughput sequencing involves the use of technology available by ABI Solid System. This genetic analysis platform can enable massively parallel sequencing of clonally-amplified DNA fragments linked to beads. The sequencing methodology is based on sequential ligation with dye-labeled oligonucleotides.

[0119] The next generation sequencing can comprise ion semiconductor sequencing

(e.g., using technology from Life Technologies (Ion Torrent)). Ion semiconductor sequencing can take advantage of the fact that when a nucleotide is incorporated into a strand of DNA, an ion can be released. To perform ion semiconductor sequencing, a high density array of micromachined wells can be formed. Each well can hold a single DNA template.

Beneath the well can be an ion sensitive layer, and beneath the ion sensitive layer can be an ion sensor. When a nucleotide is added to a DNA, H+ can be released, which can be measured as a change in pH. The H+ ion can be converted to voltage and recorded by the semiconductor sensor. An array chip can be sequentially flooded with one nucleotide after another. No scanning, light, or cameras can be required. In some cases, an IONPROTON™

Sequencer is used to sequence nucleic acid. In some cases, an IONPGM™ Sequencer is used. The Ion Torrent Personal Genome Machine (PGM) can do 10 million reads in two hours.

[0120] In some cases, high-throughput sequencing involves the use of technology available by Helicos Biosciences Corporation (Cambridge, Massachusetts) such as the Single Molecule Sequencing by Synthesis (SMSS) method. SMSS can allow for sequencing the entire human genome in up to 24 hours. SMSS, like the MIP technology, may not require a pre-amplification step prior to hybridization. SMSS may not require any amplification. SMSS is described in part in US Publication Application Nos. 20060024711; 20060024678; 20060012793; 20060012784; and 20050100932.

[0121] In some cases, high-throughput sequencing involves the use of technology available by 454 Lifesciences, Inc. (Branford, Connecticut) such as the Pico Titer Plate device which includes a fiber optic plate that transmits chemiluminescent signal generated by the sequencing reaction to be recorded by a CCD camera in the instrument. This use of fiber optics can allow for the detection of a minimum of 20 million base pairs in 4.5 hours.

[0122] Methods for using bead amplification followed by fiber optics detection are described in Marguiles, M., et al. "Genome sequencing in microfabricated high-density picolitre reactors", Nature, doi: 10.1038/nature03959; as well as in US Publication

Application Nos. 20020012930; 20030058629; 20030100102; 20030148344; 20040248161; 20050079510, 20050124022; and 20060078909.

[0123] In some cases, high-throughput sequencing is performed using Clonal Single

Molecule Array (Solexa, Inc.) or sequencing-by-synthesis (SBS) utilizing reversible terminator chemistry. These technologies are described in part in US Patent Nos. 6,969,488;

6,897,023; 6,833,246; 6,787,308; and US Publication Application Nos. 20040106110;

20030064398; 20030022207; and Constans, A., The Scientist 2003, 17(13):36.

[0124] In some cases, the next generation sequencing is nanopore sequencing (See e.g.,

Soni GV and Meller A. (2007) Clin Chem 53 : 1996-2001). A nanopore can be a small hole, e.g., on the order of about one nanometer in diameter. Immersion of a nanopore in a conducting fluid and application of a potential across it can result in a slight electrical current due to conduction of ions through the nanopore. The amount of current which flows can be sensitive to the size of the nanopore. As a DNA molecule passes through a nanopore, each nucleotide on the DNA molecule can obstruct the nanopore to a different degree. Thus, the change in the current passing through the nanopore as the DNA molecule passes through the nanopore can represent a reading of the DNA sequence. The nanopore sequencing technology can be from Oxford Nanopore Technologies; e.g., a GridlON system. A single nanopore can be inserted in a polymer membrane across the top of a microwell. Each microwell can have an electrode for individual sensing. The microwells can be fabricated into an array chip, with 100,000 or more microwells (e.g., more than 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000) per chip. An instrument (or node) can be used to analyze the chip. Data can be analyzed in real-time. One or more instruments can be operated at a time. The nanopore can be a protein nanopore, e.g., the protein alpha-hemolysin, a heptameric protein pore. The nanopore can be a solid- state nanopore, e.g., a nanometer sized hole formed in a synthetic membrane (e.g., SiNx, or Si0₂). The nanopore can be a hybrid pore (e.g., an integration of a protein pore into a solid- state membrane). The nanopore can be a nanopore with an integrated sensors (e.g., tunneling electrode detectors, capacitive detectors, or graphene based nano-gap or edge state detectors (see e.g., Garaj et al. (2010) Nature vol. 67, doi: 10.1038/nature09379)). A nanopore can be functionalized for analyzing a specific type of molecule (e.g., DNA, RNA, or protein).

Nanopore sequencing can comprise "strand sequencing" in which intact DNA polymers can be passed through a protein nanopore with sequencing in real time as the DNA translocates the pore. An enzyme can separate strands of a double stranded DNA and feed a strand through a nanopore. The DNA can have a hairpin at one end, and the system can read both strands. In some cases, nanopore sequencing is "exonuclease sequencing" in which individual nucleotides can be cleaved from a DNA strand by a processive exonuclease, and the nucleotides can be passed through a protein nanopore. The nucleotides can transiently bind to a molecule in the pore (e.g., cyclodextran). A characteristic disruption in current can be used to identify bases.

[0125] Nanopore sequencing technology from GENIA can be used. An engineered protein pore can be embedded in a lipid bilayer membrane. "Active Control" technology can be used to enable efficient nanopore-membrane assembly and control of DNA movement through the channel. In some cases, the nanopore sequencing technology is from NABsys.

Genomic DNA can be fragmented into strands of average length of about 100 kb. The 100 kb fragments can be made single stranded and subsequently hybridized with a 6-mer probe.

The genomic fragments with probes can be driven through a nanopore, which can create a current-versus-time tracing. The current tracing can provide the positions of the probes on each genomic fragment. The genomic fragments can be lined up to create a probe map for the genome. The process can be done in parallel for a library of probes. A genome-length probe map for each probe can be generated. Errors can be fixed with a process termed

"moving window Sequencing By Hybridization (mwSBH)." In some cases, the nanopore sequencing technology is from IBM/Roche. An electron beam can be used to make a nanopore sized opening in a microchip. An electrical field can be used to pull or thread DNA through the nanopore. A DNA transistor device in the nanopore can comprise alternating nanometer sized layers of metal and dielectric. Discrete charges in the DNA backbone can get trapped by electrical fields inside the DNA nanopore. Turning off and on gate voltages can allow the DNA sequence to be read.

[0126] The next generation sequencing can comprise DNA nanoball sequencing (as performed, e.g., by Complete Genomics; see e.g., Drmanac et al. (2010) Science 327: 78-81).

DNA can be isolated, fragmented, and size selected. For example, DNA can be fragmented

(e.g., by sonication) to a mean length of about 500 bp. Adaptors (Adl) can be attached to the ends of the fragments. The adaptors can be used to hybridize to anchors for sequencing reactions. DNA with adaptors bound to each end can be PCR amplified. The adaptor sequences can be modified so that complementary single strand ends bind to each other forming circular DNA. The DNA can be methylated to protect it from cleavage by a type IIS restriction enzyme used in a subsequent step. An adaptor (e.g., the right adaptor) can have a restriction recognition site, and the restriction recognition site can remain non-methylated.

The non-methylated restriction recognition site in the adaptor can be recognized by a restriction enzyme (e.g., Acul), and the DNA can be cleaved by Acul 13 bp to the right of the right adaptor to form linear double stranded DNA. A second round of right and left adaptors

(Ad2) can be ligated onto either end of the linear DNA, and all DNA with both adapters bound can be PCR amplified (e.g., by PCR). Ad2 sequences can be modified to allow them to bind each other and form circular DNA. The DNA can be methylated, but a restriction enzyme recognition site can remain non-methylated on the left Adl adapter. A restriction enzyme (e.g., Acul) can be applied, and the DNA can be cleaved 13 bp to the left of the Adl to form a linear DNA fragment. A third round of right and left adaptor (Ad3) can be ligated to the right and left flank of the linear DNA, and the resulting fragment can be PCR amplified. The adaptors can be modified so that they can bind to each other and form circular DNA. A type III restriction enzyme (e.g., EcoP15) can be added; EcoP15 can cleave the DNA 26 bp to the left of Ad3 and 26 bp to the right of Ad2. This cleavage can remove a large segment of DNA and linearize the DNA once again. A fourth round of right and left adaptors (Ad4) can be ligated to the DNA, the DNA can be amplified (e.g., by PCR), and modified so that they bind each other and form the completed circular DNA template.

[0127] Rolling circle replication (e.g., using Phi 29 DNA polymerase) can be used to amplify small fragments of DNA. The four adaptor sequences can contain palindromic sequences that can hybridize and a single strand can fold onto itself to form a DNA nanoball (DNB™) which can be approximately 200-300 nanometers in diameter on average. A DNA nanoball can be attached (e.g., by adsorption) to a microarray (sequencing flowcell). The flow cell can be a silicon wafer coated with silicon dioxide, titanium and

hexamehtyldisilazane (HMDS) and a photoresist material. Sequencing can be performed by unchained sequencing by ligating fluorescent probes to the DNA. The color of the fluorescence of an interrogated position can be visualized by a high resolution camera. The identity of nucleotide sequences between adaptor sequences can be determined.

[0128] The methods provided herein may include use of a system such as a system that contains a nucleic acid sequencer (e.g., DNA sequencer, RNA sequencer) for generating DNA or RNA sequence information. The system may include a computer comprising software that performs bioinformatic analysis on the DNA or RNA sequence information. Bioinformatic analysis can include, without limitation, assembling sequence data, detecting and quantifying genetic variants in a sample, including germline variants and somatic cell variants (e.g., a genetic variation associated with cancer or pre-cancerous condition, a genetic variation associated with infection).

[0129] Sequencing data may be used to determine genetic sequence information, ploidy states, the identity of one or more genetic variants, as well as a quantitative measure of the variants, including relative and absolute relative measures.

[0130] In some cases, sequencing of the genome involves whole genome sequencing or partial genome sequencing. The sequencing may be unbiased and may involve sequencing all or substantially all (e.g., greater than 70%, 80%, 90%) of the nucleic acids in a sample.

Sequencing of the genome can be selective, e.g., directed to portions of the genome of interest. Sequencing of select genes, or portions of genes may suffice for the analysis desired. Polynucleotides mapping to specific loci in the genome that are the subject of interest can be isolated for sequencing by, for example, sequence capture or site-specific amplification.

[0131] Applications

[0132] The methods provided herein may be used for a variety purposes, such as to diagnose or detect a condition (e.g., infection), to predict that a condition will occur, to monitor treatment, to select or modify a therapeutic regimen, or to optimize a therapy. With this approach, therapeutic and/or diagnostic regimens can be individualized and tailored according to the data obtained at different times over the course of treatment, thereby providing a regimen that is individually appropriate.

Detecting/Diagnosing/Prognosing Conditions [0133] The methods provided herein may be used to detect, diagnose, or prognose infections or diseases in patient samples, particularly human blood samples. The methods may be particularly useful to detect rare microbial nucleic acid fragments in samples that are predominantly made up of human nucleic acids. For example, cell-free DNA (cfDNA) in blood consists mostly of DNA fragments derived from the host but also contains a small amount of fragments from microbes in the body. Extraction of the cfDNA followed by deep sequencing (e.g., next-generation sequencing or NGS) may generate millions or billions of sequence reads that can be mapped against host and non-host genome databases. Likewise, the methods can also be used to detect rare populations of circulating or cell-free RNA from a particular organ. In addition, the methods can be used in settings where the target nucleic acids makes up a larger portion of the total population of nucleic acids.

[0134] The methods provided herein may be used to detect, monitor, diagnose, prognose, treat, or prevent a large variety of diseases and disorders. In particular, the methods may be used to detect one or more target nucleic acids derived from a pathogen associated with an infectious disease or disorder associated with pregnancy, such as a pathogen associated with chorioamnionitis. The pathogen may be a bacterium, virus, fungus, parasite, yeast, or other microbe, particularly an infectious microbe. Exemplary pathogens that may be detected by the present methods include but are not limited to: Escherichia coli, group B streptococcus

(Streptococcus agalactiae), anaerobic bacterium, Staphylococcus aureus, cytomegalovirus

(CMV), Mycoplasma spp. (e.g., Mycoplasma hominis), Ureaplasma spp. (e.g., Ureaplasma urealyticum, Ureaplasma parvum), human immunodeficiency virus (HIV) or other lentivirus, herpes simplex, varicella, B19 erythrovirus, Toxoplasma gondii, Treponema pallidum,

Fusobacterium nucleatum, Enterococcus faecalis, Corynebacterium aurimucosum,

Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopenumoniae ,

Streptococcus pasteurianus, Sneathia sanguinegens, Candida albicans, Neisseria

gonorrhoeae, Citrobacter koseri, Peptoniphilus harei, Klebsiella pneumoniae, Micrococcus luteus, and Flaviviruses (e.g., West Nile virus, dengue virus, tick-borne encephalitis virus, yellow fever virus, Zika virus). Exemplary diseases and disorders include any disease or disorder associated with an infection, including but not limited to: toxoplasmosis, malaria, sepsis, hepatitis (e.g., Hepatitis A, B, or C), human papilloma virus (HPV) infection, chlamydial infection, syphilitic infection, Ebola infection, and/or Staphylococcus aureus infection. In cases where the subject is pregnant, the infection may occur during any period or stage of pregnancy.

[0135] In some cases, a method described herein comprises determining if an infection is active or latent. In some cases, gene expression quantification may provide a method for detecting, predicting, diagnosing, or monitoring an active infection. In some cases, a method described herein comprises detecting an active infection. In some cases, gene expression quantification may provide a method for detecting, predicting, diagnosing, or monitoring a latent infection. In some preferred cases, a method described herein comprises detecting a latent infection.

[0136] The detection of pathogen or organ nucleic acids may involve comparing a level of pathogen or organ nucleic acids with a control or reference value to determine the presence or absence of the pathogen or organ nucleic acids and/or the quantity of pathogen or organ nucleic acids. The level may be a qualitative or a quantitative level. In some cases, the control or reference value is a predetermined absolute value indicating the presence or absence of the cell-free pathogen nucleic acids or cell-free organ-derived nucleic acids. For example, detecting a level of cell-free pathogen nucleic acids above the control value may indicate the presence of the pathogen or of an infection, while a level below the control value may indicate the absence of the pathogen or of an infection. The control value may be a value obtained by analyzing cell-free nucleic acid levels of a subject without an infection; in some cases, the control value may be a positive control value and may be obtained by analyzing cell-free nucleic acids from a subject with a particular infection, or with a particular infection of a specific organ.

[0137] In some cases, in order to determine whether an infection is present or not and often to obtain a result with precision, one or more of the following methods can be applied: (i) as described in Patent WO 2015070086 Al the totality of the reads obtained by sequencing can be aligned against a curated host genome reference database, which can be from a human, dog, cat, primate or from any other host, including for example GenBank hgl9 human reference sequences; (ii) a data processor for bioinformatics analysis can subtract or sequester the host sequences so that only non-host sequences, including pathogen- related sequences, can be further analyzed; (iii) a data processor can determine the presence of one or more pathogens by aligning the non-host sequences to a curated microbial reference sequence database, including for example reference sequences from GenBank and

Refseq; (iv) a statistical analysis framework can be applied to determine whether the presence of one or more pathogens is statistically significant; and/or (v) in some instances the data processor can quantify the amount of pathogen present based on the number of reads obtained for the pathogens as compared to the number of reads obtained by control molecules spiked into the sample at a known concentration before sequencing.

[0138] The control value may be a level of cell-free pathogen or organ-specific nucleic acids obtained from the subject (e.g., pregnant woman or woman contemplating pregnancy) at a different time point, such as a time point prior to the test time point. In such cases, comparison of the level at different time points may indicate the presence of infection, presence of infection in a particular organ, improved infection, or worsening infection. For example, an increase of cell-free pathogen nucleic acids by a certain amount over time may indicate the presence of infection or of a worsening infection, e.g., an increase of pathogen or organ-specific cell-free nucleic acids of at least 5%, 10%, 20%, 25%, 30%, 50%, 75%, 100%), 200%), 300%), or 400% compared to an original value may indicate the presence of infection, or of a worsening infection. In other examples, a reduction of pathogen or organ- specific cell-free nucleic acids by at least 5%, 10%, 20%, 25%, 30%, 50%, 75%, 100%, 200%), 300%), or 400% compared to an original value may indicate the absence of infection, or of an improved infection. Often, such measurements may be taken over a particular time period, such as every day, every other day, weekly, every other week, monthly, or every other month. For example, an increase of pathogen or organ cell-free nucleic acids of at least 50%) over a week may indicate the presence of infection.

[0139] Control or reference values may be measured as a concentration or as a number of sequencing reads. Control or reference values may be pathogen-dependent. For example, a control value for Escherichia coli may be different than a control value for Mycoplasma hominis. A database of levels or control values may be generated based on samples obtained from one or more subjects, for one or more pathogens, for one or more organs, and/or for one or more time points. Such a database may be curated or proprietary. Recommended treatment options may be based on different threshold levels. For instance, a low level may signify infection but treatment may not be necessary; a moderate level may lead to antibiotic treatment; and a high level may require immediate or serious intervention.

[0140] In some cases, analytical methods can be applied in order to obtain improved or more accurate identification of taxa. For example, control samples may be sequences in parallel with test samples in the methods provided herein. Based on sequence reads from the control samples, a baseline level of taxa (or pathogen species, variants, etc.) that may be introduced from the laboratory environment may be determined. A Poisson model or other statistical model may be used to determine whether the baseline level is significantly higher than the test samples (e.g., clinical samples). In some cases, the control is water, reagent. In some cases, the control sample may match the test sample in that they are both blood samples, plasma cells, urine samples, etc. A threshold value may be set using sequence reads from the control samples and may be compared with samples from subjects. In some cases, the threshold may be used to determine taxa that are significantly enriched above the threshold in samples from subjects. In some cases, the threshold may be used to ensure even distribution of sequence reads across reference genome database. Even distribution of sequence reads may ensure accurate identification of taxa, especially in cell-free nucleic acid samples. In some cases, the threshold can be used to make sure extra taxa were not called due to genetic similarity between two taxa. For example, if taxa A is genetically similar to taxa B, then in some instances taxa A may be called as taxa B. In one such instance, sequence reads of taxa A that are similar to sequence reads of taxa B may be overrepresented and may lead to calling taxa A and taxa B.

[0141] Analytical methods can include identification of a set of pathogens such as commensal microorganisms or natural microflora that are or are not causative of an infection using control samples from healthy individuals. In some cases, such pathogens may be from the vagina, gut, or skin. A threshold value can be set based on the set of commensal microorganisms in control samples. The threshold can be used to determine a level at which a clinical sample can be called positive for infection. A Poisson model can be used to determine significant enrichment above the threshold in a clinical sample. In some cases, the threshold value may need to be adjusted, especially when an infection is caused by multiple pathogens within a collection instead of a single pathogen. In such cases, joint distributions of pathogen abundances can be used to adjust the threshold. For example, the adjusted threshold may be lowered in an infection caused by multiple pathogens when compared with an infection caused by a single pathogen. The adjusted threshold can be used to determine if a clinical sample is indicative of an infection based on joint distributions of certain pathogens in the collection or the group.

[0142] To distinguish taxa that are most likely to be causing infection from taxa that are not such as pathogen taxa from vagina, gut or skin, one or more filters may be applied to data obtained using the methods provided herein. In some cases, the method may involve removal of samples or data relating to samples that are dominated by vaginal or gut commensal microbes that likely entered cord blood sample during collection. Specifically, samples (or associated data) with a certain number of different pathogen taxa above a threshold value may be removed from the analysis. In some cases, the threshold value may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, or more.

[0143] The methods provided herein may enable the generation of sequencing data that detects a pathogenic infection with high efficiency, high accuracy, and/or high sensitivity. Often, such methods may detect a pathogen or infection that is not detected or detectable by other methods, such as blood culture, amniocentesis, chorionic villus sampling, or polymerase chain reaction (PCR). The methods generally may have a high sensitivity, e.g., a sensitivity of greater than 80%, 85%, 90%, 95%, 99%, or 99.5%. The methods generally may have a low false positive rate, e.g., a false positive rate of less than 50%, 35%, 25%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.05%, or 0.01%.

[0144] The methods provided herein may provide high specificity, high sensitivity, high positive predictive value, and/or low negative predictive value. The methods provided herein may provide a specificity (or negative percent agreement) and/or sensitivity (or positive percent agreement) that is at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. In some cases, the nominal specificity is greater than or equal to 70%. The nominal negative predictive value (NPV) is greater than or equal to 95%. In some cases, the NPV is at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or more.

[0145] Sensitivity, Positive Percent Agreement (PPA), or true positive rate (TPR) may refer to an equation of TP/(TP+FN) x 100 or TP/(total number of infected subjects), where TP is the number of true positives and FN is the number of false negatives. When calculating the denominator for the previous equations, the value can reflect the total number of infection results based on a particular independent method of detecting infection (e.g., blood culture or PCR, cord-blood culture or PCR, histological analysis of uterine membranes after delivery).

[0146] Specificity, Negative Percent Agreement or true negative rate may refer to an equation such as TN/(TN+FP) x 100 or TN/(total number of uninfected subjects), where TN is true negative and FP is false positive. When calculating the denominator for the previous equations, the value can reflect the total number of actual "non-infections" as determined by an independent method of detecting infection (e.g., blood culture or PCR, cord-blood culture or PCR, histological analysis of uterine membranes after delivery).

[0147] In some cases, the sample is identified as infected with an accuracy of greater than 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more. In some cases, the sample is identified as infected with a sensitivity of greater than 95%. In some cases, the sample is identified as infected with a specificity of greater than 95%. In some cases, the sample is identified as infected with a sensitivity of greater than 95% and a specificity of greater than 95%. In some cases, the accuracy is calculated using a trained algorithm. The diagnosis accuracy as used herein includes specificity, sensitivity, positive predictive value, negative predictive value, and/or false discovery rate. In some cases, a method described herein has a specificity or sensitivity of greater than 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%), 96%), 97%), 98%), 99%, or 99.5%, or a positive predictive value or negative predictive value of at least 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5% or more.

[0148] When classifying a sample for diagnosis of infection, there are typically four possible outcomes from a binary classifier. If the outcome from a prediction is p and the actual value is also p, then it is called a true positive (TP); however, if the actual value is n then it is said to be a false positive (FP). Conversely, a true negative has occurred when both the prediction outcome and the actual value are n, and false negative is when the prediction outcome is n while the actual value is p. For a test that detect a disease or disorder such an infection, a false positive in this case may occur when the subject tests positive, but actually does not have the infection. A false negative, on the other hand, may occur when the subject actually does have an infection but tests negative for such infection.

[0149] The positive predictive value (PPV), or precision rate, or post-test probability of disease, is the proportion of patients with positive test results who are correctly diagnosed. It may be calculated by applying the following equation: PPV= TP/(TP+FP) x 100. The PPV may reflect the probability that a positive test reflects the underlying condition being tested for. Its value does however may depend on the prevalence of the disease, which may vary. The Negative Predictive Value (NPV) can be calculated by the following equation:

TN/(TN+FN) x 100. The negative predictive value may be the proportion of patients with negative test results who are correctly diagnosed. PPV and NPV measurements can be derived using appropriate disease prevalence estimates.

[0150] In some cases, the results of the sequencing analysis of the methods described herein provide a statistical confidence level that a given diagnosis is correct. In some cases, such statistical confidence level is above 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99% or 99.5%.

Monitoring and Treating

[0151] The methods may include monitoring whether a subject has a pregnancy -related infection over time. For example, samples may be collected serially at various times before or during the course of her pregnancy in order to determine the presence or absence of an infection. In other examples, the methods may include monitoring the course of an infection over time. In such cases, samples may be collected serially at various time points during the course of pregnancy; in some cases, the serially-collected samples are compared to each other to determine whether the infection is improving or worsening.

[0152] The methods provided herein include methods of treating a subject, e.g., a pregnant woman or woman contemplating pregnancy. The treatment may reduce, prevent or eliminate an infection in the subject in order to prevent transmission to the fetus, fetal membranes, placenta or neonate. In some cases, the treatment may reduce, prevent or eliminate infection and/or inflammation in fetal membranes (e.g., amnion, chorion), the placenta, the fetus or the neonate. The methods may include methods to treat, prevent, reduce or eliminate intra-uterine inflammation and/or infection of any type including:

chorioamnionitis, acute chorioamnionitis, sub-clinical chorioamnionitis, congenital fetal infection, and inflammation due to congenital fetal infection. In many cases, the methods provided herein may prevent, detect, or treat fetal inflammatory response syndrome (FIRS), which is generally system inflammation caused by the fetus being in direct contact with infected amniotic fluid and/or inflammatory cell transfer from the uteroplacental circulation. The FIRS may be clinical FIRS in which the fetal plasma interleukin-6 level is >1 lpg/ml. The FIRS may be subclinical FIRS, defined histologically by funisitis and fetal vasculitis. The treatment may be given to the pregnant woman or woman contemplating pregnancy and/or to the neonate after birth.

[0153] The treatment may involve administering a drug or other therapy to reduce or eliminate the inflammation and/or the infection, e.g., administration of drug or other therapy to reduce or eliminate intrauterine inflammation (e.g., inflammation in the fetus, amnion, chorion, and/or placenta) or intrauterine infection (e.g., infection of the fetus, amnion, chorion, and/or placenta). In some cases, the pregnant subject or subject contemplating pregnancy is treated prophylactically with a drug, e.g., to prevent development of an intrauterine infection, intra-uterine inflammation or maternal-fetal transmission of an infection.

[0154] Any therapy (including a drug) to improve or reduce the symptoms of an infection or inflammation may be administered to the pregnant subject or subject

contemplating pregnancy. Exemplary drugs include but are not limited to antibiotics, antiviral medication, ampicillin, sulbactam, penicillin, vancomycin, gentamycin,

aminoglycoside, clindamycin, cephalosporin, metronidazole, timentin, ticarcillin, clavulanic acid, cefoxitin, antiretroviral drugs (e.g., highly active antiretroviral therapy (HAART), reverse transcriptase inhibitors, nucleoside/nucleotide reverse transcriptase inhibitors ( RTIs), Non-nucleoside RT inhibitors, and/or protease inhibitors), and immunoglobulins.

[0155] The methods may include methods of adjusting a therapeutic regimen. For example, the subject may have a known infection and may have been administered a drug to treat the infection. The methods provided herein may be used to track or monitor the efficacy of the drug treatment. In some cases, the therapeutic regimen may be adjusted, depending on the results of such monitoring. For example, if the methods provided herein indicate that an infection is not improving as a result of the drug treatment, the therapeutic regimen may be adjusted by changing the type of drug or treatment given to the patient, discontinuing use of the previous drug, continuing use of the drug, increasing the dose of a drug treatment, or adding a new drug or other treatment to the subject's therapeutic regimen. In some cases, the therapeutic regimen may involve a particular procedure. For example, in some cases, the methods may indicate a need to induce labor. In some cases, the therapeutic regimen may involve early pregnancy termination (e.g., if the methods provided herein detect a congenital infection with serious or irreversible fetal sequelae). Likewise, if the methods indicate than an infection is improving or resolved, the adjusting may involve reducing or discontinuing the drug treatment.

[0156] The methods provided herein enable early detection and treatment of pregnancy- associated infectious disorders such as chorioamnionitis and congenital infection, and therefore may increase the duration of pregnancy and reduce the risk of preterm birth or premature labor. Preterm birth generally refers to infants delivered between 32 weeks 0 day and 35 weeks 6 days of gestation.. Marginal preterm births are of infants delivered between 36 weeks 0 days and 36 weeks 6 days of gestation. Infants born very preterm are delivered before 32 completed weeks (less than or equal to 31 weeks and 6 days); and infants born extremely preterm are delivered before 28 completed weeks of gestation. The methods provided herein may reduce the risk of preterm birth at any or all stages including reduction of the risk of preterm birth, marginal preterm birth, very preterm birth, and/or extremely preterm birth.

[0157] The methods provided herein may also increase the latency time in PPROM, e.g., the length of time from rupture of the membranes to delivery. In some cases, the latency time may increase by about or at least about 1, 2, 3, 4, 5 or more weeks.

[0158] The methods provided herein generally may result in increasing the gestational age at birth for the neonate and thus decreasing risk of neonatal complications as well as complications later in life associated with preterm birth. The complications may be acute, sub-acute, sub-clinical or chronic. In some cases, the methods reduce the risk of adverse infant outcomes such as stillbirth, premature birth, neonatal sepsis, injury to the nervous system, respiratory system, immune system, cardiovascular system, visual system and/or auditory system. Injuries to the nervous system due to chorioamnionitis, congenital fetal infections or preterm birth may include brain injury that may lead to cerebral palsy and other neurodevelopmental disabilities. In some examples, nervous system injury may include intraventricular hemorrhage, autism spectrum disorder, and neuro-cognitive difficulties. Respiratory system injury may result in respiratory distress system, persistent pulmonary hypertension of the newborn (PPHN), asthma, chronic lung disease, or other lung ailment. Cardiovascular injury may include heart damage, lowered blood pressure, increased left ventricular compliance/dilatation or other cardiac injury. Visual injury may include visual impairment, retinopathy of prematurity, or other visual disease or disorder.

[0159] A method described herein may further comprise RNA sequencing (RNA-Seq) or be combined with a method comprising RNA-Seq. Tissue damage or infection may lead to release of cell-free nucleic acids from a particular organ or tissue. For example, RNA may be released by apoptotic cells in tissues. RNA-Seq of cell-free RNA can indicate the health or status of different tissues in the body, including the uterus.

[0160] A method comprising RNA sequencing may enable detection of a specific organ or tissue that is infected and may be used to detect or monitor the health of an organ such as the uterus or the health of a fetus or neonate. RNA-Seq may be used independently to investigate uterine or fetal health or may provide increased confidence that an infection detected by a method described herein is an infection of the uterus. In some cases, an RNA- Seq test may be able to determine if an infection detected by a method described herein is a maternal infection versus a fetal infection. The RNA-Seq test may be conducted

contemporaneously with a method to detect an infection, subsequent to a method to detect an infection, or prior to a method detect and infection.

[0161] There are many potential scenarios in which a method to detect a pathogen provided herein may be combined with a method to detect the site of infection by RNA sequencing of cell-free RNA in a body fluid. For example, a method provided herein may be used to detect circulating cell-free nucleic acids from a pathogen associated with

chorioamnionitis. The method may further comprise conducting an RNA-Seq test to detect an increase in uterine cell-free RNA in the subject's blood. The combination of test results may indicate that the pathogen has infected the uterus and may even be able to determine which uterine tissue is infected (e.g., chorionic membrane, amniotic membrane, placenta). However, if an RNA-Seq test detects an increase in cell-free RNA from another organ such as the lung, the test may indicate that there is likely no uterine infection, despite positive detection of a pathogen by a method described herein; in such a case, a fetus in the pregnant subject may have a reduced risk of infection. In another scenario, a method provided herein may detect a pathogen known to infect fetal tissue. For example, the method may comprise detecting such pathogen (e.g., Zika virus) by analyzing circulating cell-free pathogen nucleic acids and further comprise conducting an RNA-Seq assay to detect whether there is a rise in circulating cell-free fetal RNA (e.g., fetal brain RNA). The health of the fetus may be in particular danger if Zika cell-free nucleic acids are detected in conjunction with rising levels of circulating cell-free fetal RNA, particularly fetal brain RNA.

[0162] An RNA-Seq test (or series of RNA-Seq tests) may sometimes be performed after a method described herein produces a positive test result (e.g., detection of a pathogen infection). The RNA-Seq test may be especially useful for confirming the infection or for identifying the location of the infection. For example, the methods may detect the presence of a pathogen in a pregnant subject by analyzing circulating cell-free nucleic acids, but the site of infection may be unclear. In such case, the method may further comprise sequencing cell -free RNA from the pregnant subject to confirm that the infection is within a uterus (such as by detection of increased levels of circulating cell-free RNA derived from uterine tissues or fetal tissues (e.g., fetal brain)). Conversely, the presence of increased levels of circulating cell -free RNA from a different organ such as the lungs of the pregnant subject may indicate that the detected pathogen has not infected the uterine tissues (e.g., chorion, amnion, placenta) or fetal tissues. In either case, the RNA sequencing test may then be repeated over time to determine whether the infection is worsening or improving in a particular organ or tissue, or whether it is spreading to different organs or tissue. Likewise, the pathogen detection assay may also be repeated over time.

[0163] In some cases, a method of detecting a pathogen described herein is conducted following the performance of an RNA-Seq test. For example, an increase in plasma levels of cell-free RNA associated with the uterus may indicate a uterine disorder such as a uterine infection. In such case, the method may further comprise detecting levels of circulating cell- free nucleic acids associated with intra-uterine infection.

[0164] A method provided herein may also be combined with a method to detect cell-free fetal DNA, particularly cell-free fetal DNA in a body fluid (e.g., blood, urine, plasma, serum). The cell-free fetal DNA test may detect chromosomal abnormalities in the fetus (e.g., chromosomal aneuploidy in chromosome 13, 18 or 21). It may also detect fetal injury, in the context of rising circulating cell-free fetal DNA.

[0165] A method described herein may be repeated, for example, to monitor an infection, treatment, or pregnancy over time. A method described herein may be repeated every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days; every 1, 2, 3, 4, 5, or 6 weeks; every 1, 2, 3, 4, 5, 6, 7, 8, or 9 months; every trimester or at routine prenatal examinations.

[0166] In some cases, when a method described herein gives a negative test result (e.g., no pathogen is detected), a method can be repeated serially over time to monitor pathogen nucleic acids in a pregnant subject or subject contemplating pregnancy. In some cases, the RNA-Seq assay is also repeated serially over time following a negative pathogen test result or negative RNA-Seq result.

[0167] In some cases, when a method described herein gives a positive test result (e.g., detection of a pathogen), a therapeutic regimen can be administered to the pregnant subject or subject contemplating pregnancy. A therapeutic regimen can include, but is not limited to, drug administration, antibiotic administration, pregnancy induction, pregnancy termination, and early pregnancy termination when irreversible sequelae of the fetus are present.

[0168] In some cases, when a method described herein gives a positive test result, a method or test can be repeated serially over time to monitor the course of infection. For example, a therapeutic regimen can be adjusted depending on upward or downward course of infection. In other cases, no therapeutic regimen may be conducted initially; for example, the infection may be monitored with a "watchful waiting" or "watch and wait" approach to see if the infection clears up without additional medical intervention. In some cases, when a method described herein gives a positive test result, a drug can be administered and the course of infection can be monitored to detect how well the drug is working or when to stop drug treatment. In some cases, the therapy can be altered as needed.

[0169] The methods described herein can detect an infection of the pregnant subject or subject contemplating pregnancy. In some cases, the infection is associated with pregnancy.

In other cases, the infection is unrelated to the pregnancy. In some cases, the infection is bacterial or viral. An infection of the pregnant subject or subject contemplating pregnancy can be monitored to see if the infection spreads to intra-uterine tissues. Similarly, an infection of the pregnant subject may be monitored to determine whether the infection has spread, or been transmitted to the fetus. In some cases, the methods provided herein can detect one or more infections of the pregnant subject or subject contemplating pregnancy, one or more intrauterine infections, and/or one or more fetal or neonatal infections. In some cases, the methods provided herein can detect one, two, or three of these conditions, in any combination.

[0170] Methods described herein can detect Zika virus. Zika virus, which is a Flavivirus, can be transmitted through mosquito bites (e.g., from an Aedes species mosquito such as Ae. aegypti or Ae. Albopictus), from a pregnant subject to a fetus, and through sex. Zika infection during pregnancy can cause fetal brain defects, including microcephaly, and other fetal birth defects such as defects of the eye, hearing deficits, and impaired growth. There have also been increased reports of Guillain-Barre syndrome in adults and other developmental disorders in newborns affected by Zika virus in the womb. Microcephaly is a condition in which the brain has not developed properly during pregnancy or has stopped growing after birth, resulting in a smaller head size. Causes of microcephaly include vertically transmitted infections such as congenital cytomegalovirus infection, toxoplasmosis, congenital rubella syndrome, and Zika virus.

[0171] Provided herein are methods of detecting a Zika virus infection in a pregnant subject or subject contemplating pregnancy comprising: (a) obtaining a sample comprising cell -free nucleic acids from a pregnant subject or subject contemplating pregnancy suspected of having an infection; (b) conducting a high-throughput sequencing assay on the cell-free nucleic acids to detect one or more Zika virus nucleic acids; and (c) quantifying an amount of the one or more Zika virus nucleic acids. In some cases, the method further comprises administering a therapeutic regimen to the pregnant subject or subject contemplating pregnancy. In some cases, the method further comprises performing an RNA-Seq test. In some cases, the RNA-Seq test is used to detect fetal tissue or organ RNA in maternal blood. In some cases, the fetal organ is the fetal brain or fetal optic nerve.

[0172] Computer control systems

[0173] The present disclosure provides computer control systems that are programmed to implement methods of the disclosure. FIG. 2 shows a computer system 201 that is programmed or otherwise configured to implement methods of the present disclosure.

[0174] The computer system 201 includes a central processing unit (CPU, also

"processor" and "computer processor" herein) 205, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 201 also includes memory or memory location 210 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 215 (e.g., hard disk), communication interface 220

(e.g., network adapter) for communicating with one or more other systems, and peripheral devices 225, such as cache, other memory, data storage and/or electronic display adapters. The memory 210, storage unit 215, interface 220 and peripheral devices 225 are in communication with the CPU 205 through a communication bus (solid lines), such as a motherboard. The storage unit 215 can be a data storage unit (or data repository) for storing data. The computer system 201 can be operatively coupled to a computer network

("network") 230 with the aid of the communication interface 220. The network 230 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in

communication with the Internet. The network 230 in some cases is a telecommunication and/or data network. The network 230 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 230, in some cases with the aid of the computer system 201, can implement a peer-to-peer network, which may enable devices coupled to the computer system 201 to behave as a client or a server.

[0175] The CPU 205 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 210. The instructions can be directed to the CPU 205, which can subsequently program or otherwise configure the CPU 205 to implement methods of the present disclosure. Examples of operations performed by the CPU 205 can include fetch, decode, execute, and writeback.

[0176] The CPU 205 can be part of a circuit, such as an integrated circuit. One or more other components of the system 201 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0177] The storage unit 215 can store files, such as drivers, libraries and saved programs. The storage unit 215 can store user data, e.g., user preferences and user programs. The computer system 201 in some cases can include one or more additional data storage units that are external to the computer system 201, such as located on a remote server that is in communication with the computer system 201 through an intranet or the Internet.

[0178] The computer system 201 can communicate with one or more remote computer systems through the network 230. For instance, the computer system 201 can communicate with a remote computer system of a user (e.g., healthcare provider). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 201 via the network 230.

[0179] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 201, such as, for example, on the memory 210 or electronic storage unit 215. The machine executable or machine readable code can be provided in the form of software.

During use, the code can be executed by the processor 205. In some cases, the code can be retrieved from the storage unit 215 and stored on the memory 210 for ready access by the processor 205. In some situations, the electronic storage unit 215 can be precluded, and machine-executable instructions are stored on memory 210.

[0180] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0181] Aspects of the systems and methods provided herein, such as the computer system 201, can be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture" typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0182] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD- ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0183] The computer system 201 can include or be in communication with an electronic display 235 that comprises a user interface (UI) 240 for providing, an output of a report, which may include a diagnosis of a subject or a therapeutic intervention for the subject. Examples of UFs include, without limitation, a graphical user interface (GUI) and web-based user interface. The analysis can be provided as a report. The report may be provided to a pregnant subject or subject contemplating pregnancy, to a health care professional, a lab- worker, or other individual.

[0184] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 205. The algorithm can, for example, facilitate the enrichment, sequencing and/or detection of pathogen nucleic acids.

[0185] Information about a patient or pregnant subject or subject contemplating pregnancy can be entered into a computer system, for example, a patient identifier such as information about pregnancy status, patient background, patient medical history, previous pregnancies, or ultrasound scans. Patient identifiers can be separated from clinical samples to obtain de-identified samples, for example by the sample sender or the sample recipient.

Patient identifiers can be replaced with accession numbers or other non-individual identifying code. Clinical samples can be sequenced using a high-throughput sequencer. De- identified sample sequence data generated by sequencer can be uploaded to a server, such as a cloud server. Using methods disclosed herein, pathogen nucleic acids within de-identified samples can be detected to obtain de-identified result data. De-identified result data can be downloaded from the server. The de-identified result data can be associated with patient identifiers, for example by the sample sender or the sample recipient. An electronic report can be generated to indicate presence or absence of pathogen. An electronic report can be generated to indicate prognosis. An electronic report can be generated to present diagnosis. If an electronic report indicates a positive test for a treatable condition or infection, the electronic report can be generated to prescribe a therapeutic regimen or a treatment plan. The computer system can be used to analyze results from a method described herein, report results to a patient or doctor, or come up with a treatment plan.

[0186] Reagents and Kits

[0187] Also provided are reagents and kits thereof for practicing one or more of the methods described herein. The subject reagents and kits thereof may vary greatly. Reagents of interest include reagents specifically designed for use in identification, detection, and/or quantitation of one or more pathogen nucleic acids in a sample obtained from a pregnant subject or subject contemplating pregnancy. The kits may comprise reagents necessary to perform nucleic acid extraction and/or nucleic acid detection using the methods described herein such as PCR and sequencing. The kit may further comprise a software package for data analysis, which may include reference profiles for comparison with the test profile, and in particular may include reference databases. The kits may comprise reagents such as buffers and water.

[0188] Such kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such kits may also include instructions to access a database. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

[0189] As used throughout the specification herein, the term "about" when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and the number or numerical range may vary from, for example, from 1% to 15% of the stated number or numerical range. In examples, the term "about" refers to ±10% of a stated number or value.

[0190] As used herein, the term "or" is used to refer to a nonexclusive or, such as "A or B" includes "A but not B," "B but not A," and "A and B," unless otherwise indicated.

[0191] As used herein, the terms "treat," "ameliorate," "treatment," and "treating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including, but are not limited to, therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit means eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder. For prophylactic benefit, treatment may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0192] Examples

[0193] Example 1 : Chorioamnionitis detection and treatment (prophetic example)

[0194] A pregnant woman is hospitalized for premature rupture of the membranes

(PPROM), defined as rupture of the membranes before 37 weeks of pregnancy, with or without other clinical symptoms of infection, such as fever. A blood draw is taken using 1 x

6 mL K2 EDTA tube (BD Vacutainer) and/or Plasma Preparation Tubes (PPT™, BD

Vacutainer) and/or 1 x 10 mL cell-free DNA BCT tube (Streck) and/or 1 x 10 mL cell-free

RNA BCT tube (Streck). Tubes are centrifuged within 24 h from the blood draw, according to manufacturer instructions, in order to separate plasma from blood cells. Plasma is collected and aliquoted in 1.5 mL polypropylene cryovial tubes and sent at room temperature to a processing laboratory to perform a method described herein, such as a sequencing assay to detect circulating cell-free pathogen DNA and/or circulating cell-free organ RNA.

[0195] While waiting for the results from the method, the pregnant woman is monitored for signs of infection and/or started under a broad-spectrum regimen of antibiotics if clinical signs of infection appear. When results from the method are obtained, the pregnant woman may (i) continue to be monitored clinically and with serial blood draws if the result is initially negative or (ii) if the test is positive, be treated by a targeted antibiotic therapy (or the initially prescribed broad-spectrum antibiotic therapy can be restricted to target the pathogens identified by the test).

[0196] A targeted antibiotic treatment is expected to increase the latency period, e.g., period between the PPROM and delivery, increasing the gestational age of the fetus at birth and decreasing neonatal complications associated with a preterm birth. A targeted antibiotic treatment may also be expected to be more effective and better tolerated with fewer side effects for the pregnant woman and the fetus. In addition, treating a patient with a targeted antibiotic therapy instead of broad-spectrum antibiotics promotes antibiotic stewardship, which is particularly important with increasing antimicrobial resistances (AMR). AMR are particularly concerning as they can jeopardize the future of the treatment of infectious diseases at large if pathogens develop resistances against many or all of the currently available antimicrobials.

[0197] Plasma processing and DNA extraction: Plasma is extracted from whole blood samples within 24 hours of sample collection, as previously described (Fan HC et al., PNAS 2008; 105(42): 16266-16271), and is stored at -80 °C. When required for analysis, plasma samples are thawed and circulating DNA is immediately extracted from 0.5-1 ml plasma.

[0198] Sequencing library preparation and sequencing: Sequencing libraries are prepared from the purified patient plasma DNA using the NEBNext DNA Library Prep Master Mix Set for Ulumina with standard lllumina indexed adapters (purchased from IDT), or using a microfluidics-based automated library preparation platform (Mondrian ST, Ovation SP Ultralow library system). Libraries are characterized using the Agilent 2100 Bioanalyzer (High sensitivity DNA kit) and quantified by qPCR.

[0199] Two control experiments can be performed to test for the presence of potential contaminants in the reagents used for DNA extraction and sequencing library preparation. In the first, two samples are prepared with a known template (Lambda gDNA, Pacbio Part no:

001 -1 19- 535), and purified DNA for sequencing using the above-described workflow

(lllumina Miseq, 3.4 and 3.5 million reads). Lambda-derived sequences are removed and the remaining sequences (0.4%) are aligned to reference database using BLAST. No evidence is found for the various infectious agents, but sequences related to the Enterobacteriaceae bacterial family (phylum Proteobacteria), primarily E. coli (> 97%) and enterobacterial phages (<1 %) are detected, which are likely a remnant of the lambda DNA culture. In a second control, a sample for sequencing is prepared from nuclease-free water. The sample is included in a sequencing run along with a sample unrelated to this work and recruited only a limited number of sequences, 15 in total, which mapped to genomes of two bacterial species. Again, no evidence is found for the infectious agents that are discussed herein.

[0200] qPCR Validation of Sequencing Results for Selected Bacterial Targets. Standard qPCR kits for the quantification of selected bacterial targets (e.g., E. coli) are used to validate the sequencing results for a subset of cell-free DNA samples. qPCR assays are run on cfDNA extracted from ~1 ml of plasma and eluted in a 100 ml Tris buffer (50 mM [pH 8.1-8.2]). The plasma extraction and PCR experiments are performed in different facilities. No-template controls are run to verify that the PCR reagents are included in every experiment.

[0201] No-Template Control. A no-template control experiment is performed. A sequencing library is prepared together with 7 additional sample libraries (cell-free human DNA) to test for possible sample-to-sample crosstalk during library preparation. To ensure formation of clusters with sufficient density on the Ulumina flow cell, the sample is sequenced together with a sample unrelated to the study. Whereas the sample unrelated to the study recruited 16 million reads, the no- template control library generated just 15 reads that mapped to two species in the reference database, the Methanocaldococcus janaschii (9 hits) and Bacillus subtilis (5 hits) genomes. No evidence is found for human related sequences, indicating that sample-to-sample contamination is low.

[0202] Example 2: Chorioamnionitis detection study

[0203] A study was conducted to evaluate whether the methods could detect pathogens in plasma obtained from cord blood and/or matched maternal samples in cases of preterm births presenting in labor and delivery with or without signs and symptoms of clinical chorioamnionitis. Secondary objectives of this study include an evaluation of the amount of cell free fetal DNA (cff DNA) sequenced concurrently and its potential correlation with pregnancy and neonatal outcomes. Chorioamnionitis is an infection of the chorion, the outer membrane of the placenta, and/or of the amnion, the fluid-filled sac surrounding the fetus. When characteristic clinical signs are present, the condition is referred to as clinical chorioamnionitis. The key clinical findings associated with clinical chorioamnionitis include fever, uterine fundal tenderness, maternal tachycardia (>100/min), fetal tachycardia (>160/min) and purulent or foul amniotic fluid. However, some of these clinical signs are non-specific and could be related to a cause other than chorioamnionitis (e.g. mild fever elicited by epidural anesthesia, other cause of infection etc.). The diagnostic can be confirmed by pathologic findings on microscopic examination of the placenta that encompasses clinically unapparent (sub-clinical) chorioamnionitis as well as clinical chorioamnionitis. Acute chorioamnionitis is the most common lesion reported in the placenta after spontaneous preterm birth. The histopathological features include amniotropic infiltration by both maternal and fetal neutrophils. Chronic chorioamnionitis is defined by the infiltration of lymphocytes in the chorioamniotic membranes and the chorionic plate, similar to that of neutrophils in acute chorioamnionitis. Funisitis, also a histopathologic diagnosis, is the extension of infection or inflammation to the umbilical cord.

[0204] Study Design and Methods

[0205] The study involved matched cord blood plasma and maternal plasma collected during labor and delivery in preterm labor (PTL) and at term cases. Participants presenting at <37 weeks of gestational age (GA) in labor and delivery were enrolled in the study as well as matched at term controls (> 37 weeks of GA). Participants were assessed clinically and stratified by the presence of clinical signs of chorioamnionitis. Participants presenting at term were enrolled as negative controls. It is not standard of care to confirm chorioamnionitis cases using microbiological tests as micro-organisms implicated in these infections are often hard to culture and the clinical symptoms resolve spontaneously in most of the cases after delivery. However, due to the high risk of neonatal sepsis related morbidity and mortality, mother and newborn are usually started on antibiotic treatment based on clinical signs of chorioamnionitis, without waiting for microbiological confirmation. This can result in overuse of antibiotics and especially broad-spectrum antibiotics.

[0206] A maternal blood sample was obtained during labor or at delivery or shortly after delivery (in K2-EDTA tubes) and cord blood was obtained immediately after delivery by milking the cord into a sterile container after cesarean deliveries and subsequently aliquoted in K2-EDTA tubes or through needlestick of the cord after vaginal deliveries and directly collected into a K2-EDTA tube. Maternal blood and cord blood samples were centrifuged at 1600 x g onsite within 24h from collection to separate maternal blood plasma and cord blood plasma, respectively. Maternal blood plasma and cord blood plasma were collected, frozen and stored at -80°C. Maternal blood plasma and cord blood plasma were centrifuged at 16,000 x g to to collect cell-free maternal plasma and cell-free cord blood plasma, respectively. Cell-free nucleic acids were extracted from plasma samples using an Omega Mag-Bind™ cfDNA kit.

[0207] Cell-free DNA was sequenced using DNA sequencing assay described herein.

Each sequencing run contained both negative and positive control samples. After removing low-quality reads, reads were mapped to the human reference genome. Remaining reads, presumed to be microbiome-derived, were mapped to a proprietary database of viral, bacterial, yeast, fungal and other eukaryote genomes. Organisms with over-represented sequences were reported as positive.

[0208] Infections were clinically confirmed without any microbiological confirmation since it is not the standard of care to obtain microbiological confirmation. Some suspected chorioamnionitis cases and most of PTL cases had histological confirmation. Some of the samples comprised frozen plasma samples whereas others, including the confirmed chorioamnionitis cases, were paraffin-embedded samples.

[0209] Quality control (QC) measures included adding an ID-spiked-in synthetic nucleic acid, which is a type of spike-in that is unique for each sample in a sequencing batch, and other synthetic nucleic acid spike-ins ("SPANK molecules") which are spiked in at a constant concentration across all libraries. Thus, the number of deduped SPANK molecules detected in a particular library is a proxy for the minimum concentration detectable in that library. This can be useful for setting a threshold based on minimum concentration of the SPANK molecules detectable in that library. The threshold can be useful to ensure sufficient sequencing depth for detection of pathogen. The threshold can also be useful in making sure that pathogen signal was not due to cross contamination from other samples. For example, enrichment of pathogens relative to the threshold set by the SPANK molecules can be compared between different samples. More generally, it is proportional to the efficiency with which that library converted DNA molecules in the original sample to reads in the DNA sequencing data. The purpose of the SPANK molecules is to help establish the relative abundance of the pathogen molecules within the mixture represented in a specimen, reported as "molecules per ml" (MPM). MPM data was used to build heatmaps and correlation plots. Sample Purity Ratio (SPR) aims to capture how significant the number of taxon-associated reads is given the estimated degree of cross-contamination in the sample. In case of failure of deduped SPANK and/or SPR, the sample was re-queued and re-run once. If QC failed twice on the same sample, the report was "no result."

[0210] Analytical methods can be applied in order to obtain improved accurate identification of taxa. For example, control samples for sequencing were prepared using nuclease-free water. Control samples were included in a sequencing run along with clinical samples from subjects. Based on sequence reads from the control samples, a baseline level of taxa that were introduced from the laboratory environment was determined. A Poisson model was used to determine whether the baseline level was significantly higher than clinical samples. A threshold was set using sequence reads from the control samples and was compared with samples from subjects. In some cases, the threshold was used to determine taxa that were significantly enriched above the threshold in samples from subjects. In some cases, the threshold was used to ensure even distribution of sequence reads across reference genome database. Even distribution of sequence reads can ensure accurate identification of taxa, especially in cell-free nucleic acid samples. In some cases, the threshold can be used to make sure extra taxa were not called due to genetic similarity between two taxa. For example, if taxa A is genetically similar to taxa B, then in some instances taxa A may be called as taxa B. In one such instance, sequence reads of taxa A that are similar to sequence reads of taxa B may be overrepresented and may lead to calling taxa A and taxa B.

[0211] Analytical methods can include identification of a set of commensal

microorganisms or natural microflora that are or are not causative of an infection using control samples from healthy individuals. A threshold can be set based on the set of commensal microorganisms in control samples. The threshold can be used to determine a level at which a clinical sample can be called positive for infection. A Poisson model can be used to determine significant enrichment above the threshold in a clinical sample. In some cases, the threshold may need to be adjusted, especially when an infection is caused by multiple pathogens within a collection instead of a single pathogen. In such cases, joint distributions of pathogen abundances can be used to adjust the threshold. For example, the adjusted threshold may be lowered in an infection caused by multiple pathogens when compared with an infection caused by a single pathogen. The adjusted threshold can be used to determine if a clinical sample is indicative of an infection based on joint distributions of certain pathogens in the collection or the group.

[0212] Results

[0213] The data include 62 pairs of matched cord blood plasma and maternal plasma and 15 cord blood samples which were missing a matched maternal sample. 9/77 cord blood samples and 1/62 maternal plasma sample returned a "no result" due to QC failure, thus ultimately 68/77 cord blood samples were retained for the analysis. (Fig. 3) Among the cord blood samples, 34 were preterm and 34 were cases of at term deliveries; among the maternal plasma samples, 29 were preterm and 32 were cases of at term labor. Overall, 52 maternal plasma and matched cord blood sample pairs were retained for analysis.

[0214] There were 24 patients that had a chorioamnionitis confirmed by histology, of which 3 had chronic chorioamnionitis and 21 others had acute chorioamnionitis. 17/24 had sub-clinical chorioamnionitis, without clinical symptoms, and was thus undetectable without microbial diagnosis or histology analysis of the placenta.

[0215] Comparison of microbial signatures in Preterm vs. At term healthy births

[0216] To identify microbial signatures that differ between preterm and at term healthy births, we compared samples from patients of those respective groups, excluding at term cases with clinical and/or histological chorioamnionitis in order to compare preterm labor deliveries to at term healthy deliveries. By applying a correlative analysis of taxa on the clinically reportable range (CRR) identified at significant level in at least one sample in cord blood, a heatmap was built of the inferred concentration based on number of reads for each sample. The taxa were ordered as consecutive leaves of a tree build from standard taxonomy and the samples were ordered, within preterm and at term births, by UPGMA (Unweighted Pair Group Method with Arithmetic Mean) hierarchical clustering using one minus the Pearson correlation coefficient as a measure of distance. Reported calls are outlined in black and significant calls that were statistically significant but filtered are outlined in grey. Fig. 3A displays the full heatmap, without any filtering to remove samples or taxa that are likely contaminated.

[0217] To distinguish taxa that are most likely to be causing infection, we applied two filters to the data. First we removed any samples that were dominated by vaginal or gut commensal microbes that likely entered cord blood sample during collection. Specifically, we removed samples for which 5 or more taxa were seen significantly above baseline

(indicated by down arrows on the heatmap in Fig. 3A). Taxa detected in control sample of nuclease-free water were used for setting a baseline. Other control samples such as a sample from heathy individual or from healthy pregnant woman can be used. The threshold of 5 was set by comparing the distribution of significance levels for all taxa in each sample. The threshold of 5 can be lowered to 4 or 3 or 2. The threshold of 5 can be elevated to 6, 7, 8, 9,

10, 15, 20, 30, or more, especially when using highly sensitive high-throughput sequencing assays. Then, to normalize the signal across taxa, so that we are not making calls for taxa that are environmental contaminants and thus present in all samples, we computed a z-score across taxa (rows). In the revised heatmap, we only display the taxa concentration if the z- score for those taxa in that sample is greater than 2, which represents being 2 standard deviations away from the mean. Fig. 3B shows the cleaned-up heatmap. These methods can be applied in further analysis of larger sample sizes and various sets of pathogens in the sample. This analysis shows clusterings which could not be resolved by a performance analysis only. From left to right: (1) a cluster of related infections due to Streptococcus spp. all in very preterm births (V), with histological chorioamnionitis, and delivered by C-section

(cesarean section) (C); (2) a cluster of Ureaplasma spp. and Mycoplasma hominis, which are closely related pathogens, also in very preterm births (V); (3) a cluster containing

Lactobacillus spp. which is enriched in At term (A) vaginal (V) deliveries. Relevant scattered infections include (1) Prevotella spp. in very preterm (V) and preterm births (P); (2) and an E.coli infection in a very preterm birth (V) associated with histological

chorioamnionitis (H).

[0218] Microbial signatures of chorioamnionitis present in cord blood and maternal plasma

[0219] Pathogens that are causing infection are expected to be elevated in the maternal blood as well as the cord blood, whereas the abundance level of contaminants should be independent from one sample to another. Therefore, to identify taxa that are causing infection, we tested each taxon for the correlation of their abundance across maternal samples to its abundance in the paired cord blood samples. Because exact abundance can be affected by contamination, the measure we used for this correlation was a p-value representing the significance of the enrichment of the taxa above expected background. To account for the fact that the signal may be weaker in the maternal blood than the cord blood, which is because the infections causing chorioamnionitis are deep tissue infections, located in the fetus, amniotic fluid or placenta, we used a Spearman rank correlation instead of a Pearson correlation. If we compare the rank of the significance of a given taxa across mothers to the rank of that taxa of the matched cord blood sample, we find a set of taxa that are significantly correlated and likely to be a cause of infection. To confirm that vaginal contaminants or contamination were not correlated across individuals; we compared the correlation of taxa across individuals with histologically confirmed chorioamnionitis to the correlation across individuals that are chorioamnionitis negative. We found 4 taxa that were significantly correlated across individuals who had chorioamnionitis {Streptococcus pseudopneumoniae p=0.007, Streptococcus mitis p=0.0023, Mycoplasma hominis p=0.00013, and Ureaplasma parvum p=0.0016) (Fig. 4A). As expected, there was not a strong signal of significant taxa in the chorioamnionitis negative group (Fig. 4B). However, there was one taxon that was significant in the chorioamnionitis negative group (Citrobacter koseri p<0.0001), which has been associated with preterm birth and neonatal sepsis in the literature. A strong correlation between the levels of this pathogen in maternal and cord blood suggests the presence of a pathogen that could be indicative of infection, even though these patients do not have confirmed chorioamnionitis. Upon further examination, the patients in which Citrobacter koseri was present had preterm deliveries, supporting the hypothesis that this pathogen contributes to risk for preterm birth. This evidence suggests that the signal relevant for infection can be identified from both the maternal and the cord blood. [0220] Further, this set of pathogens {Streptococcus mitis, Mycoplasma hominis,

Ureaplasma parvum, and Citrobacter koseri) can be used to identify women who delivered preterm from a maternal blood. Note that because of the genetic similarity of Streptococcus pseudopneumoniae to Streptococcus mitis, we chose to include only Streptococcus mitis in our test. For each individual, we summed the MPM in maternal blood for these pathogens, and then we performed a Wilcoxon rank sum test to compare the total MPM in

chorioamnionitis/funisitis negative individuals to the MPM in chorioamnionitis positive individuals as tested by histology (p=0.056). We also performed the same analysis but comparing preterm cases to at term healthy cases and found that this set of pathogens can distinguish between the two groups (p=0.05, using 10000 sets of the same number of pathogens, permuted from the set of pathogens that were present in at least one maternal plasma sample and significantly above baseline in at least one cord blood sample). By summing the MPM for this set of pathogens in the maternal blood, we identified 8 preterm cases, in which these pathogens were elevated and only 1 at term case (the pathogens were only marginally elevated in the latter). In the preterm group, two patients, one of which had histologic and clinical chorioamnionitis, would not have been called as a positive result in the cord blood or maternal blood if considering only the abundance of a single taxon. This evidence suggests that this set of taxa is predictive of preterm birth and identification using maternal blood is possible.

[0221] Based on strong correlation between the set of four taxa and chorioamnionitis, targeted PCR and/or sequencing can be performed for diagnosing the infection. The four taxa can be used to distinguish between clinical and chronic chorioamnionitis and to predict preterm birth. Based on these results, it was concluded that the microbial profile in cord blood and in maternal plasma, as measured by methods described herein, correlates with gestational age and chorioamnionitis status. It is worth noting that the maternal plasma samples available for this study were samples at the time of birth, and by that point many of the mothers had been put on antibiotics, decreasing the signal of the pathogen in the blood.

Therefore, we expect that the signal of pathogenic cfDNA seen in the maternal signal would be stronger when this test is used as intended for diagnosing infection during the pregnancy.

[0222] Pathogens that cause chorioamnionitis are enriched in cord and maternal blood

[0223] To confirm that pathogens suspected of causing chorioamnionitis were found using our method, we aggregated a list of pathogens that have been identified by culture or

PCR in amniotic fluid of individuals with PPROM (DiGuilio, et al. Am J Reprod Immunol.

2010; 64(1): 38-57, Romero et al, J Matern Fetal Neonatal Med. 2015; 28(12); 1394-1409), which included: Bacteroides fragilis, Fusobacterium nucleatum, Gardnerella vaginalsis, Haemophilus influenzae, Mycoplasma hominis, Prevotella bivia, Rothia mucilaginosa, Streptococcus mitis, Streptococcus pneumoniae, Ureaplasma parvum, and Ureaplasma urealyticum. There were three pathogens found in the literature, Sneathia sanguinegens, Candida albicans, and Neisseria gonorrhoeae that were not present above baseline levels in our data in any of our samples. This likely reflects the fact that we have a small sample of infected individuals (and thus may not have individuals infected by each of these pathogens represented in the sample) and does not demonstrate a lack of ability to detect them in other individuals who presented with infection of those pathogens.

[0224] Furthermore, although several of these pathogens are also commensal bacteria, the method described herein can distinguish levels of these pathogens that cause infection from low levels that could be due to commensals. 41 of the patients in our data were tested by histology of the placenta for chorioamnionitis, of which 24 were confirmed to be positive for chorioamnionitis, where the suspected cause is infection. We compared the MPM of the pathogens mentioned above in the cord blood samples of chorioamnionitis positive individuals to the MPM of the same pathogens in individuals that were negative for histological chorioamnionitis. Of the 10 pathogens present in at least one cord blood sample

8 were enriched in the histological chorioamnionitis individuals, which was a significantly higher fraction of enriched taxa in histological chorioamnionitis positive individuals than expected due to chance (p-value=0.0079, using Fisher's exact test) (Fig. 5A).

[0225] The pathogen cell-free nucleic acid signal is also present in maternal blood.

Using the same set of pathogens, we looked for enrichment of the pathogens in the maternal plasma samples. Because the signal of chorioamnionitis may be reduced in maternal blood, consistent with the fact that cfDNA from deep tissue infections is often less prevalent in blood, the levels of cfDNA recovered from maternal plasma are low enough to be affected by counting noise. Therefore, we calculated enrichment of these pathogens in chorioamnionitis cases by comparing the fraction of histological chorioamnionitis positive individuals in which the pathogen was present to the fraction of histological chorioamnionitis negative patients in which the pathogen was present. For all pathogens, the signal is enriched in patients with histological chorioamnionitis, which is significantly more than expected by chance (p-value=0.0061, using Fisher's exact test) (Fig. 5B). Further, using the same methods, these pathogens are enriched in patients that had preterm labor or birth (PTL) when compared to individuals with healthy, at term birth (p=0.0077, using Fisher's exact test) (Fig.

5C). This evidence suggests that the signals of pathogenic microbes are present in maternal blood and detectable by our method, despite the fact that pathogen levels are low.

Additionally, in the future, the cfDNA signal for pathogenic microbes can be enriched with DNA probes or targeted PCR amplification to detect infection in the mother before preterm birth.

[0226] Performance of the described methods in chorioamnionitis cases in preterm deliveries

[0227] Among preterm labor (PTL) deliveries, 17/39 had histological chorioamnionitis, with 2/17 chronic chorioamnionitis, 1/17 acute sub chorioamnionitis (or mild acute chorioamnionitis), 1/17 mixed acute and acute sub chorioamnionitis and 13/17 acute chorioamnionitis; 7/17 histological chorioamnionitis also had funisitis. All the cases with funisitis had a positive result using the methods described herein, in the cord blood, with one or more relevant pathogens detected in each sample (e.g. Enterococcus faecalis, Ureaplasma parvum, Streptococcus mitis, Escherichia coli, Streptococcus pneumoniae, Peptoniphilus harei, Ureaplasma urealyticum). Of the 8 cases with histological chorioamnionitis, 4 returned a positive result using the methods described herein, to one or more relevant pathogens (e.g. Fusobacterium nucleatum, Mycoplasma hominis, Coryne bacterium aurimucosum, Corynebacterium urealyticum, Klebsiella pneumoniae, Peptoniphilus harei,

Prevotella bivia); among the 4/8 negative results, 2 were associated with chronic chorioamnionitis which is a potentially sterile inflammation of the placenta, thus consistent with a negative result using the methods described herein. Seven (7/39) cases had clinical chorioamnionitis; 6/7 had also histological chorioamnionitis, while 1/7 did not have any sign of chorioamnionitis on pathology. The data obtained from the methods described herein for this latest patient returned a negative result, while 5 out of 6 clinical chorioamnionitis cases confirmed by pathology had a positive result in the cord blood, showing agreement between histological diagnostic and detection of microorganisms using the methods described. In summary, among the 34 cord blood results that were obtained from preterm deliveries, 30/34 had histology of the placenta done, whether or not the patient had a clinical chorioamnionitis.

As shown in Fig. 6A, 15/30 had confirmed chorioamnionitis by pathology and, among these,

11/15 had a positive test in the cord blood using methods of the present disclosure. Using methods of the present disclosure, sensitivity was 73%, specificity was 60%, PPV was 65% and NPV was 69%. In Fig. 6B cases were classified as chorioamnionitis if they had a clinical and/or histological chorioamnionitis. Using methods of the present disclosure, sensitivity was

73%), specificity was 52%, PPV was 55% and NPV was 71%. As chronic chorioamnionitis may not be associated with an infection but with another cause of inflammation, removing from the analysis the 2 cases of chronic chorioamnionitis, which returned negative results, increases the specificity (Sp = 60%), sensitivity (Se=60%), positive predictive value (PPV = 65%) and negative predictive value ( PV= 82%) (Fig. 6C). Overall, this data show a good sensitivity (Se) of the methods in cord blood to detect cases with chorioamnionitis among preterm births.

[0228] Performance of the described methods in cord blood in C-sections

[0229] Because the detection rate of pathogens significantly above baseline in the cord blood from vaginal deliveries was 1.5^χ higher than in cord blood from C-sections and these samples contain many taxa that are common vaginal and gut commensal microbes (e.g. Lactobacillus spp., Bacteroides spp., Streptococcus anginosus, etc.), we focused our analysis on C-sections to calculate the performance of the test in a sterile environment. All cases that underwent C-section and had clinical and/or histological chorioamnionitis had also relevant pathogens identified at levels above baseline by the described methods (Streptococcus mitis as well as Streptococcus pneumoniae, Klebsiella pneumoniae, Prevotella bivia, Micrococcus luteus and others). The calculated Se and Sp of the methods described herein were respectively 100% and 83% (Fig. 6D). There were 2 individuals without chorioamnionitis, for which we returned a positive result. However, both were at term deliveries with premature rupture of the membranes. The microorganisms identified by the method described here are taxa that are commonly found in the vaginal microbiome and could have reached the amniotic fluid after rupture of the membranes, without causing any infection during the latency period which is the period between rupture of the membranes and delivery (e.g. Lactobacillus spp.) The pattern of abundances in C-section deliveries only shows a strong correlation between presence of microorganisms and preterm birth and/or neonatal sepsis.

[0230] Detection rate of pathogens in cord blood by mode of deliver

[0231] Detection rate of pathogens between cord blood from vaginal delivery (n=49) and cord blood from C-section delivery (n=19) was compared using methods of the present disclosure (Fig. 7). Detection rate of pathogens in cord blood from vaginal delivery was 63%

(31/49 were tested positive) and from C-section delivery was 42% (8/19 were tested positive).

[0232] Performance of the described methods in maternal plasma samples in chorioamnionitis cases

[0233] Among the 29 maternal sample results in PTL deliveries, 24/29 cases had a histology of the placenta obtained, whether the patient had a clinical chorioamnionitis or not. Fig. 8A demonstrates that ten out of 24 cases had a confirmed chorioamnionitis and 3/10 had a positive result in the maternal plasma. As shown in Fig. 8B, 9 at term cases had histology of the placenta done, and adding these cases for the performance study slightly increased the specificity (Sp) and negative predictive value (NPV). Finally, if we remove the cases of chronic chorioamnionitis performance of the test is also slightly improved (Fig. 8C). The Se of the test is lower in maternal plasma as compared to cord blood and this result is closer to data reported in the biopsy replacement study which tested the performance of the methods in patients with deep tissue infections. As discussed above, Se and Sp in maternal plasma can be improved with adequate timing of collection before starting antibiotics, pull-downs or other modified molecular biology protocols as described herein and/or by applying bioinformatics analysis as shown above.

[0234] There were four cases in which specific microbes were identified significantly above baseline in both cord blood and maternal plasma (Fig. 9). Two cases were preterm births with confirmed histological chorioamnionitis. In both cases the newborn received empirical antibiotic treatment for suspicion of early onset neonatal sepsis (EONS), but blood cultures did not grow. In the first case, these methods detected Mycoplasma hominis in both cord blood and maternal plasma samples. In addition, these methods detected Fusobacterium nucleatum in cord blood plasma. In the second case, these methods detected Escherichia coli at a significant level in both maternal plasma and cord blood samples. In the two other cases,

Human Herpes Virus (see section on congenital infection) was identified. All these pathogens have been previously reported in cases of chorioamnionitis in the literature, supporting the conclusion that these methods identified relevant pathogens in cases of chorioamnionitis in both cord blood plasma and maternal plasma in preterm births.

[0235] Identifying causes of neonatal sepsis infection using maternal and cord blood

[0236] Twenty-three newborns, among the 68 cases with a cord blood result, were started on presumptive antibiotic therapy for neonatal sepsis, presumptive neonatal sepsis, respiratory distress, Premature Rupture of the Membranes (PROM) or maternal chorioamnionitis and 14/23 had a positive test in the cord blood (61%) using methods of the present disclosure. However, many of these positive results were vaginal births and may be due to contamination, we subset the data to look only at the 18 cesarean births. Two cases had a confirmed, blood-culture negative, early onset neonatal sepsis (EONS) and received antibiotics for 7 days. Our method returned a positive result for both cases {Streptococcus mitis in one case and Streptococcus mitis and Streptococcus pneumoniae in the other case).

Of the 16 cases where the fetus did not have neonatal sepsis, 1 1 were negative, and two others had a rupture of membrane before delivery and returned a result of Lactobacillus spp., which could be an ascending contamination from the vaginal microbiome as opposed to an infection. Four cases had a suspicion of EONS and received antibiotics for 2 or 3 days. Among these, all were negative. These data suggest that using the methods described herein in cord blood can be useful in the management of neonates with EONS, especially in preterm deliveries.

[0237] Identifying congenital infection using maternal and cord blood

[0238] There were two cases where our methods identified Human Herpes Virus in both the maternal plasma and cord blood (Fig. 9). One was an at-term delivery case and the methods identified Human Herpes Virus (HHV) 6A, and the other was a preterm delivery and the methods identified HHV6B. HHV6 has been reported as the most common virus detected in amniotic fluid (AF) (MT Gervasi et al.; J Matern Fetal Neonatal Med. 2012; 25(10): 2002-2013. Prevalence of Viral DNA in Amniotic Fluid of Low-Risk Pregnancies in the Second Trimester). In this literature report, corresponding viral DNA was also detected in maternal blood of six out of seven women with HHV6-positive AF and in the umbilical cord plasma, which was available in one case. In one case of HHV6 infection in the AF, the patient developed gestational hypertension at term, and in another case of HHV6 infection in the AF, the patient delivered at 33 weeks after preterm premature rupture of membranes (PPROM). Further, congenital infection with HHV6 has been reported in 1/101 births and it may have detrimental effect on neurodevelopment at 12 months of age (Caserta MT et al., Pediatrics 2014; 134 (6): 1111-1118. Early developmental outcomes of children with congenital HHV-6 infection). The other microorganisms identified in this case were probably environmental contaminants and/or vaginal delivery associated contaminants. While only two cases of congenital infection were identified among the 52 pairs analyzed, these preliminary data suggest that these methods can be useful in detecting congenital infections.

[0239] In conclusion, the results demonstrated that these methods are highly sensitive in diagnosing chorioamnionitis in preterm labor. In certain embodiments, the methods may include a limited collection or group of the most frequently encountered microorganisms in chorioamnionitis to improve sensitivity and to use the test before delivery in patients at risk of PTL in order to detect and/or predict infections and potentially prevent PTL or delay time of birth.

[0240] In neonates, very low grade bacteremia (<4 colony forming units/mL) and use of antibiotics during labor and delivery in cases of PROM and/or clinical chorioamnionitis, result in blood cultures that are often negative, making it challenging to obtain a definite microbiological diagnosis. This preliminary data suggests that these methods in cord blood can be useful for detection of microorganisms in cases of EONS or suspected EONS.

[0241] Finally, the detection, in both maternal plasma and cord blood, of HHV6A and 6B, a virus that can cause a congenital disease with potential neurodevelopmental delays, exemplifies how these methods can be used to detect other congenital diseases.

[0242] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be

understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of detecting pathogen nucleic acids in a pregnant subject comprising:

(a) obtaining a sample comprising cell-free nucleic acids from the pregnant subject, wherein the pregnant subject is at risk of having an infection; and

(b) conducting a sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids from a collection of pathogen nucleic acids, wherein the pathogen nucleic acids are specifically selected for the collection primarily based on a known association between the pathogen nucleic acids and chorioamnionitis.

2. A method of detecting pathogen nucleic acids in a pregnant subject comprising:

(b) conducting a high-throughput sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids from a collection of pathogen nucleic acids, wherein the pathogen nucleic acids are specifically selected for the collection primarily based on a known association between the pathogen nucleic acids and at least one infection selected from the group consisting of: fetal membrane infection, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection, umbilical cord infection, and neonatal infection.

3. The method of claim of any one of the preceding claims, further comprising quantifying each detected pathogen nucleic acid in order to obtain initial values for the detected pathogen nucleic acids and comparing the initial values for the detected pathogen nucleic acids with at least one reference value in order to obtain relative values for the detected pathogen nucleic acids.

4. The method of any one of the preceding claims, further comprising identifying detected pathogen nucleic acids with relative values above a threshold value, thereby obtaining a selected set of one or more pathogen nucleic acids.

5. The method of any one of the preceding claims, further comprising designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids exceeds a threshold value of different pathogen nucleic acids.

6. The method of any one of the preceding claims, wherein the threshold value is any value within the range of 2 to 20.

7. The method of any one of the preceding claims, wherein the threshold value is 2.

8. The method of any one of the preceding claims, further comprising designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids comprises nucleic acids from 20 different pathogens.

9. The method of any one of the preceding claims, further comprising designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids are pathogen nucleic acids associated with a number of different pathogens wherein the number is between 2 and 20.

10. The method of any one of the preceding claims, wherein the different pathogen nucleic acids are from different pathogens from different pathogen taxa, different pathogen genera, different pathogen species, different pathogen strains, or different pathogen variants.

11. The method of any one of the preceding claims, wherein the at least one reference value is based on levels of the pathogen nucleic acids detected in one or more samples selected from the group consisting of: water sample, blood sample, plasma sample, serum sample, urine sample, body fluid sample, reagent sample, sample from a healthy subject, sample from a healthy pregnant subject, and any combination thereof.

12. The method of any one of the preceding claims, further comprising electronically transmitting data reflecting the selected set of one or more pathogen nucleic acids to a recipient.

13. The method of any one of the preceding claims, wherein the electronically- transmitted data is used by the recipient to determine a treatment plan for the pregnant subject.

14. The method of any one of the preceding claims, wherein the electronically- transmitted data is used by the recipient to detect, monitor or diagnose a condition of the pregnant subject.

15. The method of any one of the preceding claims, wherein the obtained sample is associated with a patient identifier and the method further comprises: separating the patient identifier from the obtained sample to obtain de-identified samples; obtaining de-identified sample sequence data from the high-throughput sequencing assay; uploading the de-identified sample sequence data to a server; detecting the pathogen nucleic acids within the de-identified sample sequence data in order to obtain de-identified result data, such that the de-identified result data is on the server; down-loading the de-identified result data from the server.

16. The method of any one of the preceding claims, further comprising associating the patient identifier with the de-identified result data.

17. The method of any one of the preceding claims, further comprising administering a therapeutic regimen to the pregnant subject based on the selected set of one or more pathogen nucleic acids.

18. The method of any one of the preceding claims, wherein the administering a therapeutic regimen comprises treating the pregnant subject with a specific drug to reduce or eliminate the source of the detected pathogen nucleic acids.

19. The method of any one of the preceding claims, wherein the drug is selected from the group consisting of antibiotic, antiviral, ampicillin, sulbactam, penicillin, vancomycin, gentamycin, aminoglycoside, clindamycin, cephalosporin, metronidazole, timentin, ticarcillin, clavulanic acid, cefoxitin, antiretroviral, immunoglobulins, and any combination thereof.

20. The method of any one of the preceding claims, wherein the therapeutic regimen comprises administering a therapy to the pregnant subject and then repeating steps a and b to monitor effects of the therapy on the selected set of one or more pathogen nucleic acids.

21. The method of claim 3, further comprising terminating the pregnancy of the pregnant subject based on the selected set of one or more pathogen nucleic acids.

22. The method of claim 1, further comprising determining a risk of congenital defects in a developing fetus of the pregnant subject based on the detection results.

23. The method of claim 1, further comprising using the high-throughput sequencing assay to determine relative amounts of organ cell-free RNA of the fetus or pregnant subject compared to a control value.

24. The method of claim 16, wherein the one or more pathogen nucleic acids and increased amounts of organ cell-free RNA indicate the presence of an infection in the organ.

25. The method of claim 17, wherein the one or more pathogen nucleic acids are Zika nucleic acids and the organ is fetal brain.

26. The method of claim 17, wherein the one or more pathogen nucleic acids are pathogen nucleic acids associated with chorioamnionitis and the organ is uterus.

27. The method of any one of the preceding claims, wherein the high-throughput sequencing assay detects cell-free pathogen DNA or cell-free pathogen RNA of the pregnant subject in order to prognose a risk of preterm labor or preterm delivery.

28. The method of any one of the preceding claims, wherein the high-throughput sequencing assay detects cell-free pathogen DNA or cell-free pathogen RNA in order to prognose a risk of congenital defects in the fetus.

29. The method of any one of the preceding claims, wherein the pregnant subject has one or more clinical symptoms of chorioamnionitis or funisitis.

30. The method of any one of the preceding claims, wherein the one or more clinical symptoms of chorioamnionitis or funisitis are selected from the group consisting of fever, rapid heartbeat or tachycardia of the pregnant woman, rapid fetal heartbeat or fetal tachycardia, uterine tenderness, vaginal discharge with an unusual or foul odor or discoloration, amniotic fluid with a foul smell, maternal leukocytosis, and any combination thereof.

31. The method of any one of the preceding claims, wherein the pregnant subject has one or more risk factors for chorioamnionitis or funisitis.

32. The method of any one of the preceding claims, wherein the one or more risk factors for chorioamnionitis or funisitis are selected from the group consisting of longer duration of membrane rupture, prolonged labor, internal monitoring of labor, multiple vaginal examinations, meconium-stained amniotic fluid, smoking, alcohol abuse, drug abuse, compromised immune system, epidural anesthesia, colonization with group B streptococcus, sexually transmissible genital infections, vaginal colonization with ureaplasma, and any combination thereof.

33. The method of any one of the preceding claims, wherein the pregnant subject is a pregnant woman with one or more risk factors for premature labor and delivery.

34. The method of any one of the preceding claims, wherein the one or more risk factors for premature labor and delivery are selected from the group consisting of having a premature rupture of the membranes; having a personal or family history of premature birth, miscarriage, or stillbirth; being underweight or overweight before pregnancy; not gaining enough weight during pregnancy; having certain health conditions such as diabetes, high blood pressure, preeclampsia, or blood clot disorders; becoming pregnant after in vitro fertilization (IVF);

becoming pregnant up to 18 months after a previous birth; having a social risk associated with preterm birth; having a positive test result from an assay giving a prognosis of preterm birth, and any combination thereof.

35. The method of any one of the preceding claims, wherein the pregnant subject is a pregnant woman who previously had an amniocentesis test or chorionic villus sampling test.

36. The method of claim 28, wherein the amniocentesis test or chorionic villus sampling test indicated inflammation or infection but did not identify a specific pathogen.

37. The method of claim 1, wherein the one or more pathogen nucleic acids are derived from one or more pathogens present in the sample.

38. The method of any one of the preceding claims, wherein the one or more pathogens comprise one or more bacteria or one or more viruses.

39. The method of claim 1, wherein the one or more pathogens comprise Escherichia coli, group B streptococcus (Streptococcus agalactiae), anaerobic bacterium, Staphylococcus aureus, cytomegalovirus (CMV), Mycoplasma spp., Mycoplasma hominis, Ureaplasma spp.,

Ureaplasma urealyticum, Ureaplasma parvum, human immunodeficiency virus (HIV), lentivirus, herpes simplex, human herpes viruses, varicella, B 19 erythrovirus, Toxoplasma gondii, Treponema pallidum, Listeria monocytogenes, Plasmodium falciparum, rubella, Chlamydia trachomatis, hepatitis B virus, hepatitis E virus, Parvovirus, Enterovirus, hepatitis C virus, syphilis, gonorrhea, Fusobacterium nucleatum, Enterococcus faecalis, Corynebacterium aurimucosum, Corynebacterium urealyticum, Gardnerella vaginalis, Bacteroides fragilis, Haemophilus influenzae, Methylobacterium mesophilicum, Prevotella bivia, Rothia

mucilaginosa, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus

pseudopenumoniae, Streptococcus pasteurianus, Sneathia sanguinegens, Candida albicans, Neisseria gonorrhoeae, Citrobacter koseri, Peptoniphilus harei, Klebsiella pneumoniae, Micrococcus luteus, and Flaviviruses.

40. The method of any one of the preceding claims, wherein the one or more pathogen nucleic acids comprise cell-free pathogen DNA, cell-free pathogen RNA, or a mixture of cell-free pathogen DNA and cell-free pathogen RNA.

41. The method of any one of the preceding claims, wherein the sample is selected from the group consisting of blood, cord blood, peripheral blood, plasma, serum, cerebrospinal fluid, synovial fluid, bronchoalveolar lavage, urine, stool, saliva, nasal swab, cord blood, amniotic fluid, cell-free plasma, and any combination thereof.

42. The method of any one of the preceding claims, wherein the sample has been processed to remove at least one component from the group consisting of: cells, human cells, bacterial cells, viral particles, and exosomes.

43. The method of any one of the preceding claims, wherein the sample is a plasma sample that has been further processed to remove at least one component from the group consisting of: cells, human cells, bacterial cells, viral particles, and exosomes.

44. The method of any one of the preceding claims, wherein the method reduces the risk of preterm labor by at least 50%.

45. The method of any one of the preceding claims, wherein the detecting the pathogen nucleic acids comprises monitoring the pathogen nucleic acids over time.

46. The method of any one of the preceding claims, wherein the pathogen detected is associated with an intrauterine infection.

47. The method of any one of the preceding claims, wherein the pathogen detected is associated with a congenital fetal infection.

48. The method of any one of the preceding claims, wherein the pathogen detected is associated with a neonatal infection.

49. The method of any one of the preceding claims, wherein the intrauterine infection is acute chorioamnionitis or funisitis.

50. The method of any one of the preceding claims, wherein the intrauterine infection is chronic chorioamnionitis or funisitis.

51. The method of any one of the preceding claims, wherein the intrauterine infection is sub-clinical chorioamnionitis or funisitis.

52. The method of any one of the preceding claims, wherein the detection of one or more pathogen nucleic acids from the collection of pathogen nucleic acids is used to detect, diagnose or prognose an increased risk for infection of intrauterine tissue, chorion, amnion, umbilical cord or placenta.

53. The method of any one of the preceding claims, further comprising diagnosing the pregnant subject with at least one infection at least in part based on the detected pathogen nucleic acids, wherein the at least one infection is selected from the group consisting of: fetal membrane infection, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection, umbilical cord infection, and neonatal infection.

54. The method of any one of the preceding claims, further comprising diagnosing the pregnant subject with at least one infection at least in part based on the detected pathogen nucleic acids, wherein the at least one infection is selected from the group consisting of:

chorioamnionitis, funisitis, chronic chorioamnionitis, chronic funisitis, acute chorioamnionitis, acute funisitis, neonatal sepsis, and placental infection.

55. The method of any one of the preceding claims, further comprising diagnosing the pregnant subject or subject contemplating pregnancy with at least one infection at least in part based on the detected pathogen nucleic acids, wherein the at least one infection is selected from the group consisting of: uterine, vaginal and reproductive system infection or colonization.

56. The method of any one of the preceding claims, further comprising detecting in a subject contemplating pregnancy pathogens a presence of one or more pathogens associated with chorioamnionitis or other pregnancy-related infection.

57. The method of any one of the preceding claims, further comprising detecting, diagnosing, monitoring, or prognosing the pregnant subject with a bacterial infection or viral infection.

58. The method of any one of the preceding claims, wherein the sample is not cord blood, amniotic fluid or chorionic villus.

59. The method of any one of the preceding claims, wherein the sample is not cord blood, amniotic fluid or chorionic villus and the sample is used to detect, diagnose or prognose chorioamnionitis, fetal infection, or neonatal infection.

60. The method of any of the preceding claims, further comprising conducting an RNA sequencing assay on a sample comprising cell-free RNA from the pregnant subject in order to distinguish between extra-uterine and intra-uterine infection.

61. The method of any one of the preceding claims, further comprising detecting an infection in the cord blood in utero or after delivery of a neonate.

62. The method of any one of the preceding claims, further comprising detecting an infection from the cell-free fraction of a blood sample from the human pregnant subject.

63. The method of any of the preceding claims, wherein one or more of the steps in the method are implemented using a computer.

64. A method of predicting preterm labor in a pregnant subject comprising:

(a) obtaining a sample comprising cell-free nucleic acids from a pregnant subject or subject contemplating pregnancy suspected of having an infection; and

(b) conducting a high-throughput sequencing assay on the cell-free nucleic acids in order to detect one or more pathogen nucleic acids associated with infection of the fetal membranes, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection or neonatal infection.

65. The method of any one of the preceding claims, wherein the one or more pathogens nucleic acids derived from one or more pathogens comprising Escherichia coli, group B streptococcus (Streptococcus agalactiae), anaerobic bacterium, Staphylococcus aureus, cytomegalovirus (CMV), Mycoplasma spp., Mycoplasma hominis, Ureaplasma spp.,

Ureaplasma urealyticum, Ureaplasma parvum, human immunodeficiency virus (HIV), lentivirus, herpes simplex, varicella, B19 erythrovirus, Toxoplasma gondii, Treponema pallidum, Listeria monocytogenes, Plasmodium falciparum, rubella, Chlamydia trachomatis, hepatitis B virus, hepatitis E virus, Parvovirus, Enterovirus, hepatitis C virus, syphilis, gonorrhea,

Fusobacterium nucleatum, Enterococcus faecalis, Corynebacterium aurimucosum,

Streptococcus mitis, Streptococcus pneumoniae, Streptococcus pseudopenumoniae ,

Streptococcus pasteurianus, Sneathia sanguinegens, Candida albicans, Neisseria gonorrhoeae, Citrobacter koseri, Peptoniphilus harei, Klebsiella pneumoniae, Micrococcus luteus, and Flaviviruses.

66. The method of any one of the preceding claims, wherein the detecting the one or more pathogen nucleic acids comprises monitoring the one or more pathogen nucleic acids over time.

67. A method of treating an infection in a pregnant subject comprising administering to the pregnant subject a therapeutic regimen to treat the infection wherein the pregnant subject has been determined to have an increased level of cell-free pathogen nucleic acids and wherein the cell-free pathogen nucleic acids are associated with an infection selected from the group consisting of: fetal membrane infection, placental infection, intra-amniotic infection, intrauterine infection, congenital fetal infection and neonatal infection.

68. The method of any one of the preceding claims, wherein the determination is conducted by high-throughput sequencing or massively parallel sequencing.

69. A method of detecting pathogen nucleic acids in a pregnant subject comprising:

(a) providing a sample comprising cell-free nucleic acids from a pregnant subject; and

(b) conducting a high-throughput sequencing assay on the cell-free nucleic acids from the pregnant subject, thereby obtaining sequence reads from nucleic acids from pathogens present in a tissue or organ of the pregnant subject, wherein the tissue or organ is selected from the group consisting of: fetal membrane, chorionic tissue, intra-amniotic tissue or fluid, placenta, uterus, fetal tissue, umbilical cord, fetoplacental circulatory system, and any combination thereof.

70. The method of any one of the preceding claims, wherein the tissue or organ is chorionic or amniotic membrane tissue.

71. The method of any one of the preceding claims, wherein the tissue or organ is chorionic or amniotic membrane tissue and the sample is plasma, cord blood, amniotic fluid, serum, or urine.

72. The method of any one of the preceding claims, further comprising applying a filter to the sequence reads in order to evenly distribute coverage of the sequence reads across a pathogen genome.

73. The method of any one of the preceding claims, further comprising applying a filter to the sequence reads in order to reduce signal associated with microbes present in the vaginal canal or gut.

74. The method of any one of the preceding claims, further comprising quantifying the sequence reads from the nucleic acids from pathogens present in the tissue or organ of the pregnant subject to obtain initial pathogen values.

75. The method of any one of the preceding claims, further comprising comparing the initial pathogen values with at least one reference value in order to obtain relative pathogen values.

76. The method of any one of the preceding claims, further comprising identifying relative pathogen values above a threshold value, thereby obtaining a selected set of one or more pathogen nucleic acids.

77. The method of any one of the preceding claims, further comprising designating the sample from the pregnant subject as contaminated or inconclusive when the threshold value is any value within the range of 2 to 20.

78. The method of any one of the preceding claims, wherein the method prognoses pathogen infection of the fetus.

79. The method of any one of the preceding claims, wherein the specificity or sensitivity of the method is greater than 75%.

80. The method of any one of the preceding claims, wherein the cell-free pathogen nucleic acids are not particle-protected viral nucleic acids.

81. The method of any one of the preceding claims, further comprising designating the sample from the pregnant subject as contaminated or inconclusive when the selected set of one or more pathogen nucleic acids are associated with a number of different pathogens, wherein the number is between 2 and 20.