1111111111111111 IIIIII IIIII 1111111111 11111 11111 111111111111111 IIIII IIIII IIIIII IIII 11111111 US 20200056988Al c19) United States c12) Patent Application Publication c10) Pub. No.: US 2020/0056988 Al Butz et al. (43) Pub. Date: Feb. 20, 2020

(54) METHOD OF DETERMINING THE Publication Classification EFFICACY OF ANTIMICROBIALS (51) Int. Cl. GOIN 2113504 (2006.01) (71) Applicant: Wisconsin Alumni Research GOIN 33/497 (2006.01) Foundation, Madison, WI (US) (52) U.S. Cl. CPC ...... GOIN 2113504 (2013.01); GOIN 33/497 (72) Inventors: Daniel Elmer Butz, Madison, WI (US); (2013.01) Ann P. O'Rourke, Madison, WI (US); Mark E. Cook, Madison, WI (US) (57) ABSTRACT A method of determining efficacy of an antimicrobial treat­ ment in a subject includes calculating a breath delta value (21) Appl. No.: 16/540,627 (BDV) for each of at least six breath samples acquired from the subject over a 24 hour period starting from when the subject has been administered the antimicrobial treatment, (22) Filed: Aug. 14, 2019 calculating a mean standard deviation of BDV (SD BDV) across the six or more breath samples; and determining that the antimicrobial treatment is effective when the SD BDV is Related U.S. Application Data less than or equal to 0.46, or determining that the antimi­ crobial treatment is ineffective when the SD BDV is greater (60) Provisional application No. 62/764,874, filed on Aug. than 0.46. Also included are methods of treating a subject in 16, 2018. need of antimicrobial treatment.

r Ass~sse~.f~;·el;~!~i!lt;·~n~~·~1)···· i ,, --~----·--··--···············r··············•······-·······•_.•.•.•.•_.•.•.•.•_J______...... ,.,

; Excluded (no:299) r- ➔@!~;;;:~=:· ~···

Consented for partidpatfon {ir=32) Wi!hdrawn: • Exdusionary Criteria (ri=3) • Withdrawn by lnve.stigator(n"'2)

Analyzed {n:=27) , Excluded from analysis {inadequate breath samples} {n=7} • 20 Subjects Anaiyze

4.0 Infection Progression Y'ancom::cln q ...... ~ .. ,-.•.•··········· ...... 3.0 BDV rises and variance increases Ce,,,p;m,:, , ·if ./ . ***~ * .... '!ti-.,.•:,$' 2.0 'Iii::./ .~ •: ... ·.·•,•:::::::•· * ,..············::·.•·· ~--···O) g!?~P.Q.J~J?JQ Ir.e~trn~ot . BDV variance decreases! :A} Blue"' "Safe Zone"

B) Infection Onset ·~ BDV fails below --3,0

-4.0 FIG. 1

'···Assessed for ;§gib1li!y {n<~3i} .. I ......

' I e, ct,,Jod (~,.,i I 1 [~1f~~~~::::'.~ ~,rt, ...... , Consented for participation {n=32) Wilhdrawn: • Exduskmmy Criteria (rr=3) • Withdrawn by lnvesllgalor (!F2) I

!"fi~i~d~0iiti·~~~-;~~is (inadequate breath \ samples} (n=7) l • 20 Subjects Analyzed: o 13 Male. 7 Female o 16.2% Caucasian; 14.3% tv."rican American: 9.5% Othar ,:~,1:;;'~~~::'~,:::·,'.:'.'.'.: I

FIG. 2 Patent Application Publication Feb. 20, 2020 Sheet 2 of 7 US 2020/0056988 Al

b

a

!CU admitted no- untreated infection treated infection infection

FIG. 3

00-004 3 ,. • ::i s.o ... o.1s 3 s 2.5 ~-/~\! 'ii "' 2 .n"' a; 1.5 5.0. "0.38 -~After treatment > 0 ···•··Before treatmet)t IS '-'- 53.1% decrease in S.D. 3 0.5 0 0 1 2 3 4 s 6 7 Time since €r:roliment FIG. 4

O!J-004 3.5 s.o. "0.18 3 ?-- J .5 2.5 ~_,/ \ 'ii 2 "'ro ..0 [) 1.5 SJ). "0.38 -·After treatment > 0 1 ·• Before treatment IS 'ii 53.1% t!ecras~ In 5.D. o 0.5 0 0 1 2 3 4 6 7 ffme since enrollment FIG. 5 Patent Application Publication Feb. 20, 2020 Sheet 3 of 7 US 2020/0056988 Al

00--007 2.5

2 S.O. ~ 0.1S /·,.)~ ..., .... After treatment •. !3efon~ treatment

0 0 1 2 3 4 5 6

FIG. 6

00-011 3.S

3

··'··After treatment .,. Before treatment

0 0 .1 2 3 4 5 6 7 Time since enroi!ment FIG. 7 02-002 3 2..5

2

--After treatment s.o." 0.46 •·"·· Before treatment

1 z 7

Time since enrollment {d) FIG. 8 Patent Application Publication Feb. 20, 2020 Sheet 4 of 7 US 2020/0056988 Al

02-003 2.5 43.9% de(N,i.!Se 1!'1 S.D. 2

LS

~ •""~After tre<1tment > 1 0 · Before treatment ro ~ 0.5 0 0

❖ 0 .j. 2 3 4 5 6 Time since enmHmwt (d) FIG, 9

02-004 0 ·0.2 0.2 0.4 0.6 0.8 1 t2 1.4 LG LS -04 54.9% decrease In S.t) . ·0.6 .£ S.O. »O-!>il ~ ·0.8 11> ...... After treatment J:l .. 1 w"" > •··'··Before treatment o ·1.2 ,!!l oi -1.4 0 .. 1.6 ·l.8 Time since enrollment {d) FIG. 10

02-006 3

2.5 S.D. :0.27 IV .E 2 1, V, 15 1.5 ... ..-After treatment ,l; > S.l). "'0.49 0 1 ·•· Before tre<1tment .a 44.7% detrnas;; in SJ). ~ 0.5

0 0 0.5 1 1.5 2 2.5 3 3.5 4 T!me since enrollmeilt FIG. 11 Patent Application Publication Feb. 20, 2020 Sheet 5 of 7 US 2020/0056988 Al

04-004 2 S.D •.«0.$1 21.8% dea~ase lfi 5.D. :LS w .s (LJ 1 V, SJ'.l."0.40 (G ·"~·After treatment .0,_ <:;; ::,, o.s ··•· -Before tis!.itment 0 fl ~ 0 0 0 1 l 3 4 5 7 -{J.5 Time since enrollment FIG. 12

04-007 3

2.5

'l.' 2 ,!::, ·a; if> 1.5 S.D. ~o.86 -"-After treatment J:,"' n. 7% decrease in :LO. b 1 > ·· Before ueatment 0 s O,S ·-a; CJ 0 0 0.5 1 1.5 2 £ ,..;;C 3 -05 T1me since enrollment FIG. 13 Patent Application Publication Feb. 20, 2020 Sheet 6 of 7 US 2020/0056988 Al

4.0

3.0 Cefuroxime ::rn

Day 1 Day2 Day3 Day4 Day5 Day7 Daya Day9 Day iO

FIG. 14

4.0 'Ctlfuroxfrrie

3.0

2,0

LD

+ BAL: Klebsiarla oxytoca: endogenous flora; Aclnetobacter baumanlilca Blood: + Fungai: Aspergrnus No fumigalus B!ood: growth No SAL: No -z.o growth 9ro\11lh

Day 1 Day2 Day 3 Day4 Day 5 Day6 Day8 Day 10

FIG. 15 Patent Application Publication Feb. 20, 2020 Sheet 7 of 7 US 2020/0056988 Al

Cefepime

/\...,.__,,_,.;"'-.,.1,-.,,.,'¼;.,. ,-""•.•;,.•··'''•, ... 1.0 r·--,,,,' " f\/~\_./ . ~ . •.• ' i i, ..,J. \ ✓ \/ k Urine: No g,o.-.-th + Blood; Streptococcus coM!eila!JJs + BAL: S!aphyloooccus aureus + Brach line: Sttaptocoocus COn$tellus

Day 1 Day2 Day3 Day4 Day5 Day6 Day? Daya Day9 Day 10 FIG. 16 US 2020/0056988 Al Feb.20,2020 1

METHOD OF DETERMINING THE [0006] In addition, U.S. Pat. No. 8,512,676 describes the EFFICACY OF ANTIMICROBIALS use of oscillation modes in breath isotope ratio data to identify an "unhealthy" state in an organism. Changes in the CROSS-REFERENCE TO RELATED frequency and/or amplitude of the oscillation modes can be APPLICATIONS correlated with the health of an individual. Advantageously, advances in cavity ringdown spectrometry allow for the [0001] This application claims priority to U.S. Provisional continuous collection of breath isotope data which permits Application 62/764,874 filed on Aug. 16, 2018, which is the identification of oscillatory patterns within the breath incorporated herein by reference in its entirety. isotope data. The identified oscillation modes are particu­ larly useful in determining the transition from a healthy to an FIELD OF THE DISCLOSURE infected state in an organism within the short-term infection [0002] The present disclosure is related markers for deter­ period, e.g., 30 minutes to 2 hours. mining the efficacy of antimicrobials, and novel methods for [0007] What is needed are improved markers for the administering antibiotics, particularly to subjects in a sur­ response of subjects to antimicrobial treatment, particularly gical ICU setting. acute surgical and trauma subjects admitted to the ICU. BRIEF SUMMARY BACKGROUND [0008] In one aspect, a method of determining efficacy of [0003] Infections remain a leading cause of morbidity and an antimicrobial treatment in a subject comprises mortality in the intensive care unit (ICU). In the United [0009] calculating a breath delta value (BDV) for each of States, infectious complications develop in up to 28% of at least six breath samples acquired from the subject over a patients admitted to the ICU and require mechanical venti­ 24 hour period starting from when the subject has been lation. The current standard of care for detecting infections administered the antimicrobial treatment, wherein BDV is relies heavily on the physician to make decisions bedside, determined according to monitoring trending vitals signs and laboratory work. Hos­ pital mortality for severe infection ranges from 18% to 28% in adults and remains the leading cause of death in adult 13 12 13 12 13 ( C/ C sample- C/ c PDE) surgical ICU patients. Assessing the efficacy of antimicro­ EDV=OC= 13 C/l2CPDE xlOOO bial treatment in acute systemic inflammation and severe infection, is vital to improved patient outcomes. Moreover, unnecessary empiric antibiotic treatment is undesirable [0010] wherein PDB is a Pee Dee Belenmite reference because of the risk of antibiotic-resistant bacterial strains. standard, and BDV is expressed as parts per mil (%0); [0004] A noninvasive, non-doping, rapid stable isotope [0011] calculating a mean standard deviation of BDV (SD method to discern the onset of the catabolic state by detect­ BDV) across the six or more breath samples; and [0012] determining that the antimicrobial treatment is ing isotopic changes in the exhaled CO2 in breath was described in issued U.S. Pat. No. 5,912,178 (the '178 effective when the SD BDV is less than or equal to patent). The relative health of an organism was determined 0.46, or 13 12 [0013] determining that the antimicrobial treatment is by comparing the sampled ratio (C :C ) to a baseline ratio in the organism by testing breath samples in a mass spec­ ineffective when the SD BDV is greater than 0.46. trometer, for example. The methods disclosed in the '178 [0014] In another aspect, a method of determining efficacy patent allow for a non-invasive determination of net cata­ of an antimicrobial treatment in a subject comprises bolic processes of organisms experiencing altered organ [0015] calculating a breath delta value (BDV) for each of function or a deficit in nutrient intake. One disadvantage to at least six breath samples acquired from the subject over a the method disclosed in the '178 patent is that a comparison 24 hour period starting before the subject has been admin­ specimen is required to determine if the organism from istered the antimicrobial treatment, and calculating a breath which a breath sample is measured is in a catabolic state. delta value (BDV) for each of at least six breath samples acquired from the subject over a 24 hour period starting from [0005] Similarly, in U.S. Pat. No. 7,465,276 (the '276 when the subject has been administered the antimicrobial patent), the relative amounts of first and second breath treatment, wherein BDV is determined according to isotopes are measured over time to determine if an organism is experiencing a viral or bacterial infection. Advantages of the method of the '276 patent are that breath samples from 13 12 13 12 13 ( C/ C sample- C/ c PDE) an isotopically unenriched organism can be monitored for EDV=OC= !3C/l2CPDE xlOOO changes in isotope ratios over time to determine if the organism is experiencing a bacterial or viral infection. A disadvantage of the method is that a baseline measurement [0016] wherein PDB is a Pee Dee Belenmite reference from the healthy subject is preferred so that changes from standard, and BDV is expressed as parts per mil (%0); the baseline can be measured that are indicative of infection. [0017] calculating a mean standard deviation of BDV (SD In addition, it is generally advisable to obtain measurements BDV) across the six or more samples acquired before the over several hours or even several days so that the change in subject has been administered the antimicrobial treatment, isotope ratio from the baseline ratio can be determined. calculating an SD BDV across the six or more samples Thus, determining the transition from a healthy to an acquired after the subject has been administered the antimi­ infected organism within the short-term infection period, crobial treatment, and calculating a % decrease in BDV by e.g., 30 minutes to 2 hours, may not be possible as the subtracting the SD BDV after the antimicrobial treatment change in slope may not be measurable in this time period. from the SD BDV before the antimicrobial treatment; and US 2020/0056988 Al Feb.20,2020 2

[0018] determining that the antimicrobial treatment is [0035] determining that the antimicrobial treatment is effective when the % decrease in BDV is greater than effective when the % decrease in BDV is greater than or equal to 34%, or or equal to 34%, and continuing administering the [0019] determining that the antimicrobial treatment is initial antimicrobial treatment, or ineffective when the % decrease in BDV is less than determining that the antimicrobial treatment is ineffective 34%. when the% decrease in BDV is less than 34%, discontinuing [0020] In yet another aspect, a method of treating a subject the initial antimicrobial treatment, and administering a sub­ in need of antimicrobial treatment comprises sequent antimicrobial treatment, or adding a subsequent [0021] administering an initial antimicrobial treatment to antimicrobial treatment to the antimicrobial treatment. the subject; [0022] acquiring at least six breath samples from the BRIEF DESCRIPTION OF THE DRAWINGS subject over a 24 hour period starting from when the subject [0036] FIG. 1 depicts simulated data illustrating the inter­ has been administered the initial antimicrobial treatment; pretation of BDV from pre-infection through severe infec­ [0023] calculating a breath delta value (BDV) for each of tion. (A) Prior to infection, the baseline BDV variance is the breath samples according to typically less than 0.4%0 and is not more than 1.4%0 from the mean (left "Normal variance"). (B) As infection sets in and causes initiation of the body wide acute phase response, 13 12 13 12 13 ( C/ c sample- c/ c PDE) which can be pre-symptomatic, the BDV decreases ("Onset EDV=6 C= 13 C/l2C PDE x!OOO trend") as muscle proteins are utilized for both acute phase protein synthesis and metabolic energy needs. (C) As the infection continues to progress, there is an anaerobic shift in [0024] wherein PDB is a Pee Dee Belenmite reference metabolism that rapidly drives the BDV in positive direc­ standard, and BDV is expressed as parts per mil (%0 ); tion. An untreated, or improperly treated, infection will [0025] calculating a mean standard deviation of BDV (SD exhibit increased variance (of 0.55%0 or more) due to the BDV) across the six or more samples; and competing isotopic mechanisms. (D) When appropriate anti­ [0026] determining that the initial antimicrobial treat­ microbial treatments are administered the variance in BDV ment is effective when the SD BDV is less than or equal normalizes. to 0.46, and continuing administering the initial anti­ [0037] FIG. 2 shows a Consort Flow Diagram for study microbial treatment, or enrollment for Example 1. Subjects were enrolled when [0027] determining that the antimicrobial treatment is admitted to the ICU. The subjects were 90% blunt trauma ineffective when the SD BDV is greater than 0.46, and 10% post-operative in both the infection and non­ discontinuing the initial antimicrobial treatment, and infection groups. administering a subsequent antimicrobial treatment, or [0038] FIG. 3 shows the variation in BDV after ICU adding a subsequent antimicrobial treatment to the admission, infection or antibiotic treatment. ICU admitted antimicrobial treatment. non-infection subjects did not develop infections during the [0028] In a further aspect, a method of treating a subject study (n=9). The untreated infection represents the variation in need of antimicrobial treatment comprises in BDV before antibiotic administration in subjects that [0029] acquiring at least six breath samples from the developed infections (n=ll). The average 6 sample standard subject over a 24 hour period; deviation was calculated by determining the variation for [0030] administering an initial antimicrobial treatment to each sample with the 5 preceding samples, then averaging the subject; the variation for each timepoint for each subject then taking [0031] acquiring at least six breath samples from the the square root of the variation resulting in the average subject over a 24 hour period starting from when the subject standard deviation. The treated infection group represents has been administered the initial antimicrobial treatment; the standard deviation in BDV for the 6 samples immedi­ [0032] calculating a breath delta value (BDV) for each of ately following administration of an antibiotic treatment the breath samples acquired before and after the initial (n=ll). Error bars represent the standard error of the mean, antimicrobial treatment according to and differing letter super scripts represent significant differ­ ences between groups. Statistical analysis was performed using analysis of variance (ANOVA) approach with least

13 12 13 12 significant differences post-hoc analysis. Differences were 13 ( C/ C sample- C/ c PDE) EDV=6 C= l3C/l2C PDE x!OOO considered significant with a p-value of less than 0.05. [0039] FIGS. 4-13 show the variation in BDV over time for individual subjects. [0033] wherein PDB is a Pee Dee Belenmite reference [0040] FIG. 14 shows a representative example of a sub­ standard, and BDV is expressed as parts per mil (%0 ); ject who did not develop an infection. White blood cell count [0034] calculating a mean standard deviation of BDV (SD and core body temperature remained in the normal range per BDV) across the six or more samples acquired before the the SIRS definition for the duration of the study. C-reactive subject has been administered the initial antimicrobial treat­ protein (CRP) and procalcitonin (PCT) remained elevated ment, calculating an SD BDV across the six or more samples with an average value of 16.9(1.2) and 0.47(0.06) respec­ acquired after the subject has been administered the initial tively. Nutritionally, the subject was NPO (nil per os; unfed) antimicrobial treatment, and calculating a % decrease in for the first five days of the study and was transitioned to a BDV by subtracting the SD BDV after the initial antimi­ liquid diet on day 6. In this subject, the BDV remains within crobial treatment from the SD BDV before the initial anti­ 1%0 of the baseline sample for the duration of the study. The microbial treatment; and mean variance for this subject was 0.28%0. US 2020/0056988 Al Feb.20,2020 3

[0041] FIG. 15 shows an example of a subject who devel­ weight of the amino acid. This fractionation follows the oped an infection within the first day of study enrollment. principles of the kinetic isotope effect. Approximately 1% of Body temperature spiked out of the normal range each day the world's stable carbon has the atomic weight of 13 (1 3 C); of the study except day seven and ten with a peak tempera­ most of the remaining carbon is 12C. Amino acids and ture of 38.9 C on day one. White blood cell count (WBC) products that have incorporated 13C are less likely to be fully was out of the normal range every day of the study and metabolized to CO2 than amino acids that have incorporated 3 12 ranged from 14.2 to 22.4 cell/mm • CRP concentration C, the lighter isotope. The "heavier" carbon amino acids ranged from 3.7 mg/dL on day one to a peak of7.9 mg/dL remain as products of the APR (i.e., acute phase proteins) on day four with a low of 2.4 mg/dL on day seven. PCT while the "lighter" carbon amino acids are more likely to be peaked on day one of the study with a value of 0.25 ng/dL completely oxidized to CO2 . The result of this phenomenon 13 12 and fell to a low value of 0.08 ng/dL on day seven. Initial is that the ratio of CO2 to CO2 in breath decreases during blood and BAL cultures were negative on days one and two, the onset of infection (FIG. lB). However, as the infection but subsequent bronchioalveolar lavage (BAL) cultures progresses, patient metabolism shifts and becomes more were positive for Aspergillus fumigatus on day seven. The anaerobic, and macronutrient metabolism changes from subject was given Cefuroxime from day one to day eight of primarily a mixture of carbohydrates and lipids to primarily the study for surgical site infection prophylaxis, but when carbohydrate and body proteins. Due to isotopic discrimi­ cultures reported positive with a fungal infection on day nation against 13C at several steps in their synthesis, lipids seven cefepime was added. Cefepime treatment was contin­ are 3-5%0 lighter than carbohydrates or proteins. Thus, a ued beyond the end of the study. The progression trend is shift in macronutrient oxidation during the progression of evident on days 1 through 7 despite cefuroxime treatment infection causes a rapid increase in the 13C: 12C ratio (FIG. with a mean variation of 0.72%0, but reduced variation of lC). Because of the competing isotopic mechanisms during 0.37%0 after cefepime indicates response to treatment. an untreated infection the BDV has a higher variance than [0042] FIG. 16 shows an example wherein a subject normal (FIG. lA). After appropriate antimicribial treatment developed an infection and appropriate treatment was is administered the variance in BDV returns to normal (FIG. administered. The initially high WBC (13.1 k cells/mm3) lD). fell into the normal range on days three through five when [0046] As used herein, the breath delta value (Ii 13C) is it increased to a peak of 15.2 k cells/mm3 on day seven. calculated using the following formula with Pee Dee Belem­ Body temperatures spiked daily from day three to eight with nite (PDB) as the reference standard. a peak of 40 C on day six. Blood and BAL cultures confirm infection on day six and Vancomycin and cefepime are administered. The CRP remained between 8.1 to 12.0 mg/dL 13 12 13 12 _ 13 _ ( C/ C sample- C/ c PDE) on days one through six when it went up to 15.2 on day EDV - 6 C - 13 C/l2C PDE x 1000 seven. The initially high PCT (4.94 ng/mL) fell from day one to six, when it spiked to 8.81 ng/mL on day seven. While the BDV infection onset trend was unclear in this case, the [0047] Data are expressed as delta values in parts per ml progression trend is evident in the increasing BDV and the (%0). variability of the BDV on days 2 through 4 with a mean variance of 0.51 %0. Antibiotic treatment was begun on 5 of [0048] The standard isotope ratio of PDB is 0.0112372. A 13 the study and the BDV variability decreases to 0.15%0, positive Ii C occurs when the measured isotope ratio is 13 indicating successful antibiotic treatment. higher relative to PDB, while a negative Ii C occurs when [0043] The above-described and other features will be the measured isotope ratio is lower relative to PDB. Since 13 appreciated and understood by those skilled in the art from PDB contains the heaviest known naturally occurring C/ 12 the following detailed description, drawings, and appended C ratio, all measurements of the natural abundance of claims. carbon isotopes are negative. [0049] In one aspect, breath samples are collected in DETAILED DESCRIPTION sample bags such as 1 L Tedlar or metal foil bags. Breath [0044] Unexpectedly, it is shown herein that breath delta samples can be directly collected into an instrument value (BDV) is a marker for the response to antimicrobial designed for such collection. treatment, such as in acute surgical and trauma subjects [0050] In one embodiment, relative isotope measurements admitted to the ICU. Specifically, the variation in BDV of are made using cavity ringdown spectroscopy (CRDS). infected subjects is lower after appropriate antimicrobial CRDS uses infrared laser absorption to measure the con­ treatment than after ineffective or incorrect antimicrobial 13 12 centrations of CO2 and CO2 carbon signals, and reports treatment. 13 12 precise total CO2 levels as well as the COi CO2 ratio. An [0045] During the early onset of the acute phase response exemplary instrument is a Picarro G2101-i Isotopic CO2 to trauma, the BDV is inversely related to the severity of analyzer. Other methods to measure breath Ii 13C include trauma and, to a greater extent, the presence of a developing isotope mass spectrometry. infection. FIG. 1 illustrates simulation of BDV from pre­ [0051] In an aspect, a method of determining efficacy of an infection therough severe infection and response to treat­ antimicrobial treatment in a subject comprises ment. Pre-formed tumor necrosis factor alpha (TNFa) and interleukin-I (IL-1) induce changes in secondary metabo­ [0052] calculating a breath delta value (BDV) for each of lism. Amino acids are rapidly released from skeletal muscle at least six breath samples acquired from the subject over a where they can be used to make acute phase proteins or be 24 hour period starting from when the subject has been metabolized for fuel. Amino acids released during the acute administered the antimicrobial treatment, wherein BDV is phase response (APR) fractionate based on the molecular determined according to US 2020/0056988 Al Feb.20,2020 4

[0066] calculating a breath delta value (BDV) for each of - 13 _(13c;12Csample-13c;12CPDE)x1000 the breath samples according to EDV-6 C- 13Cj12CPDE

- 13 - (13c;12c sample-13c;12c PDE) x!O00 [0053] wherein PDB is a Pee Dee Belemnite reference EDV - 6 C - 13C/12C PDE standard, and BDV is expressed as parts per mil (%0); [0054] calculating a mean standard deviation of BDV (SD BDV) across the six or more breath samples; and [0067] wherein PDB is a Pee Dee Belemnite reference standard, and BDV is expressed as parts per mil (%0); [0055] determining that the antimicrobial treatment is effective when the SD BDV is less than or equal to [0068] calculating a mean standard deviation of BDV (SD 0.46, or BDV) across the six or more samples; and [0069] determining that the initial antimicrobial treat­ [0056] determining that the antimicrobial treatment is ment is effective when the SD BDV is less than or equal ineffective when the SD BDV is greater than 0.46. to 0.46, and continuing administering the initial anti­ [0057] In another aspect, a method of determining efficacy microbial treatment, or of an antimicrobial treatment in a subject comprises [0070] determining that the antimicrobial treatment is [0058] calculating a breath delta value (BDV) for each of ineffective when the SD BDV is greater than 0.46, at least six breath samples acquired from the subject over a discontinuing the initial antimicrobial treatment, and 24 hour period starting before the subject has been admin­ administering a subsequent antimicrobial treatment, or istered the antimicrobial treatment, and calculating a breath adding a subsequent antimicrobial treatment to the delta value (BDV) for each of at least six breath samples antimicrobial treatment. acquired from the subject over a 24 hour period starting from [0071] As shown, for example in FIG. 4, a 53.1 % decrease when the subject has been administered the antimicrobial in SD of BDV observed after antimicrobial treatment dem­ treatment, wherein BDV is determined according to onstrates that appropriate antimicrobial treatment was administered to this subject. In contrast, as shown in FIG. 5, a 17.7% decrease in SD of BDV observed after antimicro­ - 13 _(13c;12Csample-13c;12CPDE)x1000 bial treatment demonstrates that appropriate antimicrobial EDV-O C- 13C/12CPDE treatment was not administered to this subject. [0072] Exemplary subjects are mammalian subjects, spe­ cifically human subjects. In any of the methods described [0059] wherein PDB is a Pee Dee Belemnite reference standard, and BDV is expressed as parts per mil (%0); herein, the subject can be an acute surgical or trauma subject admitted to an intensive care unit and suspected of having an [0060] calculating a mean standard deviation of BDV (SD infection. BDV) across the six or more samples acquired before the [0073] Also in any of the embodiments described herein, subject has been administered the antimicrobial treatment, the subject meets at least two systemic inflammatory calculating an SD BDV across the six or more samples response syndrome criteria, infection is suspected based on acquired after the subject has been administered the antimi­ diagnostic imaging, or infection is suspected based on crobial treatment, and calculating a % decrease in BDV by culture results. Systemic inflammatory response syndrome subtracting the SD BDV after the antimicrobial treatment criteria include body temperature >38° C. or <36° C., heart from the SD BDV before the antimicrobial treatment; and rate >90 beats/min, respiratory rate >20 breaths/min, or [0061] determining that the antimicrobial treatment is 3 3 white blood cell > 12,000 cells/mm or <4,000 cells/mm , or effective when the % decrease in BDV is greater than > 10% immature neutrophils. or equal to 34%, or [0074] In any of the foregoing embodiments, the subject [0062] determining that the antimicrobial treatment is may be intubated. ineffective when the % decrease in BDV is less than [0075] Exemplary antimicrobials include antibiotics, anti­ 34%. fungals, antivirals, and antiparasitics. [0063] In the foregoing embodiments, wherein it is deter­ [0076] Exemplary antibiotics include aztreonam; cefo­ mined that the antimicrobial treatment is effective and the tetan and its disodium salt; loracarbef; cefoxitin and its antimicrobial treatment may be continued. In the foregoing sodium salt; cefazolin and its sodium salt; cefaclor; ceftib­ embodiments, wherein it is determined that that the antimi­ uten and its sodium salt; ceftizoxime; ceftizoxime sodium crobial treatment is ineffective, the antimicrobial treatment salt; cefoperazone and its sodium salt; cefuroxime and its may be discontinued and a subsequent antimicrobial treat­ sodium salt; cefuroxime axetil; cefprozil; ceftazidime; cefo­ ment may be initiated; or a subsequent antimicrobial treat­ taxime and its sodium salt; cefadroxil; ceftazidime and its ment may be added to the antimicrobial treatment. For sodium salt; cephalexin; hexachlorophene; cefamandole example, the subject may be treated with an antibiotic for a nafate; cefepime and its hydrochloride, sulfate, and phos­ suspected bacterial infection, however, upon determination phate salt; cefdinir and its sodium salt; ceftriaxone and its of a fungal infection, an antifungal may be used in addition sodium salt; cefixime and its sodium salt; cetylpyridinium to or in place of the antibiotic. chloride; ofoxacin; linexolid; temafloxacin; fleroxacin; [0064] A method of treating a subject in need of antimi­ enoxacin; gemifloxacin; lomefloxacin; astreonam; tosu­ crobial treatment comprises administering an initial antimi­ floxacin; clinafloxacin; cefpodoxime proxetil; chloroxyle­ crobial treatment to the subject; nol; methylene chloride, iodine and iodophores (povidone­ [0065] acquiring at least six breath samples from the iodine); nitrofurazone; meropenem and its sodium salt; subject over a 24 hour period starting from when the subject imipenem and its sodium salt; cilastatin and its sodium salt; has been administered the initial antimicrobial treatment; azithromycin; clarithromycin; dirithromycin; erythromycin US 2020/0056988 Al Feb.20,2020 5

and hydrochloride, sulfate, or phosphate salts ethylsucci­ feron type III, Interferon type II, Interferon type I, Inter­ nate, and stearate forms thereof, clindamycin; clindamycin feron, Lamivudine, Lopinavir, Loviride, Maraviroc, hydrochloride, sulfate, or phosphate salt; lincomycin and Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir, hydrochloride, sulfate, or phosphate salt thereof, tobramycin Nucleoside analogues, Oseltamivir (Tamiflu), Peginterferon and its hydrochloride, sulfate, or phosphate salt; streptomy­ alfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, cin and its hydrochloride, sulfate, or phosphate salt; vanco­ Protease inhibitor (pharmacology), Raltegravir, Reverse mycin and its hydrochloride, sulfate, or phosphate salt; transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, neomycin and its hydrochloride, sulfate, or phosphate salt; Pyramidine, Saquinavir, Stavudine, Synergistic enhancer acetyl sulfisoxazole; colistimethate and its sodium salt; (antiretroviral), Tea tree oil, Tenofovir, Tenofovir disoproxil, quinupristin; dalfopristin; amoxicillin; ampicillin and its Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, sodium salt; clavulanic acid and its sodium or potassium Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidara­ salt; penicillin G; penicillin G benzathine, or procaine salt; bine, Viramidine, Zalcitabine, Zanamivir (Relenza), Zido­ penicillin G sodium or potassium salt; carbenicillin and its vudine, and the like, and combinations comprising at least disodium or indanyl disodium salt; piperacillin and its one of the foregoing. sodium salt; .alpha.-terpineol; thymol; taurinamides; nitro­ [0079] Exemplary antiparasitics include mebendazole, furantoin; silver-sulfadiazine; hexetidine; methenamine; pyrantel pamoate, thiabendazole, diethylcarbamazine, iver­ aldehydes; azylic acid; silver; benzyl peroxide; alcohols; mectin, niclosamide, praziquantel, albendazole, rifampin, carboxylic acids; salts; nafcillin; ticarcillin and its disodium amphotericin B, melarsoprol, eflomithine, metronidazole, salt; sulbactam and its sodium salt; methylisothiazolone, tinidazole, miltefosine, and the like, and combinations com­ moxifloxacin; amifloxacin; pefloxacin; nystatin; carbepen­ prising at least one of the foregoing. ems; lipoic acids and its derivatives; beta-lactams antibiot­ [0080] The invention is further illustrated by the following ics; monobactams; aminoglycosides; microlides; lincos­ non-limiting examples. amides; glycopeptides; tetracyclines; chloramphenicol; quinolones; fucidines; sulfonamides; macrolides; cipro­ Methods floxacin; ofloxacin; levofloxacins; teicoplanin; mupirocin; norfloxacin; sparfloxacin; ketolides; polyenes; azoles; peni­ [0081] Study Design: cillins; echinocandines; nalidixic acid; rifamycins; oxalines; [0082] The study was conducted as a multi-center pro­ streptogramins; lipopeptides; gatifloxacin; trovafloxacin spective study, at four academic research hospitals, to assess 13 12 mesylate; alatrofloxacin mesylate; trimethoprims; sulfame­ exhaled CO2 / CO2 BDV as an indicator of infection in 20 thoxazole; demeclocycline and its hydrochloride, sulfate, or critically ill and injured adult ICU subjects. Critically ill phosphate salt; doxycycline and its hydrochloride, sulfate, or adult ICU subjects who were not suspected of having an phosphate salt; minocycline and its hydrochloride, sulfate, infection at the time of ICU admission were enrolled as or phosphate salt; tetracycline and its hydrochloride, sulfate, study subjects. or phosphate salt; oxytetracycline and its hydrochloride, [0083] Inclusion Criteria: sulfate, or phosphate salt; chlortetracycline and its hydro­ [0084] 1) age 18 years or older; chloride, sulfate, or phosphate salt; metronidazole; dapsone; [0085] 2) critically ill patient admitted to the ICU; atovaquone; rifabutin; linezolide; polymyxin B and its [0086] 3) enrolled within 48 hours of ICU admittance; hydrochloride, sulfate, or phosphate salt; sulfacetamide and [0087] 4) expected duration of hospital stay at least 120 its sodium salt; clarithromycin; and the like, and combina­ hours (five days) from time of study enrollment; and tions comprising at least one of the foregoing. [0088] 5) subject or legally authorized representative [0077] Exemplary antifungals include amphotericin B; speaks a language of which the IRB has approved a pyrimethamine; flucytosine; caspofungin acetate; flucon­ consent form. azole; griseofulvin; terbinafine and its hydrochloride, sul­ [0089] Exclusion Criteria: fate, or phosphate salt; amorolfine; triazoles (Voriconazole ); [0090] 1) known or suspected infection at time of flutrimazole; cilofungin; LY303366 (echinocandines ); pneu­ enrollment; mocandin; imidazoles; omoconazole; terconazole; flucon­ [0091] 2) known use of systemic antibiotic, antimicro­ azole; amphotericin B, nystatin, natamycin, liposomal amp­ bial and/or antifungal therapy within the seven days tericin B, liposomal nystatins; griseofulvin; BF-796; MTCH prior to hospital admission; 24; BTG-137586; RMP-7/Amphotericin B; pradimicins; benanomicin; ambisome; ABLC; ABCD; Nikkomycin Z; [0092] 3) currently active cancer, defined as receiving flucytosine; SCH 56592; ER30346; UK 9746; UK 9751; T treatment or intend to receive treatment within hospital 8581; LY121019; ketoconazole; micronazole; clotrimazole; stay for cancer (including but not limited to: radiation, econazole; ciclopirox; naftifine; itraconazole; and the like, chemotherapy, systemic orals, etc.); and combinations comprising at least one of the foregoing. [0093] 4) if not intubated, unable to cooperate with providing a breath sample; [0078] Exemplary antivirals include Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, [0094] 5) expected death within 24 hours of enrollment Arbidol, Atazanavir, Atripla (fixed dose drug), Boceprevir, or lack of commitment to aggressive treatment by Cidofovir, Combivir (fixed dose drug), Darunavir, Delavir­ family/medical team ( e.g., likely to withdraw life sup­ dine, Didanosine, Docosanol, Edoxudine, Efavirenz, port measures within 24 hrs of screening); Emtricitabine, Enfuvirtide, Entecavir, Entry inhibitors, Fam­ [0095] 6) female who was pregnant or lactating (nega­ ciclovir, Fixed dose combination (antiretroviral), Fomi­ tive serum or urine pregnancy test results within 48 virsen, Fosamprenavir, Foscamet, Fosfonet, Fusion inhibi­ hours of enrollment or to be performed during screen­ tor, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, ing); Imiquimod, Indinavir, Inosine, Integrase inhibitor, Inter- [0096] 7) prisoner; US 2020/0056988 Al Feb.20,2020 6

[0097] 8) known participation in an investigational and subjects categorized as 'high susp1c10n of infection' or interventional research study within 30 days prior to 'overt infection' were grouped and considered to have enrollment; developed an infection. [0098] 9) individuals who were directly affiliated with [0104] Breath Sample Analysis: sponsor or study staff, or their immediate families; and [0105] Breath samples were shipped to a central labora­ [0099] 10) any patient that was deemed unfit for study tory (Isomark, LLC, Madison, Wis.) within 48 hours of final participation, per the Investigator's discretion. collection from each subject. The breath samples were [0100] Study team members conducted the informed con­ 13 12 analyzed using the Canary™ device. The CO/ CO2 ratio sent discussion with the potential subject or surrogate in a of each sample was determined from direct measurement location where a private conversation could be held. The and calculated using Pee Dee Belemnite (PDB) as a standard study team member explained the study procedures, the reference: purpose of the study, and that treatment of the potential subject is not the purpose of the study. Coercion was prevented by stressing that the potential subject or surrogate does not have to agree to participate, and that the care of the potential subject will not be affected by the decision to participate. [0101] Exhaled breath samples were collected upon sub­ where BDV is expressed as parts per mil (%0). Each subject ject enrollment and every four hours thereafter until the was used as their own control for the purpose of trend subject was discharged home, transferred to a general care analysis and the first breath sample collected was considered unit/status, or after seven days of breath sample collection, the "baseline" sample. The delta over baseline (DOB) was whichever came first. Each sample was collected in four­ then calculated by subtracting the baseline sample from hour intervals, calculated from the initial sample time, with subsequent samples as described: a window of ±1 hour. Samples collected outside of the DOB~BDV,ample-BDVbaoeHne specified time interval were still analyzed. [0102] For mechanically ventilated subjects, an appropri­ DOB values for subjects with and without infection were ately trained and qualified member of the subject's clinical graphed as a function of time, and the standard error of the care team or respiratory team obtained expired breath from mean for each time point was calculated and graphed as a side port adaptor in the expiratory limb of the subject's error bars. breathing circuit. The breath sample was captured in a small [0106] Statistical Analysis: gas tight sample bag. Sample collection did not interfere [0107] The standard deviation in BDV for the uninfected with operation of the mechanical ventilator or breathing and untreated infection groups was calculated by first find­ circuit. The breath sample was collected over approximately ing the mean variance for any 6 consecutive samples, then 2-4 breath cycles. Non-mechanically ventilated subjects calculating the standard deviation by finding the square root were asked to provide a sample by exhaling into the sample of the 6 sample variance mean. For the treated infection bag. If the subject was not ventilated but had difficulty group the standard deviation was calculated using only the inflating a bag using the supplied mouthpiece, a mask 6 samples before or after administration of antimicrobial collection option was made available. Collecting a sample treatment. Response to treatment was assessed by comput­ using the mask was performed by attaching a sample bag to ing the percent change in the standard deviation of the six the breathing mask via a connector. The mask was placed samples prior to treatment with the standard deviation of the over the subject's nose and mouth during exhalation. six samples after treatment for each subject who received [0103] An endpoint adjudication committee (EAC), com­ antimicrobial treatment for infection. To determine differ­ posed of three independent senior infectious disease experts, ences in the mean standard deviation between groups an not involved in the subject clinical care, reviewed each study ANOVA analysis with least significant differences post-hoc subject's data to determine the clinical time and date of analysis was used. Differences were considered significant if infection. Each EAC member independently reviewed the p<0.05. subject cases and completed the EAC Infection Status case report form. Further, the EAC met as a group to discuss each [0108] Sample Size Determination: subject's infection status and make a majority decision. The [0109] From preliminary data collected, a sustained group decision was based on the individual reports, with a change in BDV of 1.0%0 or more was estimated to correlate two-thirds majority needed to determine status. In cases with the onset of infection when the subject was used as where an infection developed, the EAC placed a time and his-her control. The average intra-subject standard deviation date stamp for time of first suspicion of infection and (SD) across time points in critically ill adult subjects pre­ confirmation based on clinical judgement, culture, or diag­ viously studied was 0.95%0, regardless of the underlying nostic imaging. Subjects were considered to be 'suspected of medical conditions. The inter-subject SD was expected to be infection' if they met at least two systemic inflammatory 1.0%0 at most. Using the BDV measurements during the response syndrome (SIRS) criteria (i.e., body temperature breath sample monitoring period (breath sample assess­ >38° C. or <36° C., heart rate >90 beats/min, respiratory rate ments every four hours), the expected overall SD of the >20 breaths/min, or white blood cell > 12,000 cells/mm3 or mean BDV measurements ( across time points) was less than 3 <4,000 cells/mm , or > 10% immature neutrophils ), and 1.0%0. Furthermore, during the monitoring period, the mean were given antibiotics, and/or diagnostic imaging or cultures difference in the BDV between subjects who were diagnosed were ordered. For statistical analysis, subjects categorized with an infection and subjects not diagnosed with an infec­ by the EAC as 'no suspicion' or 'low suspicion of infection' tion was expected to be 1.0%0 or more (primary hypothesis). were grouped and considered to have no infection, and Assuming an overall standard deviation of 1.0%0, a total US 2020/0056988 Al Feb.20,2020 7

sample size at least eight infections was required for 80% than five days (exclusion criteria), one experienced agitation power to detect a difference of 1.0%0 with a two-sided and refused to provide samples, and in one case the sample p-value of less than 0.05. collection device was mistakenly placed on the inspiratory limb of the ventilator instead of the expiratory limb (user Results: error). Table 1 lists the ICU admitting diagnosis for each [0110] During the study, 32 subjects were consented for subject, and the evidence of infection used to classify each participation after acute trauma or surgery. Three of the subject as infected or non-infected. Of the 20 subjects consented subjects were withdrawn due to meeting exclu­ included in the analysis, infections developed in 11 of the sion criteria, and two were withdrawn by the investigator subjects, one of which was on antimicrobial treatment (FIG. 2). Seven of the analyzed subjects were excluded from before enrollment. Since each subject was used as their own analysis due to inadequate breath sample collection (FIG. 2). control for the BDV measurement, the delta over baseline Of the seven excluded subjects, five were discharged in less calculation was used for breath measurements.

TABLE 1

Clinical diagnostics for each subject analyzed in the 'infection' group based on the EAC review. Of the 9 non-infected, 8 were admitted as 'Trauma- Blunt', and 1 as 'Post-op'.

Suspected Confirmed Infection Detailed results of Infection ICU Infection Infection Category/ HR (bpm), Temp (degrees celsius), Admitting (Hrs since (Hrs since Diagnostic RR (breaths/min) Subject Diagnosis enrollment) enrollment) mechanism WBC (kcells/uL), Bands(%)

Trauma- 11.5 128.0 High HR: 121 Temp: 39.5 RR: 28 WBC: Blunt Suspicion 10.3 (75% Bands) of Infection Culture: BAL- S. aureus & Candida sp, Culture endogenous Proven Xray: no infection suspected, both atalectasis Vancomycin and Piperacillin- Tazobactarn 2 Trauma- 43.2 131.2 High HR: 93 Temp: 38.8 RR: 33 WBC: 14.2 Blunt Suspicion (81 % bands) of Infection Culture: BAL- No growth present Clinical Xray: no infection, both atelectasis, Suspicion both effusion Metronidozole and Vancomycin 3 Trauma- 75.5 103.8 Overt HR: 129 Temp 40.0 RR: 31 WBC: 12.1 Blunt Infection (80% Bands) Culture Culture: BAL- Strep. constellatu & Proven Staph. Aureus Xray: no infection, R atalectasis, R effusion, bilat pneumothorax Vancomycin and Cefepime 4 Trauma- 4.0 14.0 Overt HR: 122 Temp: 38.4 RR: 26 WBC: Blunt Infection 10.8 (76% Bands) Culture Culture: BAL- Haemophilias influenza Proven & Staph. Aureus, endogenous flora Xray: Infection suspected- subcutaneous emphysema, R atalectasis, L chest tube, L contusion Ciprofloxacin, Vancomycin and Cefepime 5 Trauma- 0.0 131.4 Overt HR: 108 Temp: 38.3 RR: 27 WBC: 21.2 Blunt Infection (52% Bands) Culture Culture: Fungal with smear- apergillus Proven fuigatus BAL- acinetobacter baumanii, klebsiella oxytoca, endogenous flora Xray: no infection, Bilat atelectasis, R effusion Cefepime 6 Trauma- 88.2 152.3 Overt HR: 113 Temp: 38.4 RR: 35 WBC: Blunt Infection 14.1 (Bands not reported) Culture Culture: BAL- P. aeruginosa Proven (>100,000) & E. coli (11,000) Cefepime 7 Trauma- 43.3 60.2 Overt HR: 139 Temp: 39.4 RR: 27 WBC: Blunt Infection 10.2 (Bands not reported) Culture Culture: Urine- enterococcus, BAL- Proven gram negative rods, coccobacilli (haemophilos influenzae) Ceftriaxone Rocephin 8 Trauma- 24.4 61.3 Overt HR: 151 Temp: 37.7 RR: 36 WBC: 8.9 Unk Infection (Bands not reported) Culture Culture: BAL-Pseudomonas Proven aeruginosa > 100,000 US 2020/0056988 Al Feb.20,2020 8

TABLE I-continued

Clinical diagnostics for each subject analyzed in tbe 'infection' group based on tbe EAC review. Of the 9 non-infected, 8 were admitted as 'Trauma- Blunt', and 1 as 'Post-op'.

Suspected Confirmed Infection Detailed results of Infection ICU Infection Infection Category/ HR (bpm), Temp (degrees celsius), Admitting (Hrs since (Hrs since Diagnostic RR (breatbs/min) Subject Diagnosis enrollment) enrollment) mechanism WBC (kcells/uL), Bands(%)

Xray: increased conspicuity of airspace opacities witbin tbe R perihilar and R upper lobe regions Cefepime 9 Trawna- 3.3 20.4 Overt HR: 101 Temp: 38.6 RR: 26 WBC: Blunt Infection 13.9 (Bands not reported) Culture Culture: BAL- Gram Negative> 100,000 Proven Serratia marcescens Xray: Pulmonary Interstitial Edema 10 Post-Op 124.0 133.8 High HR: 123 Temp: 37.1 RR: 19 WBC: Suspicion 20.4 (Bands not reported) oflnfection CT Abd/Pelv: 7 cm fluid collection CT Proven within the mesorectal region which extend to presacral Metronidazole and Ciprofloxacin 11 Trawna- 78.5 105.4 Overt HR: 113 Temp: 39.5 RR: 30 WBC: 14.8 Blunt Infection (Bands not reported) Culture Culture: BAL- MRSA" 100,000/mL Proven colonies, citrobacter koseri :c:: 100,000 colonies/mL Xray: Possible aspiration pneumonia, possible cavitary pneumonia

[0111] The variation in the BDV was examined separately [0116] For the individual in FIG. 7, the SD of BDV was in subjects without infection, with infection prior to appro­ 0.56, and after antibiotic treatment the SD was 0.37. A priate treatment, and following antimicrobial treatment. The 34.6% decrease in SD of BDV was observed after antimi­ mean standard deviation in subjects without infection was crobial treatment. Thus, the variation in BDV before and 0.40%0±0.02, while it was 0.55%0±0.02, significantly higher, after antimicrobial treatment shows that appropriate antimi­ in subjects with an untreated infection. The mean standard crobial treatment was administered to this subject. deviation in infected subjects treated with appropriate anti­ [0117] For the individual in FIG. 8, the SD of BDV was microbials was 0.36%0±0.05 and similar to uninfected sub­ 0.73, and after antibiotic treatment the SD was 0.46. A jects and significantly lower than in subjects with untreated 36.2% decrease in SD of BDV was observed after antimi­ infection (FIG. 3). For each individual who developed an crobial treatment. Thus, the variation in BDV before and infection during the study, the standard deviation was cal­ after antimicrobial treatment shows that appropriate antimi­ culated for the six samples before and after administration of crobial treatment was administered to this subject. Note that appropriate antibiotics, and the percent change in standard only 5 breath samples were taken post-treatment, and at least deviation after treatment was calculated. The mean percent 6 samples is preferred. decrease after antimicrobial treatment was 44.3%±5.5. [0118] For the individual in FIG. 9, the SD of BDV was [0112] FIGS. 4-13 show graphic representations of the 0.52, and after antibiotic treatment the SD was 0.29. A change in standard deviation after antimicrobial administra­ 43.9% decrease in SD of BDV was observed after antimi­ tion for ten individuals. crobial treatment. Thus, the variation in BDV before and [0113] For the individual in FIG. 4, the SD of BDV was after antimicrobial treatment shows that appropriate antimi­ 0.38, and after antibiotic treatment the SD was 0.18. A crobial treatment was administered to this subject. 53.1 % decrease in SD of BDV was observed after antimi­ [0119] For the individual in FIG. 10, the SD of BDV was crobial treatment. Thus, the variation in BDV before and 0.60, and after antibiotic treatment the SD was 0.27. A after antimicrobial treatment shows that appropriate antimi­ 54.9% decrease in SD of BDV was observed after antimi­ crobial treatment was administered to this subject. crobial treatment. Thus, the variation in BDV before and [0114] For the individual in FIG. 5, the SD of BDV was after antimicrobial treatment shows that appropriate antimi­ 0.76, and after antibiotic treatment the SD was 0.62. Only a crobial treatment was administered to this subject. 17.7% decrease in SD of BDV was observed after antimi­ [0120] For the individual in FIG. 11, the SD of BDV was crobial treatment. Thus, the variation in BDV before and 0.49, and after antibiotic treatment the SD was 0.27. A after antimicrobial treatment shows that appropriate antimi­ 44.7% decrease in SD of BDV was observed after antimi­ crobial treatment was not administered to this subject. crobial treatment. Thus, the variation in BDV before and [0115] For the individual in FIG. 6, the SD of BDV was after antimicrobial treatment shows that appropriate antimi­ 0.43, and after antibiotic treatment the SD was 0.15. A crobial treatment was administered to this subject. 64.7% decrease in SD of BDV was observed after antimi­ [0121] For the individual in FIG. 12, the SD of BDV was crobial treatment. Thus, the variation in BDV before and 0.51, and after antibiotic treatment the SD was 0.40. A after antimicrobial treatment shows that appropriate antimi­ 21.8% decrease in SD of BDV was observed after antimi­ crobial treatment was administered to this subject. crobial treatment. Without being held to theory it is believed US 2020/0056988 Al Feb.20,2020 9

that appropriate antimicrobial treatment was not adminis­ case, the progression trend was evident in the increasing tered to this subject. In this case, the infection was a BDV and the variability of the BDV on days 2 through 4 localized surgical site infection which did cause a dramatic with a mean variance of 0.51 %0. Antibiotic treatment began increase in white blood cell count at the time of clinical on day 5 of the study and the BDV variability decreased to diagnosis that did not resolve for at least 4 days. The 0.15%0, indicating successful antibiotic treatment. localized infection may have caused lower SD of BDV than would be expected with a more severe infection. The un­ Discussion resolving clinical signs (i.e. abnormal white blood cell [0126] This study was a pilot investigation using BDV count) indicate the treatment was not improving the infec­ technology as an adjunct for assessing the response to tion. These reasons may account for the discrepancy with the antimicrobial treatment in critically ill trauma and surgical other cases. patients with infections. We demonstrated a method that is [0122] For the individual in FIG. 13, the SD of BDV was can quickly determine if antimicrobial treatments are effec­ 0.86, and after antibiotic treatment the SD was 0.24. A tive by assessing variation in the BDV. 71.7% decrease in SD of BDV was observed after antimi­ [0127] The variation in BDV over time may be a valuable crobial treatment. Thus, the variation in BDV before and tool for determining if antimicrobial treatments are working after antimicrobial treatment shows that appropriate antimi­ to combat the infection. Since the presence of severe infec­ crobial treatment was administered to this subject. tion instigates two competing isotopic mechanisms that [0123] FIG. 14 is a representative example of a subject work in opposing directions, the variance in untreated or who did not develop an infection. White blood cell count and inappropriately treated individuals is higher than it is in core body temperature remained in the normal range per the similar individuals without infections. When antimicrobial SIRS definition for the duration of the study. CRP and PCT treatments are applied to infected individuals, the variation remained elevated with an average value of 16.9 mg/dL±l.2 in BDV following the treatment returns to a variance similar and 0.47 ng/mL±0.06 respectively. Nutritionally, this subject to the uninfected individuals. A biomarker of the appropri­ was nil per os (NPO; withholding foods and liquids) for the ateness of antimicrobial treatment will be of significant first five days of the study and was transitioned to liquids on value to aid clinicians in antimicrobial stewardship. In the day 6. In this subject, the BDV remained within 1%o of the era of competing goals of early intervention in sepsis while baseline sample for the duration of the study. The mean limiting antibiotic exposure, a tool to rapidly diagnose and variance for this subject was 0.28%0. appropriately treat individuals is truly valuable. 13 12 [0124] FIG. 15 is a representative example of a subject [0128] The exhaled CO/ CO2 breath delta value has who developed an infection within the first day of study been shown to be a marker for appropriate antimicrobial enrollment. Body temperature spiked out of the normal treatment, a tool that will aid clinicians in determining range each day of the study except days seven and ten with appropriate treatments, and assist in antimicrobial steward­ a peak temperature of38.9° C. on day one. White blood cell ship. count (WBC) was out of the normal range every day of the [0129] The use of the terms "a" and "an" and "the" and 3 study and ranged from 14.2 to 22.4 kcells/mm • CRP similar referents ( especially in the context of the following concentration ranged from 3.7 mg/dL on day one to a peak claims) are to be construed to cover both the singular and the of 7.9 mg/dL on day four with a low of 2.4 mg/dL on day plural, unless otherwise indicated herein or clearly contra­ seven. PCT peaked on day one of the study with a value of dicted by context. The terms first, second etc. as used herein 0.25 ng/dL and fell to a low value of 0.08 ng/dL on day are not meant to denote any particular ordering, but simply seven. Initial blood and bronchioalveolar (BAL) cultures for convenience to denote a plurality of, for example, layers. were negative on days one and two, but subsequent BAL The terms "comprising", "having", "including", and "con­ cultures were positive for Aspergillus fumigatus on day taining" are to be construed as open-ended terms (i.e., seven. The subject was given Cefuroxime from day one to meaning "including, but not limited to") unless otherwise day eight of the study for surgical site infection prophylaxis, noted. Recitation of ranges of values are merely intended to but when cultures reported positive with a fungal infection serve as a shorthand method of referring individually to each on day seven cefepime was added. Cefepime treatment was separate value falling within the range, unless otherwise continued beyond the end of the study. The progression indicated herein, and each separate value is incorporated into trend was evident on days 1 through 7 despite cefuroxime the specification as if it were individually recited herein. The treatment with a mean variation of 0.72%0, but reduced endpoints of all ranges are included within the range and variation of 0.37%0 after cefepime initiation, indicating independently combinable. All methods described herein response to treatment. can be performed in a suitable order unless otherwise [0125] FIG. 16 is a representative example of a subject indicated herein or otherwise clearly contradicted by con­ who developed an infection and appropriate treatment was text. The use of any and all examples, or exemplary lan­ 3 administered. The initially high WBC (13.1 kcells/mm ) fell guage (e.g., "such as"), is intended merely to better illustrate into the normal range on days three through five, then the invention and does not pose a limitation on the scope of increased to a peak of 15.2 kcells/mm3 on day seven. Body the invention unless otherwise claimed. No language in the temperatures spiked daily from day three to eight with a specification should be construed as indicating any non­ peak of 40° C. on day six. Blood and BAL cultures con­ claimed element as essential to the practice of the invention firmed infection on day six and Vancomycin and cefepime as used herein. are administered. The CRP remained between 8.1 to 12.0 [0130] While the invention has been described with ref­ mg/dL on days one through six when it went up to 15.2 on erence to an exemplary embodiment, it will be understood day seven. The initially high PCT (4.94 ng/mL) fell from by those skilled in the art that various changes may be made day one to six, when it spiked to 8.81 ng/mL on day seven. and equivalents may be substituted for elements thereof While the BDV infection onset trend was unclear in this without departing from the scope of the invention. In addi- US 2020/0056988 Al Feb.20,2020 10

tion, many modifications may be made to adapt a particular situation or material to the teachings of the invention with­ 13 12 13 12 13 ( C/ C sample- C/ c PDE) out departing from the essential scope thereof. Therefore, it EDV=OC= 13 C/l2CPDE xlOOO is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will wherein PDB is a Pee Dee Belenmite reference stan­ include all embodiments falling within the scope of the dard, and BDV is expressed as parts per mil (%0); appended claims. Any combination of the above-described calculating a mean standard deviation ofBDV (SD BDV) elements in all possible variations thereof is encompassed by across the six or more samples acquired before the the invention unless otherwise indicated herein or otherwise subject has been administered the antimicrobial treat­ clearly contradicted by context. ment, calculating an SD BDV across the six or more 1. A method of determining efficacy of an antimicrobial samples acquired after the subject has been adminis­ treatment in a subject, comprising tered the antimicrobial treatment, and calculating a % decrease in BDV by subtracting the SD BDV after the calculating a breath delta value (BDV) for each of at least antimicrobial treatment from the SD BDV before the six breath samples acquired from the subject over a 24 antimicrobial treatment; and hour period starting from when the subject has been administered the antimicrobial treatment, wherein determining that the antimicrobial treatment is effective BDV is determined according to when the % decrease in BDV is greater than or equal to 34%, or Determining that the antimicrobial treatment is inef­ 13 12 13 12 fective when the % decrease in BDV is less than 13 ( C/ c sample- c/ c PDE) EDV=6 C= 13 C/l2C PDE x!OOO 34%. 10. The method of claim 9, wherein the subject is an acute surgical or trauma subject admitted to an intensive care unit wherein PDB is a Pee Dee Belenmite reference stan­ and suspected of having an infection. dard, and BDV is expressed as parts per mil (%0 ); 11. The method of claim 10, wherein the subject meets at calculating a mean standard deviation ofBDV (SD BDV) least two systemic inflammatory response syndrome criteria, across the six or more breath samples; and infection is suspected based on diagnostic imaging, or determining that the antimicrobial treatment is effective infection is suspected based on culture results. when the SD BDV is less than or equal to 0.46, or 12. The method of claim 10, wherein the subject is determining that the antimicrobial treatment is ineffec­ intubated. tive when the SD BDV is greater than 0.46. 2. The method of claim 1, wherein the subject is an acute 13. The method of claim 9, wherein it is determined that surgical or trauma subject admitted to an intensive care unit the antimicrobial treatment is effective and the antimicrobial and suspected of having an infection. treatment is continued. 3. The method of claim 2, wherein the subject meets at 14. The method of claim 9, wherein it is determined that least two systemic inflammatory response syndrome criteria, the antimicrobial treatment is ineffective, the antimicrobial infection is suspected based on diagnostic imaging, or treatment is discontinued and a subsequent antimicrobial infection is suspected based on culture results. treatment is initiated; or a subsequent antimicrobial treat­ 4. The method of claim 1, wherein the subject is intubated. ment is added to the antimicrobial treatment. 5. The method of claim 1, wherein it is determined that the 15. The method of claim 9, wherein cavity ringdown antimicrobial treatment is effective and the antimicrobial spectroscopy is used to measure BDV. treatment is continued. 16. The method of claim 9, wherein the antimicrobial is 6. The method of claim 1, wherein it is determined that the an antibiotic, an antifungal, an antiviral, or an antiparasitic. antimicrobial treatment is ineffective, the antimicrobial 17. A method of treating a subject in need of antimicrobial treatment is discontinued and a subsequent antimicrobial treatment, comprising treatment is initiated; or a subsequent antimicrobial treat­ administering an initial antimicrobial treatment to the ment is added to the antimicrobial treatment. subject; 7. The method of claim 1, wherein cavity ringdown acquiring at least six breath samples from the subject over spectroscopy is used to measure BDV. a 24 hour period starting from when the subject has 8. The method of v, wherein the antimicrobial is an been administered the initial antimicrobial treatment; antibiotic, an antifungal, an antiviral, or an antiparasitic. calculating a breath delta value (BDV) for each of the 9. A method of determining efficacy of an antimicrobial breath samples according to treatment in a subject, comprising calculating a breath delta value (BDV) for each of at least six breath samples acquired from the subject over a 24 hour period starting before the subject has been admin­ istered the antimicrobial treatment, and calculating a breath delta value (BDV) for each of at least six breath samples acquired from the subject over a 24 hour wherein PDB is a Pee Dee Belenmite reference standard, period starting from when the subject has been admin­ and BDV is expressed as parts per mil (%0 ); istered the antimicrobial treatment, wherein BDV is calculating a mean standard deviation ofBDV (SD BDV) determined according to across the six or more samples; and US 2020/0056988 Al Feb.20,2020 11

determining that the initial antimicrobial treatment is effective when the SD BDV is less than or equal to 13 12 13 12 13 ( C/ C sample- C/ c PDE) 0.46, and continuing administering the initial anti­ BDV=OC= 13 C/l2CPDB xlOOO microbial treatment, or determining that the antimicrobial treatment is ineffec­ tive when the SD BDV is greater than 0.46, discon­ wherein PDB is a Pee Dee Belenmite reference standard, tinuing the initial antimicrobial treatment, and and BDV is expressed as parts per mil (%0 ); administering a subsequent antimicrobial treatment, calculating a mean standard deviation ofBDV (SD BDV) or adding a subsequent antimicrobial treatment to the across the six or more samples acquired before the antimicrobial treatment. subject has been administered the initial antimicrobial 18. The method of claim 17, wherein the subsequent treatment, calculating an SD BDV across the six or antimicrobial treatment targets a different class of pathogen more samples acquired after the subject has been than the initial antimicrobial treatment. administered the initial antimicrobial treatment, and 19. The method of claim 17, wherein the initial and calculating a % decrease in BDV by subtracting the SD subsequent antimicrobial treatment are an antibiotic, an BDV after the initial antimicrobial treatment from the antifungal, an antiviral, or an antiparasitic. SD BDV before the initial antimicrobial treatment; and 20. The method of method of claim 17, wherein the determining that the antimicrobial treatment is effective subject is an acute surgical or trauma subject admitted to an when the % decrease in BDV is greater than or equal intensive care unit and suspected of having an infection. to 34%, and continuing administering the initial 21. The method of claim 20, wherein the subject meets at antimicrobial treatment, or least two systemic inflammatory response syndrome criteria, determining that the antimicrobial treatment is ineffec­ infection is suspected based on diagnostic imaging, or tive when the % decrease in BDV is less than 34%, infection is suspected based on culture results. discontinuing the initial antimicrobial treatment and 22. The method of method of claim 17, wherein the administering a subsequent antimicrobial treatment, subject is intubated. or adding a subsequent antimicrobial treatment to the 23. The method of method of claim 19, wherein cavity antimicrobial treatment. ringdown spectroscopy is used to measure BDV. 26. The method of claim 24, wherein the subsequent 24. The method of method of claim 19, wherein, prior to antimicrobial treatment targets a different class of pathogen administering an initial antimicrobial treatment to the sub­ than the initial antimicrobial treatment. ject, the method comprises calculating a breath delta value 27. The method of method of claim 24, wherein the initial (BDV) for at least two pre-infection samples, and determin­ and subsequent antimicrobial treatment are an antibiotic, an ing that the subject has an infection when a change in BDV antifungal, an antiviral, or an antiparasitic. of greater than or equal to 1.0%0 is determined. 28. The method of method of claim 24, wherein the 25. A method of treating a subject in need of antimicrobial subject is an acute surgical or trauma subject admitted to an treatment, comprising intensive care unit and suspected of having an infection. acquiring at least six breath samples from the subject over 29. The method of claim 27, wherein the subject meets at a 24 hour period; least two systemic inflammatory response syndrome criteria, administering an initial antimicrobial treatment to the infection is suspected based on diagnostic imaging, or subject; infection is suspected based on culture results. acquiring at least six breath samples from the subject over 30. The method of method of claim 27, wherein the a 24 hour period starting from when the subject has subject is intubated. been administered the initial antimicrobial treatment; calculating a breath delta value (BDV) for each of the 31. The method of method of claim 24, wherein cavity breath samples acquired before and after the initial ringdown spectroscopy is used to measure BDV. antimicrobial treatment according to * * * * *