Method of detecting sepsis-related microorganisms and detecting antibiotic-resistant sepsis-related microorganisms in a fluid sample

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 61/929,175 entitled RAPID IDENTIFICATION OF MICROBIAL PATHOGEN SPECIES, VIRULENCE FACTORS AND ANTIBIOTIC RESISTANT GENE PROFILES FROM CLINICALLY SEPTIC HUMANS, filed on Jan. 20, 2014, the entirety of which is incorporated herein by reference in various non-limiting embodiments.

TECHNICAL FIELD

The present disclosure relates to methods of pathogen detection and, more specifically, to particular methods of identification of pathogenic species and their antibiotic resistance in relation to treatment of sepsis.

SEQUENCE LISTING

This disclosure, in accordance with 37 C.F.R. § 1.52, incorporates by reference the sequence listing material contained within text file titled “06854400002—ST25.text”, created on May 18, 2015 and totaling 11,311 bytes.

BACKGROUND OF THE DISCLOSURE

Sepsis, an overwhelming microbial infection of the blood, is the tenth leading cause of death in the United States and is responsible for 6% of all human deaths. Sepsis represents a large and growing burden in the healthcare setting due to increased prevalence of antibiotic resistant strains.

Delivering rapid, cutting edge molecular methods at the point-of-care (POC) can greatly enhance an ability to diagnose and treat sepsis and combat the rise of multi-drug-resistant strains by precise early determination of appropriate antibiotic coverage.

Current sepsis management is severely limited by an inability to rapidly diagnose the pathogen(s) responsible for a critically ill patient's infection. When untreated, septic patients typically have hours to live. Thus, blood cultures are drawn from a patient at the time that sepsis is suspected and 3-4 broad-spectrum intravenous antibiotics are introduced to eliminate virtually all potential pathogens. Treatments are only de-escalated three to five days later as laboratory results return, indicating the pathogenic strain and its antibiotic sensitivity profile. Due to the extended length between diagnosis and de-escalation of treatment there remains opportunity for improvement.

SUMMARY OF THE DISCLOSURE

This disclosure provides an improved method for detecting whether sepsis-related microorganisms are present in a fluid sample. The method comprises the steps of: collecting the fluid sample from a patient; fractioning the fluid sample to isolate a quantity of microorganism cells; extracting a portion of the microorganism cells from the fluid sample; lysing a portion of the microorganism cells extracted from the fluid sample to extract microorganism DNA; amplifying the microorganism DNA from the microorganism cells from a predetermined set of DNA primers to determine whether sepsis-related microorganisms are present within the fluid sample.

This disclosure also provides an alternative method comprising the steps of: collecting the fluid sample from a patient; fractioning the fluid sample to isolate a quantity of microorganism cells; extracting a portion of the microorganism cells from the fluid sample; lysing a portion of the microorganism cells extracted from the fluid sample to extract microorganism DNA therefrom; purifying the microorganism DNA; precipitating the purified microorganism DNA with an antisolvent; dissolving the precipitated microorganism DNA in a buffer solution; amplifying the microorganism DNA from the microorganism cells; hybridizing the amplified microorganism DNA to a chip with a predetermined second set of genetic markers to determine antibiotic-resistance sepsis-related microorganisms are present within the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages in the present disclosure will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings:

FIG. 1 is a representative flowchart of one embodiment of the method;

FIG. 2 is a representative flowchart of an alternate embodiment of the method;

FIG. 3 is a table of the identified microorganism species and their particular genetic markers identified by the methods of FIGS. 1 and 2;

FIG. 4 is a table of pathogens with example LAMP primer sequences; and

FIG. 5 is a table of identified microorganisms and their antibiotic resistance levels as a result of the methods shown in FIGS. 1 and 2.

Corresponding reference characters indicate corresponding parts throughout the drawings.

DETAILED DESCRIPTION OF THE DISCLOSURE

A selected embodiment of the present disclosure will now be described with reference to the drawings. It will be apparent to those skilled in the art from this disclosure that the following description of the embodiments of the present disclosure is provided for illustration only and not for the purpose of limiting the disclosure as defined by the appended claims and their equivalents. Unless other noted, all percentages and ratios are by weight. All references are expressly incorporated herein by reference in various non-limiting embodiments.

This disclosure provides a method 10, an example of which is set forth in FIG. 1. The method 10 detects sepsis-related microorganisms are present in a fluid sample. Currently, twenty microorganisms account for about 87% of sepsis-related microbial infections identified. Moreover, almost all known microbes that cause sepsis can be accounted for in a list of 50, as shown in FIG. 3. FIG. 4 indicates the pathogens as identified with example LAMP primer sequences.

The improved method 10 begins by collecting 02 the fluid sample from a patient. A patient may be a human that has been identified with sepsis. Other animals, such as livestock, that are also susceptible to sepsis may also be “patients” for the purposes of this disclosure. The most common fluid sample that can be used is blood (e.g. collected by way of a syringe from the patient). Typically, the sample size of blood may is from one to ten milliliters but can vary depending on the size of the patient and a person of skill in the art's decision to run additional analysis.

In another embodiment, the fluid sample may be any other bodily fluid type that could be used for identification of sepsis-related microorganisms. Fluid samples from urine, cerebral spinal fluid, stool, and/or mucous membranes (i.e. mammary milk, sputum and genitourinary swab) may be utilized in order to provide more localized analysis for sepsis-related organisms. This list of fluids is not meant to be limiting and any additional fluids and or combination of fluids (i.e. aspiration from an abscess or wound) may also be used. Greater than 1 ml may be needed from each biologic fluid type. No upper limit regarding sample size theoretically exists, though approximately 10 ml is typically used. Smaller sample volume is generally available for wound and cerebrospinal fluid samples, though absolute concentration of pathogen is increased, allowing detection with a lower sample abundance. Blood samples are typically diluted to 1-10% of reaction mixture. Urine samples are typically diluted to 10-25% of reaction mixture. Sputum samples are typically diluted to 10-25% of reaction mixture. Stool samples are typically diluted to 2-5% of total reaction mixture. Wound samples are typically diluted to 1-5% of total reaction mixture. Cerebrospinal fluid is typically diluted to 10-25% of reaction mixture.

Next, the fluid sample is fractioned 04 in order to isolate a quantity of microorganism cells. This promotes an initial concentration of cells and DNA in order to continue on with the amplification process. In one embodiment, centrifugal force is applied to the fluid sample in order to isolate the quantity of microorganisms within one of three visible fractions of the fluid sample. In another embodiment, additional filtration methods and or variations on isolating the microorganism cells are also applicable depending on the fluid sample type. An alternative method for enhanced bacterial concentration for improved detection includes use of a micropillar microfluidics peripheral filtration device. This would be expected to fractionate and concentrate microorganisms (size <3 micron diameter). Additional fractions of white blood cells, red blood cells, and plasma would be separated for possible use with other medical diagnostic tests. Next, the method 10 includes the step of extracting 06 a portion of the microorganism cells from the fractionalized fluid sample. This is to promote optimal concentration of microorganism cells and microorganism DNA for the remaining steps within the method 10. It is expected that >1 ng of DNA per reaction well is needed for reliable and accurate detection of microorganisms.

Next, the method 10 includes the step of lysing 08 a portion of the microorganism cells extracted from the fluid sample to extract the microorganism DNA therefrom. In one embodiment this involves heat lysis for 95 degrees C. for 5 minutes of the extracted portion of microorganism cells to break down cell membranes and suspend the microorganism DNA within the sample fraction. Other methods are possible (mechanical, liquid homogenization, sonication, freezer-thaw).

Next, the method 10 includes the step of amplifying 12 the microorganism DNA from the microorganism cells. In one embodiment, a polymerase chain reaction (PCR) is used in order to increase the amount of DNA within the extracted sample. For example, the industrial application of the method within this disclosure may utilize isothermal loop-mediated polymerase chain reaction (LAMP) DNA amplification to accurately identify and replicate the microorganism DNA within the fluid sample. LAMP typically proceeds by high temperature isothermal amplification of a microorganism DNA template at a target temperature of from 60 to 65° C. with two to three pairs of primers used and a polymerase enzyme with high strand displacement and replicative activity (the recombinant DNA polymerase is able to displace downstream DNA encountered during synthesis, and proceeds at a rapid rate). In one embodiment, the method 10 employs four primers targeted precisely to five to six distinct regions on the gene to maximize specificity to the sepsis-related microorganisms. In another embodiment, the method 10 employs six primers related to six distinct regions on a gene to maximize the specificity to the sepsis-related microorganism. Alternate PCR techniques may also be used in order to account for lab conditions and available time-frames in conducting the method 10.

In another embodiment of the present disclosure, the amplification of the microorganism DNA is conducted until an identifiable concentration is reached. Having an identified concentration of the microorganism DNA promotes identification of the possible microorganisms within the fluid sample. In various embodiments, approximately 0.5 ng DNA/reaction is needed for successful amplification.

The industrial application of the method 10 within this disclosure may utilize a Gene-Z POC analysis machine to return data related to the positive identification of microorganism based on amplification of microbial DNA within the fluid sample by specific primers in the Gene-Z plate reaction wells. For example, data may be delivered in the form of time to threshold and estimated copy number of microorganism nucleotide sequences based on calibration curves that have been generated by lab sample serial dilution testing. Baseline signal intensity can be generated during the first 6 minutes of an amplification run. The baseline signal can then be subtracted from raw signals and the difference curves are smoothed using average signal intensity from 20 consecutive points. Dividing the threshold difference by the maximum difference then normalizes curves. Time to threshold can then be calculated as the time to normalized difference in threshold exceeding an arbitrary cut-off of 0.1.

Finally, the method 10 includes the step of amplifying 16 the microorganism DNA by a predetermined set of DNA primers to determine whether sepsis-related microorganisms are present within the fluid sample. In one embodiment, many or all sepsis-related microorganisms can be determined based on particular primers. For example, Presence of Staphylococcus aureus can be detected with vicK, nuc and coA gene amplification, methicillin resistance detected by mecA amplification. Staphylococcus epidermidis can be identified by 16S RNA with methicillin resistance detected by mecA amplification. Streptococcus agalactiae species determination can be made based on ssa and mstA amplifications. Streptococcus pyogenes can be identified through mstA amplification. E.coli species can be identified based on uidA amplification and determined to be non-pathogenic (O194 strain) based on stx1, stx2 and eaeA negativity. Klebsiella pneumoniae can be identified by uge, wabB gene amplifications. Enterococcus faecalis can be identified by ef0027, and ace. In various embodiments, up to 50 sepsis-related microorganisms can have primers for testing through the methods of this disclosure. The primers are typically designed from a consensus of alleles for a gene unique to the microbial species. Primers targeting virulence and antibiotic resistance markers for bacterial pathogens can be designed using PrimerExplorer4 or retrieved from the literature. Additional methods of microbial signature identification include employment of open source resources such as the Tool for PCR Signature Identification (TOPSI) (htt://w.w.w.bhsai.org/downloads/topsi.tar.gz), the Insignia Center for Bioinformatics and Computational Biology (http://insignia.cbcb.umd.edu). High throughput primer generation is also possible using the open-source program LAVA (LAMP Assay Veratile Analysis) (http://lava-dna.googlecode.com/). Primer specificity is specifically checked against the NCBI GeneBank database using NCBI BLAST. These primers can be supplied and PCR validation reactions performed according to standard protocols for both conventional RT-PCR thermocycler analysis and the Gene-Z device.

ANTIBIOTIC RESISTANCE ANALYSIS

In another embodiment of the present disclosure, e.g. as shown in FIG. 2, the disclosure describes an alternate improved method 20 for detecting whether antibiotic-resistant sepsis-related microorganisms are present in a fluid sample.

The method 20 includes the steps of collecting 22 the fluid sample from a patient (as described at step 02 above); fractioning 24 the fluid sample to isolate a quantity of microorganism cells (as described at step 04 above); extracting 26 a portion of the microorganism cells from the fluid sample (as described at step 06 above); and lysing 28 a portion of the microorganism cells extracted from the fluid sample to extract microorganism DNA (as described at step 08 above). Any one or more of these steps may be the same or different from those described above.

Then, the method 20 further includes the step of purifying 30 the microorganism DNA. In one embodiment this is done through a phenol cholorform extraction (by mixing the sample with equal volumes of a phenol chloroform mixture), in order to concentrate the nucleic acids and reduce the presence of proteins attached to the microorganism DNA from the fluid solution.

Next, the method 20 includes the step of precipitating 40 the purified microorganism DNA with an antisolvent. In one embodiment ethanol is used as the antisolvent. This step forms precipate from the purified solution containing a higher concentration of the microorganism DNA for analysis.

Next, the method 20 continues by dissolving 38 the precipitated microorganism DNA in a buffer solution. In one embodiment, the buffer solution is 50 μl of Tris-EDTA (TE) buffer. The quantity and particular buffer may vary in based on the current conditions. Particularly, other fluid sample types may include additional purification steps as well as other buffers, such as phosphate buffered saline, in order to effectively dissolve the extracted microorganism DNA.

Next, the method 20 amplifies 36 the microorganism DNA from the microorganism cells. This is analogous to step 12 above, but may include additional or different steps as well as appreciated by those of skill in the art.

Next, the method 20 further includes the step of amplification and hybridization of 38 a nucleotide sequence of the extracted microorganism DNA. In another embodiment, this step is conducted through the use of a parallel PCR method. Other PCR methods may also be available for use during this step in the method 20.

Finally, the method further includes the step of amplifying 40 the solution and hybridization to a predetermined second set of genetic markers in order to detect the antibiotic resistance genes of the microorganism represented by the DNA within the solution. In one embodiment, the predetermined second set of genetic markers includes a plurality of antibiotic resistance genes found within sepsis-related microorganisms. In the industrial application of method 20, antibiotic-specific resistant genes most relevant to the hospital setting can be determined by profiling either the Antibiotic Resistant Gene Database with the OpenArray PCR system, or the WaferGen platform (http://w.w.w.wafergen.com/applications/gene-expression-profiling/) or creating an additional database from obtained results over time. Each sample is tested in technical triplicates. If at least two of the assays are positive, the gene will be determined as present. Resistance gene profiles will be analyzed, interrogating for resistance to certain antibiotics or classes of antibiotics in an effort to identify the drug resistance profile.

In one embodiment, the present disclosure directly addresses the need for fast and accurate diagnosis of offending pathogens in the diagnosis of sepsis. In another embodiment, the present disclosure directly addresses the need for fast and accurate diagnosis of offending pathogens by adapting a POC device to the diagnosis of sepsis. Synergistic implementation of both methods can enable physicians to identify the microorganisms responsible for a patient's septic state in 20 to 30 minutes rather than three days, and reveal an organism's genetic weaknesses in seven hours. This will maximize antibiotic utility, eradicate infection, and help conserve important antibiotics by eliminating the guesswork involved in treating septic patients.

It should be noted that the timeframes mentioned are not meant to be limiting. Although the times of 20 to 30 minutes and 7 hours are used here, the disclosure should not be restricted to any specific time period at this time, but should be viewed as changing the range from several days to a first step in a relatively short waiting time followed by a comprehensive analysis in another longer waiting time, but still relatively shorter than several days. Further, the molecular analyses conducted through these methods tend to be both more accurate and more sensitive than culture-based analysis.

FIG. 3 is a representative table listing the 50 sepsis-related microorganisms that may be identified using method 10. Along with each microorganism is also the associated genetic marker(s) that are used to identify the particular microorganism with the fluid sample. It should be noted that this list is not meant to limiting and can be modified in order to account for additional, relevant microorganisms. As discussed above, FIG. 4 indicates the pathogens as identified with example LAMP primer sequences.

FIG. 5 is a representative output table of the antibiotic resistance analysis conducted through method 20. Along with each identified microorganism are their known antibiotic resistance count, their antibiotic sensitivity count, as well as the analysis and identification of antibiotic gene resistance markers. The identified markers as compared to the prior columns are compared in order to give a percentage of resistance undetected by the identified microorganism.

The order of execution or performance of the operations in the embodiments of the disclosure illustrated and described herein is not essential, unless otherwise specified. That is, the operations described herein may be performed in any order, unless otherwise specified, and embodiments of the disclosure may include additional or fewer operations than those disclosed herein. For example, it is contemplated that executing or performing a particular operation before, contemporaneously with, or after another operation is within the scope of aspects of the disclosure.

Other aspects and features of the present disclosure may be obtained from a study of the drawings, the disclosure, and the appended claims. The methods illustrated within the disclosure may be practiced otherwise than as specifically described within the scope of the appended claims. It should also be noted that the steps and/or functions listed within the appended claims, notwithstanding the order of which steps and/or functions are listed therein, are not limited to any specific order of operation.

Although specific features of various embodiments within the disclosure may be shown in some drawings and not in others, this is for convenience only. In accordance with the principles of the disclosure, any feature of a drawing may be referenced and/or claimed in combination with any feature of any other drawing.