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( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2020/0237786 A1 USUN 20200237786A1 IN (19 ) United States (12 ) Patent Application Publication ( 10 ) Pub . No .: US 2020/0237786 A1 Hunt (43 ) Pub . Date : Jul. 30 , 2020 ( 54 ) DIAGNOSIS AND TREATMENT OF Publication Classification ENDOMETRIOSIS BY SCREENING FOR ( 51 ) Int. Ci. L - FORM BACTERIA A61K 31/65 (2006.01 ) C12Q 1/04 (2006.01 ) (71 ) Applicant: Soft Cell Biological Research , LLC , A61K 31/407 (2006.01 ) St. George , UT ( US ) A61K 31/426 (2006.01 ) A61K 9/00 ( 2006.01 ) (72 ) Inventor: John Brent Hunt, St. George , UT (US ) A61K 31/424 (2006.01 ) A61P 31/04 (2006.01 ) (52 ) U.S. CI. ( 73 ) Assignee: Soft Cell Biological Research , LLC , CPC A61K 31/65 ( 2013.01) ; C12Q 1/04 St. George , UT (US ) (2013.01 ) ; A61K 31/407 ( 2013.01 ) ; A61P 31/04 (2018.01 ) ; A61K 9/0019 ( 2013.01 ) ; (21 ) Appl. No .: A61K 9/0053 (2013.01 ) ; A61K 31/424 16 /635,608 ( 2013.01 ) ; A61K 31/426 (2013.01 ) ( 22 ) PCT Filed : Aug. 1, 2018 (57 ) ABSTRACT In some aspects , the disclosure relates to methods for ( 86 ) PCT No.: PCT /US2018 / 044885 screening clinical samples for the presence of L - form bac teria associated with endometriosis and / or ovarian fibroid $ 371 ( c ) ( 1 ) , tumors . The disclosure is based , in part , on screening (2 ) Date : Jan. 31 , 2020 methods used to diagnose a subject as likely having or likely not having endometriosis and /or an ovarian fibroid tumor. In some embodiments , the disclosure relates to methods of Related U.S. Application Data treating endometriosis by administering an antibiotic to a ( 60 ) Provisional application No. 62 / 539,688 , filed on Aug. subject that has been determined to be infected by one or 1 , 2017 . more strains of L - form bacteria . Patent Application Publication Jul. 30 , 2020 Sheet 1 of 6 US 2020/0237786 A1 Collect Sample Contact Sample To A First Growth Medium -120 bodo Incubate First Growth Medium Under A 130 First Set Of Incubation Conditions Monitor First Growth Medium For The Presence Of L - Form Bacteria Transfer A Portion of Bacteria From The First - 150 Growth Medium To A Second Growth Medium Incubate Second Growth Medium Under 160 A Second Set Of Incubation Conditions Monitor Second Growth Medium For The 170 Presence Of Bacteria Isolate Bacteria Grown On Second Growth Medium Patent Application Publication Jul. 30 , 2020 Sheet 2 of 6 US 2020/0237786 A1 210 220 230 240 250 260 270 280 FIG . 2 Patent Application Publication Jul. 30 , 2020 Sheet 3 of 6 US 2020/0237786 A1 300 Collect Sample 310 Contact Sample TO A FirstGrowih Medium 320 Within A Comminuting Container Comminute Sample 322 Incubate First Growth Medium under A First Set Of Incubation Conditions Monitor First Growth Medium For The Presence 340 Of L - Form Bacteria Transfer A Portion of Bacteria From The First 350 Growth Medium To A Second Growth Medium Incubate Second Growth Medium Under -360 A Second Set Of incubation Conditions Monitor Second Growth Medium For The -370 Presence Of Bacteria Isolaie Bacteria Grown On Second Growth Medium Patent Application Publication Jul. 30 , 2020 Sheet 4 of 6 US 2020/0237786 A1 400 Withdraw inoculani From Liquid Medium 410 Contact Inoculant To An Exposed 420 Surface Of A Solid Medium Cover looculant To Maintain 430 Hydration Of Inoculate Position Solid Medium For Incubation 440 With Inoculant Side Facing Down Incubate For A First Solid - Phase Incubation Time Period Reposition Solid Medium For -460 Incubation With InoculantSide Facing Up Incubate For A Second Solid Medium Incubation Time Period Mooddobb Patent Application Publication Jul. 30 , 2020 Sheet 5 of 6 US 2020/0237786 A1 dark color indicative of production of enzyme or other bioactive agent FIG . 5 Patent Application Publication Jul. 30 , 2020 Sheet 6 of 6 US 2020/0237786 A1 Microbacterium maritypicum Staphylococcus epidermidis FIG . 6 US 2020/0237786 A1 Jul. 30 , 2020 DIAGNOSIS AND TREATMENT OF [0006 ] Determining the presence in a patient of an L - form ENDOMETRIOSIS BY SCREENING FOR bacteria associated with endometriosis and /or ovarian L - FORM BACTERIA fibroid tumors can eliminate the need for such patients to undergo other more invasive and more costly diagnostic RELATED APPLICATIONS procedures . Methods described herein enable the culturing and detection of such L - form bacteria within a sample from [ 0001 ] This application is a national stage filing under 35 a subject having or suspected of potentially having endo U.S.C. § 371 of international PCT application , PCT/ metriosis ( e.g. , patients experiencing infertility , patients US2018 /044885 , filed Aug. 1 , 2018, which claims priority exhibiting one or more signs or symptoms or risk factors for under 35 U.S.C. $ 119 ( e ) to U.S. provisional patent appli endometriosis , etc.) , providing a means for more rapidly and cation , U.S. Ser . No. 62/ 539,688 , filed Aug. 1 , 2017, the less invasively determining the likelihood of endometriosis entire contents of each of which are incorporated herein by before proceeding with more intensive and more costly reference . diagnostic procedures. In addition , faster diagnoses of endo metriosis can ease anxiety in patients suffering from the BACKGROUND effects of endometriosis , particularly where the condition [0002 ] Endometriosis is a condition in which endome has proven difficult to diagnose using standard clinical trium tissue abnormally grows at locations outside the measures known in the art . uterus . Because such tissue growth often takes place on the [0007 ] Under culture conditions and process steps ovaries , fallopian tubes , and surrounding tissues, the condi described herein , L -form bacteria that are associated with tion can cause infertility and chronic pelvic pain . Endome endometriosis and symptoms thereof can be successfully trial tissue will bleed as part of the menstrual cycle , and cultured and isolated , even in circumstances in which the areas of endometriosis can become inflamed and scarred as sample from which the L - form bacteria are cultured is a result . It is estimated that endometriosis affects 6 to 10 % unable to produce any detectable growth of cell wall suffi of women . Among women with endometriosis , about 40 % cient bacteria using conventional bacterial culturing tech niques. In addition , embodiments of the disclosure have will have infertility issues . been used to culture bacteria for which no previous reports [ 0003 ] Diagnosis of endometriosis can be challenging . of successful culture or isolation have been made . In some Although a physician may be able to feel endometrial embodiments , L - form bacteria having no known culturable , growth during a pelvic exam , proper diagnosis cannot be cell- wall -sufficient / cell- wall -including form in nature have confirmed by exam only . Pelvic ultrasound may identify been transformed into culturable , cell -wall - sufficient form large endometriotic cysts but will not be able visualize using methods described by the disclosure . and detect smaller regions of endometriosis . Laparoscopy [0008 ] Certain embodiments relate to methods for cultur and biopsy are presently the only methods for fully diag ing L - form bacteria , methods of isolating a bacterial strain nosing endometriosis . These methods, however, are rela from a sample containing L - form bacteria , methods of tively invasive and expensive . transforming an unculturable L - form bacteria into a cultur able cell -wall - sufficient form , and methods of analyzing a BRIEF SUMMARY bacterial strain cultured or isolated from a sample in order to [0004 ] In some aspects , the disclosure relates to methods identify the bacterial strain , and /or propagate or harvest the for culturing and identifying L - form bacteria within a sub bacterial strain . ject's sample ( e.g., blood, saliva, etc. ) in order to diagnose [0009 ] In some aspects , the disclosure relates to methods the subject as having endometriosis and /or an ovarian fibroid for treating a subject having or suspected of having endo tumor. Such methods beneficially enable the detection of metriosis . The disclosure is based , in part, on administration such conditions that would otherwise potentially go unde of one or more antibiotic agents to a subject that has been tected or would require lengthier, costlier, and /or more determined to be infected by one or more L - form bacteria . invasive diagnosis . The methods described herein may Accordingly , in some embodiments , the disclosure provides therefore provide, in some embodiments , time savings, cost a method for treating endometriosis in a subject comprising savings , as well as reductions in patient/ subject anxiety obtaining (or having obtained ) a biological sample from the associated with diagnostic uncertainty surrounding the con subject ; detecting ( or having detected ) in the biological dition . sample one or more bacteria , each bacteria having a genus [ 0005 ] Endometriosis may often go undetected until pro selected from Microbacterium , Bacillus, Micrococcus, and longed periods of infertility indicate that it may be an issue . Staphylococcus; and administering to the subject an antibi Even then , an official diagnosis may require invasive lapa otic agent based upon the presence of the one or more roscopy and/ or biopsy procedures . The disclosure is based , bacteria in the sample . in part , on methods for detecting the presence of one or more [ 0010 ] In some embodiments , a subject has or is suspected types ( e.g., species ) of L - form bacterial infection within a of having endometriosis . In some embodiments , a subject is subject that has or is suspected
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