WO2023227896A1 - Identification of a bacterium of the genus bacillus - Google Patents
Identification of a bacterium of the genus bacillus Download PDFInfo
- Publication number
- WO2023227896A1 WO2023227896A1 PCT/GB2023/051379 GB2023051379W WO2023227896A1 WO 2023227896 A1 WO2023227896 A1 WO 2023227896A1 GB 2023051379 W GB2023051379 W GB 2023051379W WO 2023227896 A1 WO2023227896 A1 WO 2023227896A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- subject
- nucleic acid
- relative abundance
- urine sample
- microorganisms
- Prior art date
Links
- 241000894006 Bacteria Species 0.000 title claims description 18
- 241000193830 Bacillus <bacterium> Species 0.000 title description 2
- 244000005700 microbiome Species 0.000 claims abstract description 161
- 238000000034 method Methods 0.000 claims abstract description 128
- 210000002700 urine Anatomy 0.000 claims abstract description 119
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 87
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 86
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 86
- 239000000203 mixture Substances 0.000 claims abstract description 64
- 239000000523 sample Substances 0.000 claims description 112
- 241000186660 Lactobacillus Species 0.000 claims description 54
- 230000001580 bacterial effect Effects 0.000 claims description 51
- 238000012163 sequencing technique Methods 0.000 claims description 24
- 239000011324 bead Substances 0.000 claims description 22
- 238000000605 extraction Methods 0.000 claims description 18
- 241000218492 Lactobacillus crispatus Species 0.000 claims description 13
- 239000013068 control sample Substances 0.000 claims description 13
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 10
- 241001561398 Lactobacillus jensenii Species 0.000 claims description 5
- 241001324870 Lactobacillus iners Species 0.000 claims description 4
- 241000186606 Lactobacillus gasseri Species 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 2
- 241000894007 species Species 0.000 description 39
- 208000004926 Bacterial Vaginosis Diseases 0.000 description 36
- 208000037009 Vaginitis bacterial Diseases 0.000 description 36
- 201000007096 Vulvovaginal Candidiasis Diseases 0.000 description 36
- 208000019206 urinary tract infection Diseases 0.000 description 36
- 206010003693 Atrophic vulvovaginitis Diseases 0.000 description 35
- 206010046914 Vaginal infection Diseases 0.000 description 35
- 201000008100 Vaginitis Diseases 0.000 description 35
- 230000009245 menopause Effects 0.000 description 33
- 230000036541 health Effects 0.000 description 32
- 238000002560 therapeutic procedure Methods 0.000 description 19
- 238000003752 polymerase chain reaction Methods 0.000 description 17
- 238000005259 measurement Methods 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 15
- 241000193818 Atopobium Species 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 13
- 241000604449 Megasphaera Species 0.000 description 13
- 241000605861 Prevotella Species 0.000 description 13
- 238000003745 diagnosis Methods 0.000 description 12
- 238000003753 real-time PCR Methods 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 238000004458 analytical method Methods 0.000 description 11
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 10
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 239000000262 estrogen Substances 0.000 description 9
- 239000008188 pellet Substances 0.000 description 9
- 239000003755 preservative agent Substances 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 241000379991 Anaerococcus Species 0.000 description 8
- 241000351207 Peptoniphilus Species 0.000 description 8
- 230000007423 decrease Effects 0.000 description 8
- 239000004055 small Interfering RNA Substances 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 229940079593 drug Drugs 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000002657 hormone replacement therapy Methods 0.000 description 7
- 238000007481 next generation sequencing Methods 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 241000207202 Gardnerella Species 0.000 description 6
- 244000052616 bacterial pathogen Species 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 230000002101 lytic effect Effects 0.000 description 6
- 241000588921 Enterobacteriaceae Species 0.000 description 5
- 241001135215 Prevotella bivia Species 0.000 description 5
- 241000193990 Streptococcus sp. 'group B' Species 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 5
- 239000004327 boric acid Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 239000004310 lactic acid Substances 0.000 description 5
- 235000014655 lactic acid Nutrition 0.000 description 5
- 238000012544 monitoring process Methods 0.000 description 5
- 230000002335 preservative effect Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 210000001215 vagina Anatomy 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000495778 Escherichia faecalis Species 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 230000002485 urinary effect Effects 0.000 description 4
- 210000002229 urogenital system Anatomy 0.000 description 4
- 108020005544 Antisense RNA Proteins 0.000 description 3
- 241000222122 Candida albicans Species 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 239000007984 Tris EDTA buffer Substances 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000002313 adhesive film Substances 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 229940095731 candida albicans Drugs 0.000 description 3
- 239000003184 complementary RNA Substances 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 230000002934 lysing effect Effects 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 244000000010 microbial pathogen Species 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- 206010006784 Burning sensation Diseases 0.000 description 2
- 208000004483 Dyspareunia Diseases 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 108700039887 Essential Genes Proteins 0.000 description 2
- 229920002527 Glycogen Polymers 0.000 description 2
- 108020005004 Guide RNA Proteins 0.000 description 2
- 108700011259 MicroRNAs Proteins 0.000 description 2
- 108091007412 Piwi-interacting RNA Proteins 0.000 description 2
- 208000003251 Pruritus Diseases 0.000 description 2
- 108090000621 Ribonuclease P Proteins 0.000 description 2
- 102000004167 Ribonuclease P Human genes 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 108091007415 Small Cajal body-specific RNA Proteins 0.000 description 2
- 102000039471 Small Nuclear RNA Human genes 0.000 description 2
- 108020003224 Small Nucleolar RNA Proteins 0.000 description 2
- 102000042773 Small Nucleolar RNA Human genes 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 108091032917 Transfer-messenger RNA Proteins 0.000 description 2
- 206010069055 Vulvovaginal pain Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002654 craniosacral therapy Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000005713 exacerbation Effects 0.000 description 2
- 210000004392 genitalia Anatomy 0.000 description 2
- 229940096919 glycogen Drugs 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000007803 itching Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000002679 microRNA Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000016087 ovulation Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002250 progressing effect Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 108020004418 ribosomal RNA Proteins 0.000 description 2
- 108091029842 small nuclear ribonucleic acid Proteins 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 108010057210 telomerase RNA Proteins 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 150000007971 urates Chemical class 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- DBTMGCOVALSLOR-UHFFFAOYSA-N 32-alpha-galactosyl-3-alpha-galactosyl-galactose Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(OC2C(C(CO)OC(O)C2O)O)OC(CO)C1O DBTMGCOVALSLOR-UHFFFAOYSA-N 0.000 description 1
- 108091034151 7SK RNA Proteins 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000203809 Actinomycetales Species 0.000 description 1
- 241000193798 Aerococcus Species 0.000 description 1
- 241001135756 Alphaproteobacteria Species 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000611330 Chryseobacterium Species 0.000 description 1
- RXVWSYJTUUKTEA-UHFFFAOYSA-N D-maltotriose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(O)C(CO)O1 RXVWSYJTUUKTEA-UHFFFAOYSA-N 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108091008102 DNA aptamers Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241001535083 Dialister Species 0.000 description 1
- 241000688137 Diaphorobacter Species 0.000 description 1
- 208000027244 Dysbiosis Diseases 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 241000207201 Gardnerella vaginalis Species 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 206010027304 Menopausal symptoms Diseases 0.000 description 1
- 241000588653 Neisseria Species 0.000 description 1
- 206010030247 Oestrogen deficiency Diseases 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 241000122116 Parvimonas Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000095588 Ruminococcaceae Species 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- 108091061750 Signal recognition particle RNA Proteins 0.000 description 1
- 108091092920 SmY RNA Proteins 0.000 description 1
- 241001237710 Smyrna Species 0.000 description 1
- 241000424747 Sneathia Species 0.000 description 1
- 108020003213 Spliced Leader RNA Proteins 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- LUEWUZLMQUOBSB-UHFFFAOYSA-N UNPD55895 Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(OC3C(OC(O)C(O)C3O)CO)C(O)C2O)CO)C(O)C1O LUEWUZLMQUOBSB-UHFFFAOYSA-N 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 206010047791 Vulvovaginal dryness Diseases 0.000 description 1
- 206010056530 Vulvovaginal pruritus Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 108091029474 Y RNA Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000007140 dysbiosis Effects 0.000 description 1
- 206010013990 dysuria Diseases 0.000 description 1
- 238000007848 endpoint PCR Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 238000007849 hot-start PCR Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- UYQJCPNSAVWAFU-UHFFFAOYSA-N malto-tetraose Natural products OC1C(O)C(OC(C(O)CO)C(O)C(O)C=O)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(O)C(CO)O2)O)C(CO)O1 UYQJCPNSAVWAFU-UHFFFAOYSA-N 0.000 description 1
- LUEWUZLMQUOBSB-OUBHKODOSA-N maltotetraose Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O[C@@H]3[C@@H](O[C@@H](O)[C@H](O)[C@H]3O)CO)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-OUBHKODOSA-N 0.000 description 1
- FYGDTMLNYKFZSV-UHFFFAOYSA-N mannotriose Natural products OC1C(O)C(O)C(CO)OC1OC1C(CO)OC(OC2C(OC(O)C(O)C2O)CO)C(O)C1O FYGDTMLNYKFZSV-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003821 menstrual periods Effects 0.000 description 1
- 108010068249 mitochondrial RNA-processing endoribonuclease Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 108040009109 ribonuclease MRP activity proteins Proteins 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000007841 sequencing by ligation Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 230000036559 skin health Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000005353 urine analysis Methods 0.000 description 1
- 206010046901 vaginal discharge Diseases 0.000 description 1
- 210000003905 vulva Anatomy 0.000 description 1
- 238000007482 whole exome sequencing Methods 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
- FYGDTMLNYKFZSV-BYLHFPJWSA-N β-1,4-galactotrioside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@H](CO)O[C@@H](O[C@@H]2[C@@H](O[C@@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-BYLHFPJWSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Definitions
- the present invention relates to a method of determining genitourinary microbiome composition from a urine sample of a subject. More specifically the invention relates to a method of determining genitourinary or vaginal health in a subject using the method. In particular, the method of the present invention can be used to determine menopausal status in a subject.
- the microbiome is the collection of microorganisms, such as bacteria, viruses and fungi which live on and inside our bodies. It has come to light that these microorganisms contribute to our health in a number of ways, for example in development of the immune system, protecting the body against pathogenic microorganisms, maintaining skin health and maintaining good digestion. A person’s core microbiome is formed during the early years of life. However, over time, the composition of the microbiome can change, whether that be in response to external factors, diet, drugs, illness or internal factors such as hormonal changes.
- the genitourinary microbiome refers to the microorganisms present in the genitourinary system, which includes the urinary and genital organs. This includes the vaginome or vaginal microbiome (the microorganism species present in the vagina). Similar to the microbiome as a whole, the composition of the genitourinary microbiome and vaginome have been linked to health and disease. For example, the microorganisms present in the vagina maintain vaginal homeostasis, and an imbalance (or dysbiosis) can be associated with poor vaginal health. The vaginal microbiome is also implicated in the progression of the menopause.
- oestrogen levels decrease which results in changes in the vaginome.
- oestrogen leads to the production of glycogen.
- Amylase breaks down glycogen into products including maltose, maltotriose and maltotetraose. Lactobacilli can grow in these products and produce lactic acid. The presence of lactic acid lowers the pH and impedes growth of potentially pathogenic bacteria.
- oestrogen deficiency (for example as a result of menopause) means that Lactobacilli can no longer proliferate and pathogenic bacteria may become dominant.
- vaginal swabs are invasive and are often not well tolerated by patients. Such samples may also be difficult to obtain without assistance from a medical professional. Therefore, determining the microorganisms present in the genitourinary microbiome and particularly the vaginome can be problematic. It is an object of the present invention to obviate or mitigate one or more of the abovementioned problems.
- the present invention relates to a method of determining the genitourinary microbiome composition of a subject and is based, in part, on studies by the inventors in which they have shown that the composition of the genitourinary microbiome, obtained by the analysis of the urine sample, can be used to determine vaginal health and in particular can be used to determine the menopausal status of a subject.
- a method of determining the menopausal status of a subject by assessing genitourinary microbiome composition of the subject comprising:
- the term “genitourinary microbiome composition” as used herein means the composition of microorganisms present in the genitourinary system, which includes the urinary and genital organs. This includes, for example, the vaginome or vaginal microbiome (the microorganisms present in the vagina).
- composition of the genitourinary microbiome and particularly the vaginome composition is usually determined using a vaginal swab.
- the present inventors have surprisingly shown that it is possible to obtain highly accurate information regarding the composition of the genitourinary microbiome through analysis of a urine sample from the subject. This is particularly advantageous as urine samples are less invasive than vaginal swabs and therefore tolerated more by subjects. What is more, such samples can be much more easily obtained, which is particularly important for those unable to perform vaginal self-swabbing.
- urine samples provide information about a greater area of the genitourinary tract and therefore the results obtained can be used to indicate the genitourinary health of the subject, particularly vaginal health and menopause status.
- the inventors have shown that they are able to accurately predict the menopausal status of a subject by analysing the relative abundance of microorganisms present in a urine sample from the subject.
- the present inventors have surprisingly found that it is possible to obtain highly accurate information regarding the composition of the genitourinary microbiome through urine analysis. This is unexpected.
- the composition of microorganisms in the urinary microbiome differs from that of the vaginal microbiome as the bladder and vagina are anatomically distinct.
- the method of the present invention further comprises comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the menopausal status of a subject.
- the present invention therefore also provides a method of determining the menopausal status of a subject by assessing genitourinary microbiome composition of the subject, the method comprising:
- menopausal status is used to mean whether the subject is premenopausal (i.e. has not undergone permanent cessation of ovulation), peri-menopausal (the 5 years preceding menopause, when ovaries begin to make less oestrogen) or post-menopausal (in which at least 12 months has passed without a menstrual period/ovulation has permanently ceased).
- a clinician may also consider a female subject’s age and medical history when assessing their menopausal status. For example the date of the subject’s last period and their genitourinary symptoms (such as recurrent UTIs, vulva and/or vaginal soreness, burning or irritation, vaginal dryness or reduced discharge) may be relevant.
- genitourinary symptoms such as recurrent UTIs, vulva and/or vaginal soreness, burning or irritation, vaginal dryness or reduced discharge
- the oestrogen levels in a female subject decrease which results in subsequent pH changes in the genitourinary system and consequent changes in the genitourinary microbiome composition.
- Lactobacillus species particularly Lactobacillus crispatus
- oestrogen levels begin to decline and so Lactobacillus species become less dominant in the genitourinary microbiome.
- oestrogen levels are low, pH is high and the genitourinary microbiome has a high diversity of bacterial species with low levels of Lactobacillus and higher levels of pathogenic bacteria. Diversity of microorganisms increases during the transition from pre-menopause to perimenopause and post-menopause.
- the present inventors have found that by determining the genitourinary microbiome composition from a urine sample they can accurately predict the menopausal status of the subject.
- the urine sample can be any suitable sample, for example the first urine of the day or a subsequent urine sample.
- the sample may be a first-stream urine sample (i.e. the first part of the urine to be passed), or a mid-stream or late-stream urine sample.
- the sample is a first-stream urine sample.
- the inventors have found that a first stream-urine sample surprisingly results in improved ease of nucleic acid extraction and nucleic acid yield. Furthermore, and as shown below, the inventors have shown that when using the results of the method to indicate the menopausal status of the subject, for example, the results using first- stream urine more accurately reflect the menopausal status of the subject.
- the present invention involves (a) extracting nucleic acid from a urine sample from the subject.
- extracting nucleic acid from a urine sample means removing the nucleic acid from the urine sample.
- the nucleic acid extracted may be free in the urine or may be present in cells, for example microorganisms (e.g. bacterial species) present in the urine sample.
- the present invention involves extracting nucleic acid from cells and/or microorganisms present in a urine sample from the subject.
- nucleic acid refers to macromolecules formed by a chain of nucleotides which stores genetic information. Nucleic acids include DNA and RNA.
- the nucleic acid comprises DNA.
- the nucleic acid comprises RNA.
- the nucleic acid comprises DNA and RNA.
- RNA messenger RNA
- rRNA ribosomal RNA
- SRP RNA signal recognition particle RNA
- tRNA transfer messenger RNA
- tmRNA transfer messenger RNA
- snRNA small nuclear RNA
- snoRNA small nucleolar RNA
- SmY RNA small Cajal body specific RNA
- gRNA guide RNA
- ribonuclease P RNase P
- ribonuclease MRP RNase MRP
- DNA and/or RNA from microorganisms may be from bacteria, fungi or viruses.
- step (a) comprises preparing a cell pellet from the urine.
- the cells are the microorganism cells present in the urine, particularly including the microorganisms whose relative abundance is to be assessed.
- the method may comprise diluting the urine before preparing the cell pellet. Dilution of the urine may be achieved using any suitable means.
- the urine is diluted by addition of a buffer, for example TE buffer, boric acid or EDTA.
- the urine is diluted by addition of TE buffer.
- the inventors have surprisingly found that by diluting urine, the collection of impurities is reduced. What is more, the addition of the buffer dissolves any crystals such as urates to avoid collection of such impurities with the cell pellet.
- step (a) comprises lysing cells present in the urine sample.
- lysing the cells may be achieved through chemical lysis or physical lysis, for example bead beating or sonication.
- lysing the cells is achieved by contacting the sample with a chemical lytic agent, for example a lysis buffer.
- the chemical lytic agent may include lysozyme or another commercially available lytic enzyme. The present inventors have found that the use of lysozyme gives improved nucleic acid yield and the most accurate measurement of bacterial communities.
- nucleic acid can be extracted from cells.
- column-based extraction methods may be used in which nucleic acids bind to a solid phase of silica under specific binding conditions. Nucleic acids are then subsequently eluted from the solid phase.
- Kits are available for performing such extraction and include, for example, Qiagen DNeasy Blood and Tissue Kit.
- nucleic acid extraction could take place by way of phenol-chloroform extraction or bead-based extraction, for example through the use of magnetic beads.
- nucleic acids bind to the magnetic beads under specific binding conditions, leaving contaminants in solution. The nucleic acid can be subsequently removed from the beads under elution conditions.
- Kits are available for performing such extraction and include, for example, Thermo MagMAXTM Microbiome Ultra Nucleic Acid Isolation Kit.
- the nucleic acid may be extracted by column-based or magnetic bead-based extraction methods.
- the method comprises extracting nucleic acid from the urine sample using a magnetic bead-based extraction method. The inventors have found that using a magnetic bead-based approach, rather than a column approach, addresses the problem of the column becoming blocked with impurities from the urine and thereby improves the nucleic acid quality obtained.
- step (a) further comprises isolating the nucleic acid from the sample, suitably after extraction thereof.
- the present invention may comprise a step of providing a urine sample from a subject.
- the invention may comprise adding a preservative to the urine sample, for example EDTA, boric acid or other commercially available preservatives. These may be lytic or non-lytic.
- EDTA EDTA
- boric acid or other commercially available preservatives.
- Preferred preservatives include boric acid.
- the urine sample may be refrigerated once the preservative has been added.
- the present invention may comprise a step of obtaining a urine sample from a subject.
- Step (b) of the method of the present invention involves analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample.
- step (b) may involve amplifying the extracted nucleic acid.
- the skilled person will appreciate that there are various methods by which the nucleic acid can be amplified. Methods include, for example, polymerase chain reaction (PCR). PCR techniques include basic PCR, reverse transcription (RT)-PCR, hot-start PCR, long PCR, quantitative PCR, quantitative endpoint PCR, quantitative real-time PCR, rapid amplified polymorphic DNA analysis, nested PCR and high-fidelity PCR. In embodiments, the amplification takes place using quantitative PCR (qPCR) or PCR.
- qPCR quantitative PCR
- step (b) comprises the addition of at least one reagent for performing the PCR or quantitative PCR.
- the at least one reagent is added to the extracted nucleic acid of step (a).
- the at least one reagent is selected from: DNA polymerase, buffer, dNTPs, or a source of magnesium.
- step (b) comprises the addition of all three of these components, referred to as a PCR master mix.
- the master mix may suitably be sourced from any manufacturer.
- the PCR Master Mix may be added to the extracted nucleic acid in any suitable amount, for example an amount of around 5-15 pl, around 7-12 pl, or around 10 pl.
- step (b) further comprises the addition of primers and probes as required for PCR or quantitative PCR amplification of the extracted nucleic acid.
- primers are added in equal amounts to the extracted nucleic acid of step (a) of the method.
- Step (b) may further comprise amplifying and indicating the level of a control nucleic acid together with the extracted nucleic acid, the control nucleic acid being definitively present within the urine sample.
- the control nucleic acid is a human nucleic acid when the urine sample is of human origin. More preferably the control nucleic acid is a human nucleic acid, preferably the control human nucleic acid is of a housekeeping gene or genes that are substantially conserved in human nucleic acid.
- PCR or quantitative PCR takes place using standard procedures known in the art and using standard machinery known in the art.
- qPCR is described in Higuchi R et al. Kinetic PCR analysis: Real time Monitoring of DNA amplification reactions. Bio/Technology 1993; 11 :1026-30.
- PCR and indeed qPCR involves a set program of heating cycles.
- the heating cycle may comprise any of the following stages: mix activation, initial denaturation, denaturation, annealing, extension, final extension, or cooling.
- the heating cycle comprises at least initial denaturation, denaturation, and annealing.
- any of the stages may be repeated, alone or in combination with any other stage.
- the denaturation and annealing stages are repeated, more preferably these stages are repeated for between 15 and 45 cycles, for example 20 cycles.
- step (b) of the method comprises amplifying nucleic acid encoding bacterial 16S rRNA present in the extracted nucleic acid.
- the primers used in the PCR or qPCR reaction are specific to the bacterial 16S rRNA genes.
- the method comprises amplifying nucleic acid encoding bacterial 16S variable regions 1-9 (V1- 9), more preferably bacterial 16S variable regions 1-4 (V1-4), more preferably bacterial 16S variable regions 3-4 (V3-4).
- the amplification may take place by PCR, in embodiments using qPCR.
- the One 16S NGS Library Preparation Kit (by YouSeq) is a suitable kit for amplifying these bacterial 16S variable regions and may be used in the method of the present invention.
- Targeting amplification and sequencing of the nucleic acid encoding bacterial 16S rRNA gene is known in the art as a method for studying bacterial diversity. However, the accuracy may depend on the choice of primers, the bacterial species contained in the sample and the areas of 16S rRNA targeted.
- the present inventors have found that by targeting 16S rRNA variable regions 3-4, they can accurately identify bacterial species found in urine. They can also correctly identify potentially pathogenic species such as Gardnerella vaginalis which can be useful for indicating vaginal health.
- step (b) of the method may further comprise sequencing the extracted nucleic acid.
- step (b) of the method may comprise sequencing the amplified nucleic acid, preferably the amplified nucleic acid encoding bacterial 16S rRNA, preferably nucleic acid encoding variable regions 1-9, 1-4 or 3-4 of the bacterial 16S rRNA.
- Such sequencing can be used to identify the bacterial species present in the urine sample. Amplification of the nucleic acid by qPCR allows the relative abundance of the bacterial species in the urine sample to be determined by subsequent sequencing.
- each primer used in amplification may have a partial adapter sequence, for facilitating sequencing of said sequence.
- Any suitable sequencing method known in the art may be used, for example traditional sequencing methods such as Sanger sequencing, or next generation sequencing methods.
- Sequencing methods include whole genome sequencing, whole exome sequencing, targeted sequencing and metagenomic sequencing.
- high throughput/next generation sequencing methods which are capable of rapidly generating large amounts of sequence data, are preferred since they allow rapid and accurate identification of large numbers of microorganisms.
- the sequencing comprises next generation sequencing.
- Next generation sequencing methods enable a large number of distinct nucleic acid sequences to be sequenced simultaneously and at a high density, including, for example, sequencing-by-synthesis, sequencing by ligation, pyrosequencing and ion semiconductor sequencing.
- a preferred sequencing method is sequencing-by-synthesis, for example using the Illumina iSeq 100 or MiSeq systems.
- a fluorescently labelled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias.
- the end result is true base- by-base sequencing that enables accurate data for a broad range of applications.
- the method virtually eliminates errors and missed calls associated with strings of repeated nucleotides (homopolymers)’ (from lllumina.com).
- a bioinformatics method may be used to determine the microorganisms present in the urine sample.
- the method of the present invention involves analysing the extracted nucleic acid to identify the relative abundance of microorganisms in the urine sample.
- relative abundance means the percentage of a microorganism of a particular kind relative to the total number of microorganisms in the sample.
- the microorganisms may include bacteria, fungi, viruses, parasites and protozoa for example.
- the microorganisms may include commensal microorganisms and/or pathogenic microorganisms.
- the microorganisms comprise bacteria. Accordingly, the method may comprise analysing the extracted nucleic acid to identify the relative abundance of bacterial species in the urine sample.
- the bacterial species may include, for example, Lactobacilli, Gardnerella, Prevotella, Pseudomonas, Peptoniphilus, Chryseobacterium, Neisseria, Bergeyelia, Acinetobacter, Alphaproteobacteria,Anaerococcus, Actinomycetales, Atopobium, Escherichia, Shigella, Flavobacterium, Dialister, Streptococcus, Aerococcus, Campylobacter, Shuttlewothia, Staphylococcus, Megasphaera, Parvimonas, Ruminococcaceae/Oscillococcaceae, Sneathia and/or Diaphorobacter.
- the present invention is used to determine the menopausal status of a subject.
- the method comprises identifying the relative abundance of Lactobacilli in the urine sample.
- the Lactobacilli may comprise, for example, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.
- the inventors have found that by determining the relative abundance of Lactobacilli in the sample, the menopausal stage can be predicted. For example, pre-menopausal subjects have a high relative abundance of Lactobacilli (for example greater than 70%) with L. crispatus dominant. Therefore, a relative abundance of Lactobacilli of greater than 70% may be indicative of pre-menopausal status.
- Lactobacilli are still dominant, but to a lesser degree. Therefore, a relative abundance of Lactobacilli of between 40 and 70% may be indicative of peri-menopausal status. What is more peri-menopausal subjects show a greater extent of non-Lactobacillus crispatus spp as compared to pre-menopausal subjects. On the other hand, post-menopausal subjects have low levels of Lactobacilli and a high diversity of bacterial species (no dominance of a single species), with pathogenic bacteria often present. Therefore, a relative abundance of Lactobacilli of less than 40% may be indicative of post-menopausal status.
- the method comprises identifying the relative abundance of Lactobacillus crispatus in the urine sample. As mentioned above, pre-menopausal subjects have a dominance of Lactobacillus crispatus within the Lactobacilli present. In embodiments, the method may comprise determining a vaginal microbiome measurement (VMM) based on the genitourinary microbiome composition and the relative abundance of the microorganisms in the urine sample.
- VMM vaginal microbiome measurement
- VMM vaginal microbiome measurement
- bacterial species may be awarded values as follows:
- Non-crispatus Lactobacillus species including but not limited to L. jensenii, gasserii and iners i.e. lactic acid bacteria) and other potentially protective species- 0.5-4
- VMM vaginal microbiome measurement
- the method may further comprise assigning a community state type (CST) to the subject, based on the genitourinary microbiome composition/relative abundance of microorganisms in the urine sample.
- CST community state type
- Vaginal CSTs are known to be linked to vaginal health and disease and menopause status (Brotman et al).
- a CST may be assigned to the subject based on the relative abundance of microorganisms in the urine sample.
- the CSTs may be assigned according to the following criteria:
- the term “dominant” as used herein means a microorganism or bacterial species which is present as a significant proportion of the total microorganisms or bacterial species, for example, over 30%, 40%, 50%, 60% or 70% of the total microorganisms or bacterial species present.
- the method of the present invention may comprise determining the menopausal status based on one or more of Lactobacilli relative abundance, vaginal microbiome measurement (VMM) and/or assigned CST type.
- VMM vaginal microbiome measurement
- the present invention can also be used to determine progression of menopause.
- the present invention therefore also provides a method for determining the menopause status in a subject, determining progression of menopause or assessing response to therapy of a subject with menopause by assessing genitourinary microbiome composition of the subject.
- the method comprises the steps of:
- the present invention also provides a method for determining the menopause status in a subject, determining progression of menopause or assessing response to therapy of a subject with menopause by assessing genitourinary microbiome composition of the subject.
- the method comprises the steps of:
- the method may comprise identifying the relative abundance of Lactobacilli in the urine sample, assigning a community state type (CST) to the subject and/or determining a vaginal microbiome measurement (VMM), as described in more detail above.
- CST community state type
- VMM vaginal microbiome measurement
- the method may comprise monitoring over time to determine whether menopause status has progressed.
- an initial genitourinary microbiome composition may be compared with a genitourinary microbiome composition in a sample obtained later in time.
- the genitourinary microbiome composition obtained may change over time, to be more indicative of peri-menopause or post-menopause for example.
- the genitourinary microbiome composition may be indicative of pre-menopause, but a later analysis may be indicative of peri- or post-menopause. For example, the dominance of Lactobacilli in the genitourinary microbiome composition may decrease, indicative of progression of menopause status.
- the present inventors have also found that in post-menopause subjects treated with hormone replacement therapy (HRT) the genitourinary microbiome composition after treatment shows an increased level of Lactobacilli. Therefore, the method can also be used to determine whether a subject is responding to therapy.
- the method may comprise monitoring over time to determine whether a subject is responding to therapy.
- an initial genitourinary microbiome composition may be compared with one or more genitourinary microbiome composition analyses undertaken on samples obtained later in time (for example after therapy has commenced).
- the genitourinary microbiome composition may change over time, for example the relative abundance of Lactobacilli in the sample may increase which may indicate that the subject is responding to therapy.
- the method of the present invention may also be used to determine whether a drug is effective at treating menopause, in a similar manner to that described above in relation to determining whether a subject is responding to therapy.
- the use of “effective” is used to indicate that a treatment reduces or alleviates signs or symptoms of menopause, improves the clinical course of the disease, decreases the number or severity of exacerbations or reduces any other objective or subjective indicia of the disease.
- the method of the present invention can be used to determine whether drugs used to treat menopause, in addition to other drugs developed to treat menopause are effective.
- the method of the present invention may be used to determine whether a subject is more likely than not to have menopause based on the genitourinary microbiome composition.
- a subject with a putative diagnosis of peri-menopause or post-menopause may be diagnosed as being “more likely” or “less likely” to be peri-menopause or post-menopause in light of the information provided by the method of the present invention.
- the present invention may therefore be used to assist a clinician with the diagnosis of menopause.
- the method of the present invention may, in certain embodiments, comprise detecting other signs or symptoms of peri-menopause or menopause, conducting clinical tests of perimenopause or menopause and/or measuring other peri-menopause or menopause markers.
- the above description is not limited to making an initial identification (or diagnosis) of menopause in a subject but is also applicable to confirming a provisional diagnosis of menopause or “ruling out” such a diagnosis.
- the method of the present invention can be used to determine menopausal status, the results obtained by the method of the present invention can be used to determine a suitable treatment for the subject. Therefore, the method of the present invention may further comprise a step of:
- step (d) of the method of the present invention may be used to determine a suitable treatment, for example hormone replacement therapy.
- the present invention may further comprise providing the subject with a suitable treatment based on the relative abundance of microorganisms in the urine sample.
- the present inventors have also found that by determining the relative abundance of microorganisms in a urine sample, the genitourinary health, particularly the vaginal health of a subject can be determined.
- the method of the present invention may be used to determine the genitourinary or vaginal health of a subject.
- the present invention therefore provides a method of determining the genitourinary health or vaginal health of a subject by assessing the genitourinary microbiome composition of the subject, the method comprising:
- the method makes use of the genitourinary microbiome composition to determine genitourinary and particularly vaginal health.
- the present inventors have found that by determining the composition of the genitourinary microbiome from a urine sample, it is possible to identify the genitourinary or vaginal health of the subject and, in particular, whether the subject has any of the following conditions: vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis.
- the term “genitourinary health” and “vaginal health” as used herein are used to mean the state of the genitourinary system or vagina in terms of whether illness or injury is present, for example.
- the method of the present invention further comprises comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the genitourinary health or vaginal health of a subject.
- the present invention provides a method of determining the genitourinary health or vaginal health of a subject by assessing genitourinary microbiome composition of the subject, the method comprising:
- the present inventors have found that by determining the composition of the genitourinary microbiome from a urine sample, it is possible to identify the genitourinary or vaginal health of the subject and, in particular, whether the subject has any of the following conditions: vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis.
- the inventors have shown that they are able to accurately predict the menopausal status of a subject by analysing the relative abundance of bacterial species present in a urine sample from the subject.
- the method of the present invention can also be used to determine other aspects of genitourinary or vaginal health.
- subjects suffering from vulvovaginal atrophy which causes dysuria, dyspareunia, bleeding after intercourse, vaginal discharge, sorenes, itching or burning sensations
- Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella species are also often present.
- the method comprises identifying the relative abundance of Lactobacilli in the urine sample.
- the method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella.
- a relative abundance of Lactobacilli of less than 50% may be indicative of vulvovaginal atrophy, particularly in combination with the presence of one or more of the following bacterial species: Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella.
- a relative abundance of greater than 25% of Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella may be indicative of vulvovaginal atrophy.
- Subjects suffering from bacterial vaginosis exhibit low levels of Lactobacilli in the genitourinary microbiome. Gardnerella, Atopobium, Megasphaera, and Prevotella species are also often present.
- the method comprises identifying the relative abundance of Lactobacilli in the urine sample.
- the method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: Gardnerella, Atopobium, Megasphaera, and Prevotella.
- a relative abundance of Lactobacilli of less than 50% may be indicative of bacterial vaginosis, particularly in combination with the presence of one or more of the following bacterial species: Gardnerella, Atopobium, Megasphaera, and Prevotella.
- Gardnerella, Atopobium, Megasphaera, and Prevotella A relative abundance of greater than 25% of Gardnerella, Atopobium, Megasphaera, and/or Prevotella may be indicative of bacterial vaginosis.
- the method comprises identifying the relative abundance of Lactobacilli in the urine sample.
- the method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: E. coli, K. pneumoniae and E. faecalis.
- a relative abundance of Lactobacilli of less than 50% may be indicative of urinary tract infections, particularly in combination with the presence of one or more of the following bacterial species: E. coli, K. pneumoniae and E. faecalis.
- a relative abundance of greater than 25% of E. coli, K. pneumoniae and/or E. faecalis may be indicative of urinary tract infections.
- Subjects suffering from aerobic vaginitis exhibit low levels of Lactobacilli in the genitourinary microbiome.
- Group B Streptococcus, P. bivia, Enterobacteriaceae are also often present.
- the method comprises identifying the relative abundance of Lactobacilli in the urine sample.
- the method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: Group B Streptococcus, P. bivia and Enterobacteriaceae.
- a relative abundance of Lactobacilli of less than 50% may be indicative of aerobic vaginitis, particularly in combination with the presence of one or more of the following bacterial species: Group B Streptococcus, P. bivia and Enterobacteriaceae.
- a relative abundance of greater than 25% of Group B Streptococcus, P. bivia and/or Enterobacteriaceae may be indicative of aerobic vaginitis.
- the method comprises identifying the relative abundance of Lactobacilli iners in the urine sample.
- the method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: Group B Streptococcus, P.
- a relative abundance of Lactobacilli of less than 50% may be indicative of vulvovaginal candidiasis, particularly in combination with the presence of one or more of the following bacterial species: Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella.
- the presence of Candida albicans is also indicative of vulvovaginal candidiasis.
- a relative abundance of greater than 25% of Atopobium, Megasphaera, Anaerococcus, Peptoniphilus, Prevotella and/or Candida albicans may be indicative of vulvovaginal candidiasis.
- the method may comprise determining a vaginal microbiome measurement (VMM) based on the genitourinary microbiome composition and the relative abundance of the microorganisms in the urine sample.
- VMM vaginal microbiome measurement
- VMM vaginal microbiome measurement
- microorganisms may be awarded values as follows:
- Non-crispatus Lactobacillus species including but not limited to L. jensenii, gasserii and iners i.e. lactic acid bacteria) and other potentially protective species- 0.5-4
- VMM vaginal microbiome measurement
- the present invention therefore also provides a method for determining the presence of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis, determining progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis or assessing response to therapy of a subject with vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis by assessing genitourinary microbiome composition of the subject.
- the method comprises the steps of:
- the present invention also provides a method for determining the presence of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis in a subject, determining progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis or assessing response to therapy of a subject with vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis.
- the method comprises the steps of:
- the method may comprise identifying the relative abundance of Lactobacilli in the urine sample, as described in more detail above.
- the method may further comprise identifying the relative abundance of the bacterial species outlined above as being associated with the presence of these respective conditions.
- the method may comprise monitoring over time to determine whether vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis has progressed.
- an initial genitourinary microbiome composition may be compared with a genitourinary microbiome composition in a sample obtained later in time.
- the genitourinary microbiome composition obtained may change over time, to be more indicative of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis for example.
- the relative abundance of Lactobacilli in the genitourinary microbiome composition may decrease, indicative of progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis.
- the relative abundance of certain pathogenic microorganisms may increase, indicative of progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis.
- the method can also be used to determine whether a subject is responding to therapy.
- the method may comprise monitoring over time to determine whether a subject is responding to therapy.
- an initial genitourinary microbiome composition may be compared with one or more genitourinary microbiome composition analyses undertaken on samples obtained later in time (for example after therapy has commenced).
- the genitourinary microbiome composition may change over time, for example the relative abundance of Lactobacilli in the sample may increase which may indicate that the subject is responding to therapy. What is more, the relative abundance of certain pathogenic bacteria, as discussed above, may decrease, which may indicate that the subject is responding to therapy.
- the method of the present invention may also be used to determine whether a drug is effective at treating vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, in a similar manner to that described above in relation to determining whether a subject is responding to therapy.
- the use of “effective” is used to indicate that a treatment reduces or alleviates signs or symptoms of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, improves the clinical course of the disease, decreases the number or severity of exacerbations or reduces any other objective or subjective indicia of the disease.
- the method of the present invention can be used to determine whether drugs used to treat vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis are effective. Since a diagnosis of a disease is often not based on the results of a single test alone, the method of the present invention may be used to determine whether a subject is more likely than not to have vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis based on the genitourinary microbiome composition.
- a subject with a putative diagnosis of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis may be diagnosed as being “more likely” or “less likely” to have vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis in light of the information provided by the method of the present invention.
- the present invention may therefore be used to assist a clinician with the diagnosis of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis.
- the method of the present invention may, in certain embodiments, comprise detecting other signs or symptoms of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, conducting clinical tests of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis and/or measuring other vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis markers.
- the above description is not limited to making an initial identification (or diagnosis) of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis in a subject but is also applicable to confirming a provisional diagnosis of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis or “ruling out” such a diagnosis.
- the method of the present invention can be used to determine whether a subject has vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, the results obtained by the method of the present invention can be used to determine a suitable treatment for the subject. Therefore, the method of the present invention may further comprise a step of:
- step (d) of the invention may include determining a suitable treatment, e.g. antibiotic treatment, for the subject.
- the present invention may further comprise providing the subject with a suitable treatment based on the relative abundance of microorganisms in the urine sample.
- the subject is a human subject, preferably a female human subject.
- the subject may be a female human subject with reduced oestrogen levels.
- reduced oestrogen levels we mean that the level of oestrogen is reduced compared to a control sample, for example a pre-menopausal control sample.
- control sample As will be appreciated by the skilled person, the origin of the control sample as used herein, will depend upon the particular subject being tested. However, the control sample may be obtained from an age-matched subject and/or a subject of the same sex. Furthermore, since the comparison of relative abundance of microorganisms may be used to determine genitourinary health or vaginal health of a subject, the control sample may be derived, for example, from a subject with good genitourinary and/or vaginal health.
- the control sample may be derived from a subject who is pre-menopausal or does not have vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis.
- the method of the present invention is an in vitro method carried out on a urine sample obtained from a subject.
- Figure 1 Pie charts showing the relative abundance of bacterial species in urine samples from the following subjects: A) Pre-menopausal; B) Peri-menopausal; C) Post-menopausal; and D) Post-menopausal with HRT.
- the present inventors developed the following method to efficiently extract DNA from urine samples.
- the cell pellet is resuspended in 180 pl enzymatic lytic agent (ELB recipe).
- Tris base 20 mM final
- HCI 0.4846 g
- step 17 Repeat step 17 to step 20 using 1 ml of Wash Buffer
- step 17 Repeat step 17 to step 20 using 1 ml of 80% Ethanol.
- step 17 Repeat step 17 to step 20 using 1 ml of 80% Ethanol.
- the inventors undertook 16S library preparation, sequencing and bioinformatics as follows.
- the inventors amplified the 16S rRNA genes of all bacteria present in each of the extracted DNA samples, simultaneously adding sequencing adapters and sample-specific indexes.
- the 16S variable regions targeted were V3 and V4. qPCR was performed on Applied Biosystems StepOnePlus instrument.
- the inventors have found that by focussing their sequencing and subsequent analysis on the V3 and V4 variable regions of 16S, improved identification of bacterial species are obtained. Many bacterial species associated with vaginal health are not correctly identified using analysis of the V1 , V2, V5, V6, V7, V8 and V9 variable regions and as such it is advantageous to focus sequencing on the V3 and V4 variable regions.
- the inventors were able to accurately predict the menopausal status of the subjects with high accuracy based on composition of the genitourinary microbiome, and in particular the relative abundance of bacterial species in the genitourinary microbiome.
- Table 1 Initial results comparing first-steam, mid-stream and vaginal swab samples
- Figure 1 demonstrates the relative abundance of bacterial species in example subjects: A) Pre- menopausal; B) Peri-menopausal; C) Post-menopausal; and D) Post-menopausal with HRT.
- A Pre- menopausal
- B Peri-menopausal
- C Post-menopausal
- D Post-menopausal with HRT.
- Lactobacillus species are dominant, with Lactobacillus gasseri, Lactobacillus crispatus and Lactobacillus iners showing dominance, forming between 87% and 93% of the bacteria present.
- Lactobacillus crispatus is the dominant Lactobacillus in pre-menopausal subjects.
- VMM vaginal microbiome measurement
- Non-crispatus Lactobacillus species including but not limited to L. jensenii, gasserii and iners i.e. lactic acid bacteria) and other potentially protective species- 0.5-4
- Potentially pathogenic species - -2 include but not limited to L. jensenii, gasserii and iners i.e. lactic acid bacteria.
- VMM vaginal microbiome measurement
- VMM vaginal microbiome measurement
- VMM vaginal microbiome measurement
- the vaginal microbiome measurement (VMM) for the premenopausal subjects of Figure 1A were 3.51 and 3.85 respectively.
- the vaginal microbiome measurement (VMM) for the peri-menopausal subject of Figure 1 B was 1.97 and the vaginal microbiome measurement (VMM) for the post-menopausal subjects of Figure 1 C were 0.86 and 1 .43 respectively.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a method of determining the menopausal status of a subject by assessing genitourinary microbiome composition of the subject, the method comprising: (a) extracting nucleic acid from a urine sample from the subject; and (b) analysing the extracted nucleic acid to identify the relative abundance of microorganisms in the urine sample.
Description
IDENTIFICATION OF A BACTERIUM OF THE GENUS BACILLUS
Field of the Invention
The present invention relates to a method of determining genitourinary microbiome composition from a urine sample of a subject. More specifically the invention relates to a method of determining genitourinary or vaginal health in a subject using the method. In particular, the method of the present invention can be used to determine menopausal status in a subject.
Background to the Invention
The microbiome is the collection of microorganisms, such as bacteria, viruses and fungi which live on and inside our bodies. It has come to light that these microorganisms contribute to our health in a number of ways, for example in development of the immune system, protecting the body against pathogenic microorganisms, maintaining skin health and maintaining good digestion. A person’s core microbiome is formed during the early years of life. However, over time, the composition of the microbiome can change, whether that be in response to external factors, diet, drugs, illness or internal factors such as hormonal changes.
The genitourinary microbiome refers to the microorganisms present in the genitourinary system, which includes the urinary and genital organs. This includes the vaginome or vaginal microbiome (the microorganism species present in the vagina). Similar to the microbiome as a whole, the composition of the genitourinary microbiome and vaginome have been linked to health and disease. For example, the microorganisms present in the vagina maintain vaginal homeostasis, and an imbalance (or dysbiosis) can be associated with poor vaginal health. The vaginal microbiome is also implicated in the progression of the menopause. During the menopause, oestrogen levels decrease which results in changes in the vaginome. In particular, oestrogen leads to the production of glycogen. Amylase breaks down glycogen into products including maltose, maltotriose and maltotetraose. Lactobacilli can grow in these products and produce lactic acid. The presence of lactic acid lowers the pH and impedes growth of potentially pathogenic bacteria. On the other hand, oestrogen deficiency (for example as a result of menopause) means that Lactobacilli can no longer proliferate and pathogenic bacteria may become dominant. By studying vaginal swab samples, studies have shown that alterations in the microbiome are associated with specific menopausal symptoms. However, vaginal swabs are invasive and are often not well tolerated by patients. Such samples may also be difficult to obtain without assistance from a medical professional. Therefore, determining the microorganisms present in the genitourinary microbiome and particularly the vaginome can be problematic.
It is an object of the present invention to obviate or mitigate one or more of the abovementioned problems.
Summary of the Invention
The present invention relates to a method of determining the genitourinary microbiome composition of a subject and is based, in part, on studies by the inventors in which they have shown that the composition of the genitourinary microbiome, obtained by the analysis of the urine sample, can be used to determine vaginal health and in particular can be used to determine the menopausal status of a subject.
In a first aspect of the present invention there is provided a method of determining the menopausal status of a subject by assessing genitourinary microbiome composition of the subject, the method comprising:
(a) extracting nucleic acid from a urine sample from the subject; and
(b) analysing the extracted nucleic acid to identify the relative abundance of microorganisms in the urine sample.
As will be appreciated by the skilled person, the term “genitourinary microbiome composition” as used herein means the composition of microorganisms present in the genitourinary system, which includes the urinary and genital organs. This includes, for example, the vaginome or vaginal microbiome (the microorganisms present in the vagina).
The composition of the genitourinary microbiome and particularly the vaginome composition is usually determined using a vaginal swab. However, the present inventors have surprisingly shown that it is possible to obtain highly accurate information regarding the composition of the genitourinary microbiome through analysis of a urine sample from the subject. This is particularly advantageous as urine samples are less invasive than vaginal swabs and therefore tolerated more by subjects. What is more, such samples can be much more easily obtained, which is particularly important for those unable to perform vaginal self-swabbing. Furthermore, urine samples provide information about a greater area of the genitourinary tract and therefore the results obtained can be used to indicate the genitourinary health of the subject, particularly vaginal health and menopause status.
The inventors have shown that they are able to accurately predict the menopausal status of a subject by analysing the relative abundance of microorganisms present in a urine sample from the subject.
As mentioned above, the present inventors have surprisingly found that it is possible to obtain highly accurate information regarding the composition of the genitourinary microbiome through urine analysis. This is unexpected. In the art it is known that there are difficulties obtaining nucleic acids from urine due to the impurities present and many variables affect the composition of the urine, such as fluid intake, diet and disease. Furthermore, it would be expected that the composition of microorganisms in the urinary microbiome differs from that of the vaginal microbiome as the bladder and vagina are anatomically distinct.
In embodiments, the method of the present invention further comprises comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the menopausal status of a subject. The present invention therefore also provides a method of determining the menopausal status of a subject by assessing genitourinary microbiome composition of the subject, the method comprising:
(a) extracting nucleic acid from a urine sample from the subject;
(b) analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample; and
(c) comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the menopausal status of the subject.
The term “menopausal status” as used herein is used to mean whether the subject is premenopausal (i.e. has not undergone permanent cessation of ovulation), peri-menopausal (the 5 years preceding menopause, when ovaries begin to make less oestrogen) or post-menopausal (in which at least 12 months has passed without a menstrual period/ovulation has permanently ceased).
A clinician may also consider a female subject’s age and medical history when assessing their menopausal status. For example the date of the subject’s last period and their genitourinary symptoms (such as recurrent UTIs, vulva and/or vaginal soreness, burning or irritation, vaginal dryness or reduced discharge) may be relevant.
As described above, during the transition from pre-menopause to peri-menopause and postmenopause, the oestrogen levels in a female subject decrease which results in subsequent pH changes in the genitourinary system and consequent changes in the genitourinary microbiome composition. In pre-menopausal subjects, Lactobacillus species (particularly Lactobacillus crispatus) are dominant and due to the low pH pathogenic bacteria cannot grow. However,
during transition to peri-menopause, oestrogen levels begin to decline and so Lactobacillus species become less dominant in the genitourinary microbiome. In post-menopausal subjects, oestrogen levels are low, pH is high and the genitourinary microbiome has a high diversity of bacterial species with low levels of Lactobacillus and higher levels of pathogenic bacteria. Diversity of microorganisms increases during the transition from pre-menopause to perimenopause and post-menopause. The present inventors have found that by determining the genitourinary microbiome composition from a urine sample they can accurately predict the menopausal status of the subject.
The urine sample can be any suitable sample, for example the first urine of the day or a subsequent urine sample. The sample may be a first-stream urine sample (i.e. the first part of the urine to be passed), or a mid-stream or late-stream urine sample. However, in embodiments, the sample is a first-stream urine sample. The inventors have found that a first stream-urine sample surprisingly results in improved ease of nucleic acid extraction and nucleic acid yield. Furthermore, and as shown below, the inventors have shown that when using the results of the method to indicate the menopausal status of the subject, for example, the results using first- stream urine more accurately reflect the menopausal status of the subject.
The present invention involves (a) extracting nucleic acid from a urine sample from the subject. As will be appreciated by the skilled person the term “extracting nucleic acid” from a urine sample means removing the nucleic acid from the urine sample. The nucleic acid extracted may be free in the urine or may be present in cells, for example microorganisms (e.g. bacterial species) present in the urine sample. In particular, the present invention involves extracting nucleic acid from cells and/or microorganisms present in a urine sample from the subject.
As will be appreciated by the skilled person, the term nucleic acid as used herein refers to macromolecules formed by a chain of nucleotides which stores genetic information. Nucleic acids include DNA and RNA.
In some embodiments the nucleic acid comprises DNA.
In some embodiments the nucleic acid comprises RNA.
In some embodiments the nucleic acid comprises DNA and RNA.
Suitable types of DNA include genomic DNA from a microorganism, plasmid DNA, DNA aptamers, viral DNA and cosmid DNA, for example.
Suitable types of RNA include messenger RNA (mRNA), ribosomal RNA (rRNA), signal recognition particle RNA (SRP RNA), transfer RNA (tRNA), transfer messenger RNA (tmRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), SmY RNA, small Cajal body specific RNA (scaRNA), guide RNA (gRNA), ribonuclease P (RNase P), ribonuclease MRP (RNase MRP), Y RNA, telomerase RNA component (TERC), spliced leader RNA (SL RNA), antisense RNA (asRNA), cis-natural antisense RNA (cis-NAT), long noncoding RNA (IncRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), small interfering RNA (siRNA), trans-acting siRNA (tasiRNA), repeat associated siRNA (rasiRNA), 7SK RNA (7SK), viral RNA and RNA aptamers. Suitably these types of RNA may be from a microorganism.
DNA and/or RNA from microorganisms may be from bacteria, fungi or viruses.
In embodiments, step (a) comprises preparing a cell pellet from the urine. As will be appreciated the cells are the microorganism cells present in the urine, particularly including the microorganisms whose relative abundance is to be assessed. In such embodiments, the method may comprise diluting the urine before preparing the cell pellet. Dilution of the urine may be achieved using any suitable means. However, in embodiments, the urine is diluted by addition of a buffer, for example TE buffer, boric acid or EDTA. In embodiments the urine is diluted by addition of TE buffer. The inventors have surprisingly found that by diluting urine, the collection of impurities is reduced. What is more, the addition of the buffer dissolves any crystals such as urates to avoid collection of such impurities with the cell pellet.
In embodiments, step (a) comprises lysing cells present in the urine sample. The skilled person will appreciate that there are various methods by which cell lysis can be achieved. For example, lysing the cells may be achieved through chemical lysis or physical lysis, for example bead beating or sonication. In embodiments, lysing the cells is achieved by contacting the sample with a chemical lytic agent, for example a lysis buffer. The chemical lytic agent may include lysozyme or another commercially available lytic enzyme. The present inventors have found that the use of lysozyme gives improved nucleic acid yield and the most accurate measurement of bacterial communities.
The skilled person will appreciate that there are various methods by which nucleic acid can be extracted from cells. For example, column-based extraction methods may be used in which nucleic acids bind to a solid phase of silica under specific binding conditions. Nucleic acids are then subsequently eluted from the solid phase. Kits are available for performing such extraction and include, for example, Qiagen DNeasy Blood and Tissue Kit. Alternatively, nucleic acid extraction could take place by way of phenol-chloroform extraction or bead-based extraction, for example through the use of magnetic beads. In magnetic bead-based extraction, nucleic acids bind to the magnetic beads under specific binding conditions, leaving contaminants in solution.
The nucleic acid can be subsequently removed from the beads under elution conditions. Kits are available for performing such extraction and include, for example, Thermo MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit. In embodiments, the nucleic acid may be extracted by column-based or magnetic bead-based extraction methods. In embodiments, the method comprises extracting nucleic acid from the urine sample using a magnetic bead-based extraction method. The inventors have found that using a magnetic bead-based approach, rather than a column approach, addresses the problem of the column becoming blocked with impurities from the urine and thereby improves the nucleic acid quality obtained.
Preferably step (a) further comprises isolating the nucleic acid from the sample, suitably after extraction thereof.
In embodiments, the present invention may comprise a step of providing a urine sample from a subject. In such embodiments, the invention may comprise adding a preservative to the urine sample, for example EDTA, boric acid or other commercially available preservatives. These may be lytic or non-lytic. The inventors have found that the addition of a preservative to the urine sample results in improved nucleic acid extraction and subsequent amplification. Preferred preservatives include boric acid. Suitably, the urine sample may be refrigerated once the preservative has been added.
In embodiments, the present invention may comprise a step of obtaining a urine sample from a subject.
Step (b) of the method of the present invention involves analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample.
In embodiments, step (b) may involve amplifying the extracted nucleic acid. The skilled person will appreciate that there are various methods by which the nucleic acid can be amplified. Methods include, for example, polymerase chain reaction (PCR). PCR techniques include basic PCR, reverse transcription (RT)-PCR, hot-start PCR, long PCR, quantitative PCR, quantitative endpoint PCR, quantitative real-time PCR, rapid amplified polymorphic DNA analysis, nested PCR and high-fidelity PCR. In embodiments, the amplification takes place using quantitative PCR (qPCR) or PCR.
Preferably step (b) comprises the addition of at least one reagent for performing the PCR or quantitative PCR. Suitably the at least one reagent is added to the extracted nucleic acid of step (a).
Preferably the at least one reagent is selected from: DNA polymerase, buffer, dNTPs, or a source of magnesium. More preferably step (b) comprises the addition of all three of these components, referred to as a PCR master mix. The master mix may suitably be sourced from any manufacturer. The PCR Master Mix may be added to the extracted nucleic acid in any suitable amount, for example an amount of around 5-15 pl, around 7-12 pl, or around 10 pl.
Suitably step (b) further comprises the addition of primers and probes as required for PCR or quantitative PCR amplification of the extracted nucleic acid. Preferably the primers are added in equal amounts to the extracted nucleic acid of step (a) of the method.
Step (b) may further comprise amplifying and indicating the level of a control nucleic acid together with the extracted nucleic acid, the control nucleic acid being definitively present within the urine sample. Preferably the control nucleic acid is a human nucleic acid when the urine sample is of human origin. More preferably the control nucleic acid is a human nucleic acid, preferably the control human nucleic acid is of a housekeeping gene or genes that are substantially conserved in human nucleic acid.
Suitably the PCR or quantitative PCR takes place using standard procedures known in the art and using standard machinery known in the art. qPCR is described in Higuchi R et al. Kinetic PCR analysis: Real time Monitoring of DNA amplification reactions. Bio/Technology 1993; 11 :1026-30.
As will be appreciated by the skilled person, PCR, and indeed qPCR involves a set program of heating cycles. The heating cycle may comprise any of the following stages: mix activation, initial denaturation, denaturation, annealing, extension, final extension, or cooling. Suitably the heating cycle comprises at least initial denaturation, denaturation, and annealing. Optionally, any of the stages may be repeated, alone or in combination with any other stage. Preferably the denaturation and annealing stages are repeated, more preferably these stages are repeated for between 15 and 45 cycles, for example 20 cycles.
In preferred embodiments, step (b) of the method comprises amplifying nucleic acid encoding bacterial 16S rRNA present in the extracted nucleic acid. As such, the primers used in the PCR or qPCR reaction, for example, are specific to the bacterial 16S rRNA genes. In embodiments, the method comprises amplifying nucleic acid encoding bacterial 16S variable regions 1-9 (V1- 9), more preferably bacterial 16S variable regions 1-4 (V1-4), more preferably bacterial 16S variable regions 3-4 (V3-4). In such embodiments, the amplification may take place by PCR, in embodiments using qPCR. The One 16S NGS Library Preparation Kit (by YouSeq) is a suitable kit for amplifying these bacterial 16S variable regions and may be used in the method of the present invention. Targeting amplification and sequencing of the nucleic acid encoding bacterial
16S rRNA gene is known in the art as a method for studying bacterial diversity. However, the accuracy may depend on the choice of primers, the bacterial species contained in the sample and the areas of 16S rRNA targeted. The present inventors have found that by targeting 16S rRNA variable regions 3-4, they can accurately identify bacterial species found in urine. They can also correctly identify potentially pathogenic species such as Gardnerella vaginalis which can be useful for indicating vaginal health.
In embodiments of the present invention, step (b) of the method may further comprise sequencing the extracted nucleic acid. In embodiments, step (b) of the method may comprise sequencing the amplified nucleic acid, preferably the amplified nucleic acid encoding bacterial 16S rRNA, preferably nucleic acid encoding variable regions 1-9, 1-4 or 3-4 of the bacterial 16S rRNA. Such sequencing can be used to identify the bacterial species present in the urine sample. Amplification of the nucleic acid by qPCR allows the relative abundance of the bacterial species in the urine sample to be determined by subsequent sequencing.
In embodiments in which sequencing of the extracted nucleic acid takes place, each primer used in amplification may have a partial adapter sequence, for facilitating sequencing of said sequence. Any suitable sequencing method known in the art may be used, for example traditional sequencing methods such as Sanger sequencing, or next generation sequencing methods. Sequencing methods include whole genome sequencing, whole exome sequencing, targeted sequencing and metagenomic sequencing. Given the potentially large number and variety of microorganisms which may be present in the urine sample of the present invention, high throughput/next generation sequencing methods, which are capable of rapidly generating large amounts of sequence data, are preferred since they allow rapid and accurate identification of large numbers of microorganisms. In embodiments, the sequencing comprises next generation sequencing. Next generation sequencing methods enable a large number of distinct nucleic acid sequences to be sequenced simultaneously and at a high density, including, for example, sequencing-by-synthesis, sequencing by ligation, pyrosequencing and ion semiconductor sequencing. A preferred sequencing method is sequencing-by-synthesis, for example using the Illumina iSeq 100 or MiSeq systems. In this method, a fluorescently labelled reversible terminator is imaged as each dNTP is added, and then cleaved to allow incorporation of the next base. Since all 4 reversible terminator-bound dNTPs are present during each sequencing cycle, natural competition minimizes incorporation bias. The end result is true base- by-base sequencing that enables accurate data for a broad range of applications. The method virtually eliminates errors and missed calls associated with strings of repeated nucleotides (homopolymers)’ (from lllumina.com).
In an embodiment a bioinformatics method may be used to determine the microorganisms present in the urine sample.
The method of the present invention involves analysing the extracted nucleic acid to identify the relative abundance of microorganisms in the urine sample. As will be appreciated by the skilled person, the term “relative abundance” means the percentage of a microorganism of a particular kind relative to the total number of microorganisms in the sample.
The microorganisms may include bacteria, fungi, viruses, parasites and protozoa for example. The microorganisms may include commensal microorganisms and/or pathogenic microorganisms. In particular embodiments, the microorganisms comprise bacteria. Accordingly, the method may comprise analysing the extracted nucleic acid to identify the relative abundance of bacterial species in the urine sample.
The bacterial species may include, for example, Lactobacilli, Gardnerella, Prevotella, Pseudomonas, Peptoniphilus, Chryseobacterium, Neisseria, Bergeyelia, Acinetobacter, Alphaproteobacteria,Anaerococcus, Actinomycetales, Atopobium, Escherichia, Shigella, Flavobacterium, Dialister, Streptococcus, Aerococcus, Campylobacter, Shuttlewothia, Staphylococcus, Megasphaera, Parvimonas, Ruminococcaceae/Oscillococcaceae, Sneathia and/or Diaphorobacter.
The present invention is used to determine the menopausal status of a subject. In embodiments, the method comprises identifying the relative abundance of Lactobacilli in the urine sample. The Lactobacilli may comprise, for example, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri. The inventors have found that by determining the relative abundance of Lactobacilli in the sample, the menopausal stage can be predicted. For example, pre-menopausal subjects have a high relative abundance of Lactobacilli (for example greater than 70%) with L. crispatus dominant. Therefore, a relative abundance of Lactobacilli of greater than 70% may be indicative of pre-menopausal status. In peri-menopausal subjects, Lactobacilli are still dominant, but to a lesser degree. Therefore, a relative abundance of Lactobacilli of between 40 and 70% may be indicative of peri-menopausal status. What is more peri-menopausal subjects show a greater extent of non-Lactobacillus crispatus spp as compared to pre-menopausal subjects. On the other hand, post-menopausal subjects have low levels of Lactobacilli and a high diversity of bacterial species (no dominance of a single species), with pathogenic bacteria often present. Therefore, a relative abundance of Lactobacilli of less than 40% may be indicative of post-menopausal status.
In embodiments, the method comprises identifying the relative abundance of Lactobacillus crispatus in the urine sample. As mentioned above, pre-menopausal subjects have a dominance of Lactobacillus crispatus within the Lactobacilli present.
In embodiments, the method may comprise determining a vaginal microbiome measurement (VMM) based on the genitourinary microbiome composition and the relative abundance of the microorganisms in the urine sample.
The vaginal microbiome measurement (VMM) may be determined based on the microorganisms present in the genitourinary microbiome. For example, bacterial species may be awarded values as follows:
Lactobacillus crispatus - 6
Non-crispatus Lactobacillus species (including but not limited to L. jensenii, gasserii and iners i.e. lactic acid bacteria) and other potentially protective species- 0.5-4
Potentially pathogenic species - -2
Other (defined as the sum total of bacterial species with less than 1 % relative abundance each) = 0.75.
The values may be multiplied by the relative abundance of each microorganism, the totals added together and divided by 100 to give a vaginal microbiome measurement (VMM). A VMM of less than around 1 may be indicative of post-menopause. A VMM of between around 1 and 3.5 may be indicative of peri-menopause. A VMM of greater than around 3.5 may be indicative of premenopause.
In embodiments, the method may further comprise assigning a community state type (CST) to the subject, based on the genitourinary microbiome composition/relative abundance of microorganisms in the urine sample. The term “community state type” (CST) is used in microbial ecology to describe a group of community states with similar microbial phylotype composition and abundance. Vaginal CSTs are known to be linked to vaginal health and disease and menopause status (Brotman et al). A CST may be assigned to the subject based on the relative abundance of microorganisms in the urine sample. The CSTs may be assigned according to the following criteria:
As will be appreciated by the skilled person, the term “dominant” as used herein means a microorganism or bacterial species which is present as a significant proportion of the total microorganisms or bacterial species, for example, over 30%, 40%, 50%, 60% or 70% of the total microorganisms or bacterial species present.
The method of the present invention may comprise determining the menopausal status based on one or more of Lactobacilli relative abundance, vaginal microbiome measurement (VMM) and/or assigned CST type.
Based on the above, the present invention can also be used to determine progression of menopause. The present invention therefore also provides a method for determining the menopause status in a subject, determining progression of menopause or assessing response to therapy of a subject with menopause by assessing genitourinary microbiome composition of the subject. The method comprises the steps of:
(a) extracting nucleic acid from a urine sample from the subject; and
(b) analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample.
The present invention also provides a method for determining the menopause status in a subject, determining progression of menopause or assessing response to therapy of a subject with menopause by assessing genitourinary microbiome composition of the subject. The method comprises the steps of:
(a) extracting nucleic acid from a urine sample from the subject;
(b) analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample; and
(c) comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine menopause status in a subject, determining progression of menopause or assessing response to therapy of a subject with menopause.
In such embodiments the method may comprise identifying the relative abundance of Lactobacilli in the urine sample, assigning a community state type (CST) to the subject and/or determining a vaginal microbiome measurement (VMM), as described in more detail above.
In embodiments where the method is being used to determine whether menopause is progressing, the method may comprise monitoring over time to determine whether menopause status has progressed. In such an embodiment, an initial genitourinary microbiome composition
may be compared with a genitourinary microbiome composition in a sample obtained later in time. The genitourinary microbiome composition obtained may change over time, to be more indicative of peri-menopause or post-menopause for example. In an initial analysis, the genitourinary microbiome composition may be indicative of pre-menopause, but a later analysis may be indicative of peri- or post-menopause. For example, the dominance of Lactobacilli in the genitourinary microbiome composition may decrease, indicative of progression of menopause status.
The present inventors have also found that in post-menopause subjects treated with hormone replacement therapy (HRT) the genitourinary microbiome composition after treatment shows an increased level of Lactobacilli. Therefore, the method can also be used to determine whether a subject is responding to therapy. In embodiments where the method is being used to determine whether a subject is responding to therapy, the method may comprise monitoring over time to determine whether a subject is responding to therapy. In such an embodiment, an initial genitourinary microbiome composition may be compared with one or more genitourinary microbiome composition analyses undertaken on samples obtained later in time (for example after therapy has commenced). The genitourinary microbiome composition may change over time, for example the relative abundance of Lactobacilli in the sample may increase which may indicate that the subject is responding to therapy.
The method of the present invention may also be used to determine whether a drug is effective at treating menopause, in a similar manner to that described above in relation to determining whether a subject is responding to therapy. The use of “effective” is used to indicate that a treatment reduces or alleviates signs or symptoms of menopause, improves the clinical course of the disease, decreases the number or severity of exacerbations or reduces any other objective or subjective indicia of the disease. The method of the present invention can be used to determine whether drugs used to treat menopause, in addition to other drugs developed to treat menopause are effective.
Since a diagnosis of a disease is often not based on the results of a single test alone, the method of the present invention may be used to determine whether a subject is more likely than not to have menopause based on the genitourinary microbiome composition. Thus, for example, a subject with a putative diagnosis of peri-menopause or post-menopause may be diagnosed as being “more likely” or “less likely” to be peri-menopause or post-menopause in light of the information provided by the method of the present invention. The present invention may therefore be used to assist a clinician with the diagnosis of menopause.
The method of the present invention may, in certain embodiments, comprise detecting other signs or symptoms of peri-menopause or menopause, conducting clinical tests of perimenopause or menopause and/or measuring other peri-menopause or menopause markers.
As will be appreciated by the skilled person, the above description is not limited to making an initial identification (or diagnosis) of menopause in a subject but is also applicable to confirming a provisional diagnosis of menopause or “ruling out” such a diagnosis.
Since the method of the present invention can be used to determine menopausal status, the results obtained by the method of the present invention can be used to determine a suitable treatment for the subject. Therefore, the method of the present invention may further comprise a step of:
(d) determining a suitable treatment for the subject based on the relative abundance of microorganisms in the urine sample.
For example, if the method of the present invention determines that the subject is postmenopausal, step (d) of the method of the present invention may be used to determine a suitable treatment, for example hormone replacement therapy. In embodiments, the present invention may further comprise providing the subject with a suitable treatment based on the relative abundance of microorganisms in the urine sample.
The present inventors have also found that by determining the relative abundance of microorganisms in a urine sample, the genitourinary health, particularly the vaginal health of a subject can be determined.
Therefore, in embodiments, the method of the present invention may be used to determine the genitourinary or vaginal health of a subject.
The present invention therefore provides a method of determining the genitourinary health or vaginal health of a subject by assessing the genitourinary microbiome composition of the subject, the method comprising:
(a) extracting nucleic acid from a urine sample from the subject; and
(b) analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample.
The method makes use of the genitourinary microbiome composition to determine genitourinary and particularly vaginal health. The present inventors have found that by determining the
composition of the genitourinary microbiome from a urine sample, it is possible to identify the genitourinary or vaginal health of the subject and, in particular, whether the subject has any of the following conditions: vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis. The term “genitourinary health” and “vaginal health” as used herein are used to mean the state of the genitourinary system or vagina in terms of whether illness or injury is present, for example.
In embodiments, the method of the present invention further comprises comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the genitourinary health or vaginal health of a subject.
Thus, the present invention provides a method of determining the genitourinary health or vaginal health of a subject by assessing genitourinary microbiome composition of the subject, the method comprising:
(a) extracting nucleic acid from a urine sample from the subject;
(b) analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample; and
(c) comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the genitourinary health or vaginal health of the subject.
As mentioned above, the present inventors have found that by determining the composition of the genitourinary microbiome from a urine sample, it is possible to identify the genitourinary or vaginal health of the subject and, in particular, whether the subject has any of the following conditions: vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis. In particular, the inventors have shown that they are able to accurately predict the menopausal status of a subject by analysing the relative abundance of bacterial species present in a urine sample from the subject.
The method of the present invention can also be used to determine other aspects of genitourinary or vaginal health. For example, subjects suffering from vulvovaginal atrophy (which causes dysuria, dyspareunia, bleeding after intercourse, vaginal discharge, sorenes, itching or burning sensations) exhibit low levels of Lactobacilli in the genitourinary microbiome. Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella species are also often present. In embodiments in which the method is used to determine the presence of vulvovaginal atrophy in a subject, the method comprises identifying the relative abundance of Lactobacilli in the urine sample. The method may further comprise identifying the relative abundance of one or more of
the following bacterial species in the urine sample: Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella. A relative abundance of Lactobacilli of less than 50% may be indicative of vulvovaginal atrophy, particularly in combination with the presence of one or more of the following bacterial species: Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella. A relative abundance of greater than 25% of Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella may be indicative of vulvovaginal atrophy.
Subjects suffering from bacterial vaginosis exhibit low levels of Lactobacilli in the genitourinary microbiome. Gardnerella, Atopobium, Megasphaera, and Prevotella species are also often present. In embodiments in which the method is used to determine the presence of bacterial vaginosis in a subject, the method comprises identifying the relative abundance of Lactobacilli in the urine sample. The method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: Gardnerella, Atopobium, Megasphaera, and Prevotella. A relative abundance of Lactobacilli of less than 50% may be indicative of bacterial vaginosis, particularly in combination with the presence of one or more of the following bacterial species: Gardnerella, Atopobium, Megasphaera, and Prevotella. A relative abundance of greater than 25% of Gardnerella, Atopobium, Megasphaera, and/or Prevotella may be indicative of bacterial vaginosis.
Subjects suffering from urinary tract infections exhibit low levels of Lactobacilli in the genitourinary microbiome. E. coli, K. pneumoniae and E. faecalis are also often present. In embodiments in which the method is used to determine the presence of urinary tract infections in a subject, the method comprises identifying the relative abundance of Lactobacilli in the urine sample. The method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: E. coli, K. pneumoniae and E. faecalis. A relative abundance of Lactobacilli of less than 50% may be indicative of urinary tract infections, particularly in combination with the presence of one or more of the following bacterial species: E. coli, K. pneumoniae and E. faecalis. A relative abundance of greater than 25% of E. coli, K. pneumoniae and/or E. faecalis may be indicative of urinary tract infections.
Subjects suffering from aerobic vaginitis (which causes dyspareunia, burning sensation, itching, odourless discharge, redness, swelling) exhibit low levels of Lactobacilli in the genitourinary microbiome. Group B Streptococcus, P. bivia, Enterobacteriaceae are also often present. In embodiments in which the method is used to determine the presence of aerobic vaginitis in a subject, the method comprises identifying the relative abundance of Lactobacilli in the urine sample. The method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: Group B Streptococcus, P. bivia and Enterobacteriaceae. A relative abundance of Lactobacilli of less than 50% may be indicative of aerobic vaginitis, particularly in combination with the presence of one or more of the following
bacterial species: Group B Streptococcus, P. bivia and Enterobacteriaceae. A relative abundance of greater than 25% of Group B Streptococcus, P. bivia and/or Enterobacteriaceae may be indicative of aerobic vaginitis.
In subjects suffering from vulvovaginal candidiasis (which causes vaginal itching, discharge, burning, pain and redness) the genitourinary microbiome is dominated by Lactobacillus iners and is most commonly caused by Candida albicans. Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella species are also often present. In embodiments in which the method is used to determine the presence of vulvovaginal candidiasis in a subject, the method comprises identifying the relative abundance of Lactobacilli iners in the urine sample. The method may further comprise identifying the relative abundance of one or more of the following bacterial species in the urine sample: Group B Streptococcus, P. bivia and Enterobacteriaceae. A relative abundance of Lactobacilli of less than 50% may be indicative of vulvovaginal candidiasis, particularly in combination with the presence of one or more of the following bacterial species: Atopobium, Megasphaera, Anaerococcus, Peptoniphilus and Prevotella. The presence of Candida albicans is also indicative of vulvovaginal candidiasis. A relative abundance of greater than 25% of Atopobium, Megasphaera, Anaerococcus, Peptoniphilus, Prevotella and/or Candida albicans may be indicative of vulvovaginal candidiasis.
When referring to a relative abundance of greater than 25% of various bacterial species, for example Gardnerella, Atopobium, Megasphaera, and/or Prevotella, we mean a relative abundance of one of these bacterial species alone or a combination of one or more of these bacterial species.
In embodiments, the method may comprise determining a vaginal microbiome measurement (VMM) based on the genitourinary microbiome composition and the relative abundance of the microorganisms in the urine sample.
The vaginal microbiome measurement (VMM) may be determined based on the microorganisms present in the genitourinary microbiome. For example, microorganisms may be awarded values as follows:
Lactobacillus crispatus - 6
Non-crispatus Lactobacillus species (including but not limited to L. jensenii, gasserii and iners i.e. lactic acid bacteria) and other potentially protective species- 0.5-4
Potentially pathogenic species - -2
Other (defined as the sum total of bacterial species with less than 1 % relative abundance each) = 0.75.
The values may be multiplied by the relative abundance of each microorganism, the totals added together and divided by 100 to give a vaginal microbiome measurement (VMM).
Therefore, by determining the genitourinary microbiome composition of the subject, it is possible to predict whether a subject is suffering from one of the abovementioned conditions.
The present invention therefore also provides a method for determining the presence of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis, determining progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis or assessing response to therapy of a subject with vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis by assessing genitourinary microbiome composition of the subject. The method comprises the steps of:
(a) extracting nucleic acid from a urine sample from the subject; and
(b) analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample.
The present invention also provides a method for determining the presence of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis in a subject, determining progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis or assessing response to therapy of a subject with vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis. The method comprises the steps of:
(a) extracting nucleic acid from a urine sample from the subject;
(b) analysing the nucleic acid to identify the relative abundance of microorganisms in the urine sample; and
(c) comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the presence of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis, determining progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis or assessing response to therapy of a subject with vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis.
In such a method, the method may comprise identifying the relative abundance of Lactobacilli in the urine sample, as described in more detail above. The method may further comprise
identifying the relative abundance of the bacterial species outlined above as being associated with the presence of these respective conditions.
In embodiments where the method is being used to determine whether vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis is progressing, the method may comprise monitoring over time to determine whether vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis has progressed. In such an embodiment, an initial genitourinary microbiome composition may be compared with a genitourinary microbiome composition in a sample obtained later in time. The genitourinary microbiome composition obtained may change over time, to be more indicative of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis for example. For example, the relative abundance of Lactobacilli in the genitourinary microbiome composition may decrease, indicative of progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis. What is more, the relative abundance of certain pathogenic microorganisms, as discussed above, may increase, indicative of progression of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis.
The method can also be used to determine whether a subject is responding to therapy. In embodiments where the method is being used to determine whether a subject is responding to therapy, the method may comprise monitoring over time to determine whether a subject is responding to therapy. In such an embodiment, an initial genitourinary microbiome composition may be compared with one or more genitourinary microbiome composition analyses undertaken on samples obtained later in time (for example after therapy has commenced). The genitourinary microbiome composition may change over time, for example the relative abundance of Lactobacilli in the sample may increase which may indicate that the subject is responding to therapy. What is more, the relative abundance of certain pathogenic bacteria, as discussed above, may decrease, which may indicate that the subject is responding to therapy.
The method of the present invention may also be used to determine whether a drug is effective at treating vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, in a similar manner to that described above in relation to determining whether a subject is responding to therapy. The use of “effective” is used to indicate that a treatment reduces or alleviates signs or symptoms of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, improves the clinical course of the disease, decreases the number or severity of exacerbations or reduces any other objective or subjective indicia of the disease. The method of the present invention can be used to determine whether drugs used to treat vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis, vulvovaginal candidiasis are effective.
Since a diagnosis of a disease is often not based on the results of a single test alone, the method of the present invention may be used to determine whether a subject is more likely than not to have vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis based on the genitourinary microbiome composition. Thus, for example, a subject with a putative diagnosis of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis may be diagnosed as being “more likely” or “less likely” to have vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis in light of the information provided by the method of the present invention. The present invention may therefore be used to assist a clinician with the diagnosis of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis.
The method of the present invention may, in certain embodiments, comprise detecting other signs or symptoms of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, conducting clinical tests of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis and/or measuring other vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis markers.
As will be appreciated by the skilled person, the above description is not limited to making an initial identification (or diagnosis) of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis in a subject but is also applicable to confirming a provisional diagnosis of vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis or “ruling out” such a diagnosis.
Since the method of the present invention can be used to determine whether a subject has vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, the results obtained by the method of the present invention can be used to determine a suitable treatment for the subject. Therefore, the method of the present invention may further comprise a step of:
(d) determining a suitable treatment for the subject based on the relative abundance of microorganisms in the urine sample.
For example, if the present invention determines that the subject has bacterial vaginosis, for example, step (d) of the invention may include determining a suitable treatment, e.g. antibiotic treatment, for the subject. In embodiments, the present invention may further comprise providing
the subject with a suitable treatment based on the relative abundance of microorganisms in the urine sample.
In embodiments of the present invention, the subject is a human subject, preferably a female human subject. In embodiments of the present invention, the subject may be a female human subject with reduced oestrogen levels. By reduced oestrogen levels we mean that the level of oestrogen is reduced compared to a control sample, for example a pre-menopausal control sample.
As will be appreciated by the skilled person, the origin of the control sample as used herein, will depend upon the particular subject being tested. However, the control sample may be obtained from an age-matched subject and/or a subject of the same sex. Furthermore, since the comparison of relative abundance of microorganisms may be used to determine genitourinary health or vaginal health of a subject, the control sample may be derived, for example, from a subject with good genitourinary and/or vaginal health. Since the comparison of relative abundance of microorganisms may be used to determine the menopausal status of a subject, and/or whether a subject has vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis, the control sample may be derived from a subject who is pre-menopausal or does not have vulvovaginal atrophy, bacterial vaginosis, urinary tract infections, aerobic vaginitis or vulvovaginal candidiasis.
As will be appreciated by the skilled person, the method of the present invention is an in vitro method carried out on a urine sample obtained from a subject.
The described and illustrated embodiments are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.
The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments.
It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it
may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.
Detailed Description of the Invention
The present invention will now be further described with reference to the following figure which shows:
Figure 1 : Pie charts showing the relative abundance of bacterial species in urine samples from the following subjects: A) Pre-menopausal; B) Peri-menopausal; C) Post-menopausal; and D) Post-menopausal with HRT.
Example 1
Sample collection
The inventors collected urine samples from female subjects. These were stored at room/ambient temperature (typically 16-30°C) and at 2-8°C with and without preservative. When no preservative was used, extraction of the nucleic acid was relatively poor. On the other hand, use of boric acid, EDTA and other commercially available proprietary preservatives allowed successful nucleic acid extraction, with boric acid performing best. Storing samples at 2-8°C with preservatives further improved subsequent extraction. All samples were centrifuged to collect a cell pellet and the supernatant discarded. Pellets were stored at -70°C until required. First- stream and mid-stream urine samples and vaginal swab samples were compared.
DNA extraction from urine
The present inventors developed the following method to efficiently extract DNA from urine samples.
1 . Add 3 ml TE buffer (10:1 , pH8.0) to 10 ml urine sample, mix. This dissolves any crystals such as urates which can form during urine storage. Prevents collections of crystals with cell pellet.
2. Centrifuge at 4°C for 15 mins at 5000 ref to create cell pellet. This faster speed than recommended in the literature improves pellet size.
3. The cell pellet is resuspended in 180 pl enzymatic lytic agent (ELB recipe).
ELB recipe:
0.4846 g Tris base (20 mM final)
150 ml PCR water to pH 8.0 with HCI (approx 200 pl)
0.149 g EDTA (2 mM final)
2.4 ml Tween 20/TritonX -100 (1 .2% final)
A200 ml PCR water
Add 20 mg Lysozyme to 1 ml of above mix
4. Incubate at 37°C for 1 hour.
Next, a Qiagen DNeasy Blood and Tissue Kit (column-based extraction) or Thermo MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit (magnetic bead-based kit) was used.
Qiagen DNeasy Blood and Tissue Kit
5. Add 200 pl Buffer AL, mix.
6. Add 25 pl Proteinase K, mix.
7. Incubate at 56°C for 30 mins.
8. Add 200 pl ethanol (96-100%), mix.
9. Add mixture to spin column, spin at 6000xg for 1 min, discard flow-through and collection tube.
10. Place in new collection tube, add 500 pl buffer AW1 , spin at 6000xg for 1 min, discard flowthrough and collection tube.
11. Place in new collection tube, add 500 pl buffer AW2, spin at 20,000xg for 3min, discard flowthrough and collection tube.
12. Place in a new 1.5 ml microcentrifuge tube and add 100 pl buffer AE onto column membrane.
13. Incubate at room temp for 1 min, spin at 6000xg for 1 min.
Thermo MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit
5. T ransfer the Sample to the appropriate wells of a new deep-well plate.
6. Add 220 pl Lytic agent.
7. Add 40 pl of Proteinase K to each sample.
8. Seal the plate with MicroAmp™ Clear Adhesive Film, ensuring that it is adequately sealed around the individual wells.
9. Shake the sealed plate at 900 rpm for 5 minutes.
10. Place the plate in an incubator at 65°C for 20 minutes.
Note: Ensure that there is free air flow to the bottom of the plate for proper sample heating.
11 . Remove plate from incubator.
12. Invert Binding Bead Mix to mix, then add 520 pl to each sample in the Sample Plate.
Note: Remix the Binding Bead Mix by inversion frequently during pipetting to ensure even distribution of beads to all samples or wells. The mixture containing the Binding Beads is viscous. Therefore, pipet slowly to ensure that the correct amount is added. DO NOT use a repeat pipette to add to the samples as the high viscosity will cause variations in volume added.
Note: Centrifuge the plate at 800 rpm for 1 min to collect liquid to bottom of plate if needed.
13. Seal the plate with MicroAmp™ Clear Adhesive Film, ensuring that it is adequately sealed around the individual wells.
14. Shake the sealed plate at 900 rpm for 5 minutes.
15. Place the sealed plate on the magnetic stand for at least 5 minutes, or until all the beads have collected.
16. Keeping the plate on the magnet, carefully remove the cover, then discard the supernatant from each well.
IMPORTANT! Avoid disturbing the beads.
17. Remove the plate from the magnetic stand, then add 1 ml of Wash Buffer to each sample.
18. Reseal the plate, then shake at 800 rpm for 30 seconds.
19. Place the plate back on the magnetic stand for 3 minutes, or until all the beads have collected.
20. Keeping the plate on the magnet, carefully remove the cover, then discard the supernatant from each well.
IMPORTANT! Avoid disturbing the beads.
21 . Repeat step 17 to step 20 using 1 ml of Wash Buffer
22. Repeat step 17 to step 20 using 1 ml of 80% Ethanol.
23. Repeat step 17 to step 20 using 1 ml of 80% Ethanol.
24. Dry the beads by shaking the plate (uncovered) at 800 rpm for 2 minutes.
25. Add 50 pl of Elution Solution to each sample, then seal the plate with MicroAmp™ Clear Adhesive Film.
26. Place the plate in an incubator at 75°C for 5 minutes.
27. Remove the plate from the incubator, then place on a shaker at 800 rpm for 5 minutes.
28. Place the sealed plate on the magnetic stand for 3 minutes or until all beads are collected against the magnets.
29. Keep the plate on the magnet and carefully remove the seal, then transfer the eluates to a fresh standard (not deep-well) plate.
IMPORTANT! To prevent evaporation, seal the plate containing the eluate immediately after the transfers are complete.
It has previously been suggested that a column-based extraction approach gives the most accurate representation of urinary microbiome. However, the inventors have found that using a magnetic bead-based approach, rather than a column approach, addresses the problem of the
column becoming blocked with impurities from the urine and thereby improves the DNA quality obtained.
Example 2
The inventors undertook 16S library preparation, sequencing and bioinformatics as follows.
YouSeq The One 16s NGS Library Prep Kit
Setup following The ONE 16S NGS Library Preparation Kit handbook Version 1 .0. The inventors amplified the 16S rRNA genes of all bacteria present in each of the extracted DNA samples, simultaneously adding sequencing adapters and sample-specific indexes. The 16S variable regions targeted were V3 and V4. qPCR was performed on Applied Biosystems StepOnePlus instrument.
16S Sequencing
Illumina iSeqI OO instrument with iSeq 100 i1 Reagents.
Bioinformatics
1 . Export fastq files from Illumina iSeqI OO instrument.
2. Convert to fasta format and analyse against ALS database (updated from STIRRUPS database (Fettweis et al) to include recently identified bacteria, new taxonomies and shifted to V3/V4 from V1/3).
3. Generate reports.
The inventors have found that by focussing their sequencing and subsequent analysis on the V3 and V4 variable regions of 16S, improved identification of bacterial species are obtained. Many bacterial species associated with vaginal health are not correctly identified using analysis of the V1 , V2, V5, V6, V7, V8 and V9 variable regions and as such it is advantageous to focus sequencing on the V3 and V4 variable regions.
Example 3
Following extraction of DNA from the urine of subjects and subsequent 16S sequencing and analysis, the inventors were able to accurately predict the menopausal status of the subjects with high accuracy based on composition of the genitourinary microbiome, and in particular the relative abundance of bacterial species in the genitourinary microbiome.
The results are shown in Table 1 below:
Table 1 : Initial results comparing first-steam, mid-stream and vaginal swab samples
Using first-steam urine samples the inventors were able to successfully predict menopausal status in 81% of subjects. Mid-steam urine samples successfully predicted menopausal status in 67% of subjects and vaginal swab samples successfully predicted menopausal status in 78% of subjects. These results show that urine is a suitable alternative to vaginal swab for determination of menopausal phase and genitourinary health via the microbiome. The percentage of correct calls increases once confounding variables such as pregnancy and medication (e.g. HRT) are taken into account as spurious results are explainable and data illustrates their impact on vaginal health.
Figure 1 demonstrates the relative abundance of bacterial species in example subjects: A) Pre- menopausal; B) Peri-menopausal; C) Post-menopausal; and D) Post-menopausal with HRT. As can be seen, in pre-menopausal subjects (A), Lactobacillus species are dominant, with
Lactobacillus gasseri, Lactobacillus crispatus and Lactobacillus iners showing dominance, forming between 87% and 93% of the bacteria present. Lactobacillus crispatus is the dominant Lactobacillus in pre-menopausal subjects. In peri-menopausal subjects (B), Lactobacillus species are still dominant, but not to the same extent forming around 50% of the bacterial species present. Strikingly, post-menopausal subjects (C) have a high diversity of bacterial species with low levels of Lactobacillus. In post-menopausal subjects (D) who are treated with HRT Lactobacillus species are once again dominant forming over 78% of the bacteria present, indicating that HRT improves the vaginal microbiome.
Example 4
The subjects were assigned a vaginal microbiome measurement (VMM) based on the presence of certain bacteria and their relative abundance.
The bacteria were awarded values as follows:
Lactobacillus crispatus - 6
Non-crispatus Lactobacillus species (including but not limited to L. jensenii, gasserii and iners i.e. lactic acid bacteria) and other potentially protective species- 0.5-4 Potentially pathogenic species - -2
Other (defined as the sum total of bacterial species with less than 1 % relative abundance each) = 0.75.
These values were multiplied by the relative abundance of each bacteria:
For example, the subject of Figure 1 A
• Lactobacillus crispatus 6 x 43% = 258,
• non-crispatus Lactobacillus species 4 x 14% = 56,
• non-crispatus Lactobacillus species 1 x 30% = 30,
• non-lactobacillus species -2 x 1 % = -2
• ‘Other’ (defined as the sum total of bacteria with <1 .0% relative abundance) 0.75 x 12% = 9
These totals were added together and divided by 100 to give the vaginal microbiome measurement (VMM):
258+56+30-2+9 = 351/100 = 3.51
Menopausal status was assigned as follows based on the vaginal microbiome measurement (VMM):
Post-menopause <1.0
Peri-menopause 1 .0 - 3.5
Pre-menopause >3.5
The inventors have found that the calculated vaginal microbiome measurement (VMM) (based on the bacterial species present in the urine sample) correlates highly with the menopausal status of the subject. For example, the vaginal microbiome measurement (VMM) for the premenopausal subjects of Figure 1A were 3.51 and 3.85 respectively. The vaginal microbiome measurement (VMM) for the peri-menopausal subject of Figure 1 B was 1.97 and the vaginal microbiome measurement (VMM) for the post-menopausal subjects of Figure 1 C were 0.86 and 1 .43 respectively.
References
Brotman, R.M et al. Association between the vaginal microbiota, menopause status, and signs of vulvovaginal atrophy. Menopause, 2014 May;21 (5):450-8.
Fettweis J et al. Species-level classification of the vaginal microbiome. BMC Genomics, 2012;
13 Suppl 8(Suppl 8):S17.
Higuchi R et al. Kinetic PCR analysis: Real time Monitoring of DNA amplification reactions. Bio/Technology 1993; 11 :1026-30.
Claims
1 . A method of determining the menopausal status of a subject by assessing genitourinary microbiome composition of the subject, the method comprising:
(a) extracting nucleic acid from a urine sample from the subject; and
(b) analysing the extracted nucleic acid to identify the relative abundance of microorganisms in the urine sample.
2. The method according to claim 1 , wherein the urine sample is a first-stream urine sample.
3. The method according to claim 1 or 2, wherein extracting nucleic acid from the urine sample comprises extracting nucleic acid using magnetic bead-based extraction.
4. The method according to any preceding claim, wherein step (b) further comprises amplifying the extracted nucleic acid.
5. The method according to any preceding claim, wherein step (b) comprises amplifying nucleic acid encoding bacterial 16S rRNA present in the extracted nucleic acid.
6. The method according to claim 5, wherein the nucleic acid encoding 16S rRNA comprise the nucleic acid encoding 16S variable regions 1-9, preferably 1-4, preferably 3-4.
7. The method according to any preceding claim, wherein step (b) further comprises sequencing the extracted nucleic acid.
8. The method according to claim 1 , wherein the method further comprises:
(c) comparing the relative abundance of microorganisms in the urine sample with the relative abundance of microorganisms in a control sample to determine the menopausal status of a subject.
9. The method according to any preceding claim, wherein the microorganisms comprise bacteria.
10. The method according to any preceding claim, wherein the method comprises identifying the relative abundance of Lactobacilli in the urine sample.
11 . The method according to claim 10, wherein the Lactobacilli comprise Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii and Lactobacillus gasseri.
12. The method according to claim 10 or 11 , wherein:
(i) a relative abundance of Lactobacilli of greater than 70% indicates of premenopausal status;
(ii) a relative abundance of Lactobacilli of between 40 and 70% indicates peri- menopausal status; and/or
(iii) a relative abundance of Lactobacilli of less than 40% indicates post-menopausal status.
13. The method according to any preceding claim, wherein the method comprises assigning a community state type (CST) to the subject.
14. The method according to any preceding claim, wherein the subject is a human female.
15. The method according to any preceding claim, wherein the method is an in vitro method.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB2207807.5 | 2022-05-26 | ||
GBGB2207807.5A GB202207807D0 (en) | 2022-05-26 | 2022-05-26 | Methods |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023227896A1 true WO2023227896A1 (en) | 2023-11-30 |
Family
ID=82323995
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2023/051379 WO2023227896A1 (en) | 2022-05-26 | 2023-05-25 | Identification of a bacterium of the genus bacillus |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB202207807D0 (en) |
WO (1) | WO2023227896A1 (en) |
-
2022
- 2022-05-26 GB GBGB2207807.5A patent/GB202207807D0/en not_active Ceased
-
2023
- 2023-05-25 WO PCT/GB2023/051379 patent/WO2023227896A1/en unknown
Non-Patent Citations (8)
Title |
---|
AMMITZBØLL NADIA ET AL: "Pre- and postmenopausal women have different core urinary microbiota", SCIENTIFIC REPORTS, vol. 11, no. 1, 26 January 2021 (2021-01-26), XP093059261, Retrieved from the Internet <URL:https://www.nature.com/articles/s41598-021-81790-8> DOI: 10.1038/s41598-021-81790-8 * |
BROTMAN, R.M ET AL.: "Association between the vaginal microbiota, menopause status, and signs of vulvovaginal atrophy", MENOPAUSE, vol. 21, no. 5, May 2014 (2014-05-01), pages 450 - 8, XP055706447, DOI: 10.1097/GME.0b013e3182a4690b |
BROWN SARAH E ET AL: "Assessing the Concordance Between Urogenital and Vaginal Microbiota: Can Urine Specimens Be Used as a Proxy for Vaginal Samples?", FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY,, vol. 1, 30 November 2020 (2020-11-30), pages 1 - 11, XP009545124, ISSN: 2235-2988, DOI: 10.3389/FCIMB.2021.671413 * |
FETTWEIS J ET AL.: "Species-level classification of the vaginal microbiome", BMC GENOMICS, vol. 13, 2012, pages S17, XP021127995, DOI: 10.1186/1471-2164-13-S8-S17 |
FRANCE MICHAEL ET AL: "Towards a deeper understanding of the vaginal microbiota", NATURE MICROBIOLOGY, NATURE PUBLISHING GROUP UK, LONDON, vol. 7, no. 3, 1 March 2022 (2022-03-01), pages 367 - 378, XP037708637, DOI: 10.1038/S41564-022-01083-2 * |
HIGUCHI R ET AL.: "Kinetic PCR analysis: Real time Monitoring of DNA amplification reactions", BIO/TECHNOLOGY, vol. 11, 1993, pages 1026 - 30, XP000197685, DOI: 10.1038/nbt0993-1026 |
MUHLEISEN ALICIA L ET AL: "Menopause and the vaginal microbiome", MATURITAS, ELSEVIER, AMSTERDAM, NL, vol. 91, 1 June 2016 (2016-06-01), pages 42 - 50, XP029649867, ISSN: 0378-5122, DOI: 10.1016/J.MATURITAS.2016.05.015 * |
REBECCA M. BROTMAN ET AL: "Association between the vaginal microbiota, menopause status, and signs of vulvovaginal atrophy :", MENOPAUSE: THE JOURNAL OF THE NORTH AMERICAN MENOPAUSE SOCIETY, vol. 21, no. 5, 1 May 2014 (2014-05-01), US, pages 450 - 458, XP055706447, ISSN: 1072-3714, DOI: 10.1097/GME.0b013e3182a4690b * |
Also Published As
Publication number | Publication date |
---|---|
GB202207807D0 (en) | 2022-07-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Forney et al. | Comparison of self-collected and physician-collected vaginal swabs for microbiome analysis | |
Dols et al. | Microarray-based identification of clinically relevant vaginal bacteria in relation to bacterial vaginosis | |
Bostwick et al. | Antimicrobial resistance genes and modelling of treatment failure in bacterial vaginosis: clinical study of 289 symptomatic women | |
CN114898808B (en) | Method and system for predicting sensitivity of Klebsiella pneumoniae to cefepime | |
Chen et al. | Vaginal microbiome variances in sample groups categorized by clinical criteria of bacterial vaginosis | |
CN110904250B (en) | Multiplex fluorescent quantitative PCR primer, kit and detection method for detecting multiple bacteria | |
WO2012117431A1 (en) | Method and reagent kit for the identification of biological fluids in a sample | |
Komiya et al. | MinION, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting | |
KR20170085931A (en) | Method for Gut Microbiota Analysis Using Real-time PCR | |
CN103276099A (en) | Primer and kit for fluorescent quatititive PCR (polymerase chain reaction) detection of helicobacter pylori | |
Westaway et al. | Methods for exploring the faecal microbiome of premature infants: a review | |
JP7445980B2 (en) | Methods and kits for predicting outcomes of assisted reproductive techniques | |
WO2023227896A1 (en) | Identification of a bacterium of the genus bacillus | |
WO2023227897A1 (en) | Identification of a bacterium of the genus bacillus | |
CN115747361A (en) | Real-time fluorescent MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method | |
CN113564269B (en) | Probe composition for preventing reverse transcription of bacterial conserved region and application thereof | |
Hartmeyer et al. | Selecting PCR for the Diagnosis of Intestinal Parasitosis: Choice of Targets, Evaluation of In‐House Assays, and Comparison with Commercial Kits | |
JP2017189166A (en) | Method for diagnosing chronic pyoderma and diagnostic kit for chronic pyoderma | |
KR20220012896A (en) | Methods for detecting rare DNA sequences in fecal samples | |
WO2019116234A1 (en) | Methods for determining microbiome ribosomal rna composition changes | |
CN117265140B (en) | Burkholderia cepacia complex PCR detection primer and detection method thereof | |
EP1281087A2 (en) | Early diagnosis of bladder tumor in urine samples | |
CN114058695B (en) | Application of urinary tract flora detection in female urinary tract calculus diagnosis | |
CN114606317B (en) | Flora marker for predicting lymph node metastasis of gastric cancer and application thereof | |
McClure et al. | Strategies for global RNA sequencing of the human pathogen Neisseria gonorrhoeae |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23729460 Country of ref document: EP Kind code of ref document: A1 |