Methods for detecting and distinguishing treponema pallidum subspecies and uses thereof

Abstract

The present disclosure relates to a method for detecting and distinguishing between Treponema pallidum subspecies in a samples and distinguishing between venereal syphilis and non-venereal disease (e.g., yaws or bejel). Compositions, assays, and kits for use in the methods are also disclosed.

Description

Docket No. 15947WOO1 / 43913.601METHODS FOR DETECTING AND DISTINGUISHING TREPONEMA PALLIDUM SUBSPECIES AND USES THEREOFRELATED APPLICATION INFORMATION

[0001] This application claims priority to provisional application 63 / 716328, filed November 5, 2024, which is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to methods, assays, compositions and kits for distinguishing Treponema pallidum subspecies in samples and differentiating between subjects having venereal syphilis and non-venereal disease (e.g., yaws or bejel).SEQUENCE LISTING STATEMENT

[0003] The contents of the electronic sequence listing titledABBTL_43913_601_SequenceListing.xml (Size: 13,448 bytes; and Date of Creation: November 5, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0004] Syphilis is diagnosed with serologic testing of blood or CSF. Tests for non-treponemal antigens such as RPR need to be confirmed with treponemal antigens (e.g. IFA with specific T. pallidum antibodies). Primary and secondary syphilis can be confirmed with dark field or phase-contrast microscopy. Syphilis cases have surged 80% in the last 5 years and there is an urgent need for new diagnostics.

[0005] There are three subspecies of T. pallidum which induce similar, but distinct diseases. T. pallidum pallidum (TP A) causes the venereal disease syphilis, which is transmitted by sexual contact and has a worldwide distribution. It can also be passed on congenitally to infants of infected pregnant mothers. Primary lesions on genitals / skin are followed years later by involvement of bone, viscera, CNS, and cardiovascular systems. T. pallidum endemicum (TEN) causes the non-venereal disease bejel. It is an acute disease of limited geographic distribution, typically occurring in areas with poor socioeconomic or sanitary conditions. Skin lesions and rashes resemble that of venereal syphilis, but in this case, bacteria only incubates for weeks to months as opposed to years. The only way to distinguish it from syphilis is by molecular testing. T. pallidum pertenue (TPE) causes the non-venereal disease yaws. It is a chronic, relapsing disease primarily found in warm, humid, tropical areas. Primary and secondary cutaneous lesions are highly contagious and can be debilitating and disfiguring.Docket No. 15947WOO1 / 43913.601Unlike venereal syphilis, the disease does not involve the brain, eyes, heart, etc. and is confined to lesions of the skin and bones.

[0006] The 3 subspecies of T. pallidum are very close genetically, to such an extent that they cannot be distinguished serologically (e.g. antigens cross-react) or by microscopy. Prolonged periods of latency in between eruptions of skin lesions make it difficult to detect and diagnose syphilis by molecular means. However, performing molecular testing after an initial exposure when lesions (and treponemal bacteria) are present would facilitate an accurate diagnosis.

[0007] Assays based on nucleic acid amplification using either conventional or fluorescent PCR technologies for the diagnosis of venereal syphilis have been recently summarized in Table 1 by Zhou et al. (C. Zhou et al. 2019), the data of which has been combined with data from Luo et al. (Luo et al. 2020) and summarized in Table 1 below.

[0008] Table 1 - Data summarized from Zhou et al. 2020 and Luo et al. 2020Docket No. 15947WOO1 / 43913.601

[0009] The primers and probes commonly used by the different research groups have consistently targeted three major genes individually: polA, ttp47, and bmp (Luo et al. 2020). It is important to note that these targets were selected prior to pan-genome analysis of Treponema pallidum (Jaiswal et al. 2020), therefore, none of these regions had been determined a genomic island nor a pathogenic island of Treponema pallidum subspecies pallidum. Therefore polA,Docket No. 15947WOO1 / 43913.601 ttp47 and bmp genes cannot be considered specific to Treponema pallidum subspecies pallidum (Luo et al. 2020), nor do they play a unique structural or metabolic function in this bacteria (Jaiswal et al. 2020). Considering the poor diagnostic sensitivity of previous assays, it has been suggested that these targets lack specificity for selective amplification of Treponema pallidum subspecies pallidum (Gayet-Ageron et al. 2015a). A more recent approach described by Salle et al. (Salle et al. 2022) used the combination of the previously selected targets polA and ttp47 with the aim of increasing the diagnostic sensitivity. Despite the improvements reached with this multiplex approach compared to corresponding singlet assays, the sensitivity assessed in -300 clinical samples was still as low as 89%, considered low for PCR technology (Kralik and Ricchi 2017).

[0010] Accordingly, there is a need for an improved molecular assay that can distinguish T. pallidum subspecies and aid in the differential diagnosis of venereal syphilis and non-venereal disease (e.g., bejel or yaws).SUMMARY

[0011] Aspects of the present disclosure relate to methods, assays and kits for detecting T. pallidum and distinguishing between Treponema pallidum subspecies in samples, for example, to differentiate between venereal and non-venereal disease (e.g., syphilis) in subjects from whom the samples were obtained.

[0012] In an aspect, the present disclosure provides a method for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the method comprising: (a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises: (i) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (ii) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; (b) detecting the Treponema pallidum bacteria in the sample if the nucleic acid amplification assay generates the first signal; and (c) distinguishing the subspecies of Treponema pallidum present in the sample if the nucleic acid amplification assay generates the second target second signal.Docket No. 15947WOO1 / 43913.601

[0013] In another aspect, the present disclosure provides a nucleic acid amplification assay for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the assay comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample.

[0014] In yet another aspect, the present disclosure provides a kit for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the kit comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies,; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum,' and (c) instructions for performing a nucleic acid amplification assay for detecting the Treponema pallidum bacteria in the sample and distinguishing the subspecies of Treponema pallidum present in the sample.

[0015] In some aspects, the subspecies are selected from the group consisting of T. pallidum pallidum (TP A), T. pallidum endemicum (TEN), and T. pallidum pertenue (TPE).

[0016] In some aspects, the first target bacterial nucleic sequence comprises a region of the 16s rRNA gene that is conserved across the subspecies. In some aspects, the conserved nucleic acid comprises a pathogenicity island for Treponema pallidum.

[0017] In some aspects, the first probe hybridizes to a polynucleotide sequencing corresponding to nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene. In yet other embodiments, the first probe comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that differs from SEQ ID NO: 1 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In certain embodiments, the first primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene. In certain other embodiments, the first primer pair comprises a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 orDocket No. 15947WOO1 / 43913.601 nucleotide sequences that differ from SEQ ID NO: 2 and SEQ ID NO: 3 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

[0018] In some aspects, the second target bacterial nucleic acid sequence comprises a region of the 50SL35 gene that is unique for one subspecies of Treponema pallidum. In some embodiments, the second probe hybridizes to a polynucleotide sequence corresponding nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene. In other embodiments, the second probe comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence that differs from SEQ ID NO: 4 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In yet other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene. In still other embodiments, the second primer pair comprises a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 or nucleotide sequences that differ from SEQ ID NO: 5 and SEQ ID NO: 6 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

[0019] In other aspects, the subspecies is identified as subspecies T. pallidum pallidum if the first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pallidum. In such aspects, identification of the presence of the subspecies T. pallidum pallidum in the sample is indicative that the subject from whom the sample was obtained has venereal syphilis.

[0020] In still other aspects, the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum pertenue. In some embodiments, the subspecies is identified as subspecies T. pallidum pertenue if the first signal is generated and at least a second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pertenue.

[0021] In yet still other aspects, the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum endemicum. In some embodiments, the subspecies is identified as subspecies T. pallidum endemicum if the first signal is generated and at least a second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum endemicum.Docket No. 15947WOO1 / 43913.601

[0022] In further aspects, the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum. In some embodiments, the subspecies is identified as subspecies T. pallidum endemicum or T. pallidum pertenue if the first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in T. pallidum pallidum. In other embodiments, identification of the presence of the subspecies T. pallidum endemicum in the sample is indicative that the subject from whom the sample was obtained has bejel. In yet other embodiments, identification of the presence of the subspecies T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained has yaws. In certain embodiments, identification of the presence of the subspecies T. pallidum endemicum or T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained does not have venereal syphilis.

[0023] In yet another aspect, the present disclosure provides a method for identifying a subject as more likely than not having venereal syphilis, the method comprising: (a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises: (i) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across Treponema pallidum subspecies pallidum, pertenue, and endemicum, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (ii) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; (b) identifying the subject has more likely than not having venereal syphilis if the nucleic acid amplification assay generates both the first signal and the second signal.

[0024] In yet another aspect, the present disclosure provides a nucleic acid amplification assay for identifying a subject as more likely than not having venereal syphilis, the assay comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, pertenue , and endemicum, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (b) at least a second probe and / orDocket No. 15947WOO1 / 43913.601 primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; and (c) a nucleic acid amplification system for amplifying the first and second target bacterial nucleic acid sequences, if present in the sample, using the first probe and / or primer pair and at least the second probe and / or primer pair.

[0025] In yet another aspect, the present disclosure provides a kit for identifying a subject as more likely than not having venereal syphilis, the kit comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, per tenue , and endemicunr, (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum,' and (c) instructions for performing a nucleic acid amplification assay for identifying the subject has more likely than not having venereal syphilis if the nucleic acid amplification assay generates both a first and second signal due to the amplification of the first and second target bacterial nucleic acid sequences within a sample obtained from the subject.

[0026] In some aspects, the method, assay, or kit further comprises: (i) recommending a treatment for venereal syphilis if the subject is indicated as having or identified as more likely than not having venereal syphilis; (ii) recommending a treatment for yaws if the subject is indicated as having yaws; or (iii) recommending a treatment for bejel if the subject is indicated as having bejel.

[0027] In another aspect, the disclosure provides a composition comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the first probe comprises a first detectable label that generates a first signal when the first probe and / or primer pair hybridize to and amplify the first target bacterial nucleic acid sequence; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the at least the second probe comprises a second detectable label that generates a second signal when the at least the second probe and / or primer pair hybridize to and amplify the at least the second target bacterial nucleic acid sequence.Docket No. 15947WOO1 / 43913.601BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The patent or application file contains drawings executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0029] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0030] FIG. 1 (SEQ ID NO: 7) shows a 16RNA gene sequence alignment for Treponema pallidum subspecies perlenue. endemicus and pallidum, showing the primer pair designed for the detection of Treponema pallidum from Centurion-Lara et al. (1997) in orange and an exemplary first probe and primer pair of the present disclosure in red.

[0031] FIG. 2 A (SEQ ID NO: 8) and FIG. 2B (SEQ ID NO: 9) show locations of Treponema probes and primers. FIG. 2A shows an alignment between subspecies pallidum, endemicum, and pertenue indicating that the exemplary embodiment of first probe and primer pair for hybridizing to and amplifying the exemplary first target bacterial nucleic acid (e.g., 16S rRNA) targets a region of the target nucleic acid that is conserved across the subspecies. FIG. 2B shows the exemplary embodiment of second probe and primer pair for hybridizing to and amplifying the exemplary second target bacterial nucleic acid (e.g., 50SL35) targets a nucleic acid sequence that is unique in subspecies pallidum.

[0032] FIG. 3 shows the sequences of the exemplary embodiments of the first and second probe and primer pairs depicted in the alignments shown in FIG. 2 A and FIG. 2B.

[0033] FIG. 4A (SEQ ID NO: 10) and FIG. 4B (SEQ ID NO: 11) shows that an exemplary first target nucleic acid comprising the 16S rRNA gene yielded a linear range from 109-102copies / pL and amplification efficiency 101.78% in an exemplary nucleic acid amplification assay of the present disclosure (e.g., a qPCR assay).

[0034] FIG. 5 shows the plasmid curve constructed using the data in Table 10.

[0035] FIG. 6 shows a dose-response plot constructed using the data from Tables 10 and 11.

[0036] FIG. 7 shows a dose-response plot constructed using the data from Tables 12 and 13.

[0037] FIG. 8 shows the results of a curve constructed from the data in Table 14 for CY5-16S, which was used to demonstrate the linearity of cultured syphilis shown in FIG. 10

[0038] FIG. 9 shows the results of a curve constructed from the data in Table 14 for FAM-35L, which was also used to demonstrate the linearity of cultured syphilis shown in FIG. 10.

[0039] FIG. 10 is a graph showing the linearity of cultured syphilis constructed using the data in Table 14 and FIG. 9 and FIG. 10.Docket No. 15947WOO1 / 43913.601

[0040] FIG. 11 shows dose-response plots for FAM-35L-venereal and C5-16S-non-venereal syphilis.

[0041] FIG. 12 and FIG. 13 respectively show the linearity of data from the Alinity® sensitivity study, including all LOD data (FIG. 12) and average CT data (FIG. 13).

[0042] FIG. 14 shows assay comparisons of Trina samples run on the m2000 and Alinity® m platforms.

[0043] FIG. 15 and FIG. 16 respectively show a comparison of m2000 and Alinity® m assay results for FAM.

[0044] FIG. 17 and FIG. 18 respectively show a comparison of m2000 and Alinity® m assay results for VIC IC.

[0045] FIG. 19 and FIG. 20 respectively show a comparison of m2000 and Alinity® m assay results for CY5.

[0046] FIG. 21 shows a comparison of m2000 and Alinity® m assay results for linearity.

[0047] FIG. 22 shows the evaluation of analytical sensitivity of the duplex-qPCR using the TPA strain Nichols in 10-fold dilution series. Values represent the number of replicates detected for each assay on each instrument (orange: m2000, purple: Alinity® m) numbers between brackets represent the mean CT-values obtained for each assay.

[0048] FIG. 23 shows a comparison of the probit dose response for the m2000 and Alinity® m assays. The upper row shows that the m2000-LOD determined by 95% detectability is 22.1 cps / mL, whereas the lower row shows that the Alinity® m-Lod determined by 95% detectability is 34.8 cps / mL.

[0049] FIG. 24 shows results of assay performance on reference strain material TPP Nichols which was propagated in rabbit.

[0050] FIG. 25 and FIG. 26 show conditions for Alinity® m. The exemplary first target bacterial nucleic acid (e.g., 50SL35 gene)-set yielded a linear range from 109-101copies / pL and amplification efficiency 97.28%. The exemplary second target bacterial nucleic acid (16S gene)-set yielded a linear range from 109- 102copies / pL and amplification efficiency 101.78%.

[0051] FIG. 27A and FIG. 27B show linear range and sensitivity for the exemplary first target bacterial nucleic acid (e.g., 16S rRNA)

[0052] FIG. 28A and FIG. 28B show linear range and sensitivity for the exemplary second target bacterial nucleic acid (e.g., 50SL35).

[0053] FIG. 29A and FIG. 29B shows analytical sensitivity results for the exemplary second target bacterial nucleic acid (e.g., 50SL35 gene)-set, which yielded a linear range from 109- 102copies / pL and amplification efficiency 101.78%.Docket No. 15947WOO1 / 43913.601

[0054] FIG. 30A and FIG. 3 OB show the results of an evaluation of the exemplary embodiment of an assay of the present disclosure (e.g., multiplex qPCR) for diagnostic of venereal syphilis on clinical swabs samples collected from Trina.

[0055] FIG. 31A-D shows design and feasibility evaluation of the duplex qPCR assay for detection of pathogenic Treponeme and discrimination of Treponema pallidum sub pallidum. A) Genomic targets selected: an 80 bp region within the rplm pathogenicity island specific to T. pallidum subsp. pallidum, and a 75 bp region of the 16S rRNA gene conserved across all three T. pallidum subspecies. B) Sequence alignment confirmed assay specificity in silico: rplm was absent from T. pallidum subsp. pertenue and endemicum, while 16S primer-probe set showed 100% identity across the three pathogenic T. pallidum subspecies. C) Duplex qPCR schematic showing fluorescence channels used for detection: rplm (FAM), 16S rRNA (Cy5), and an internal control (VIC). (D) Feasibility testing with defined input: amplification plots illustrate detection at high, medium, and low concentrations, with clear separation from negative controls in each channel, confirming applicability of the assay.

[0056] FIG. 32A-D shows optimization of MgCh concentration and primer / probe ratios for the duplex Treponema qPCR assay. Evaluation of MgCE concentration (A and B) to a gradient of 4, 5, and 6 mM, for (A) 16S rRNA and (B) rpml targets. Optimization of primer / probe concentrations (C and D), with different combination of the concentration of the primer forward / primer reverse / probe for (C) 16S rRNA and (D) rpml targets gradient combinations (primers:probes = 0.6 / 0.6 / 0.5, 0.5 / 0.5 / 0.4, 0.4 / 0.4 / 0.3, 0.3 / 0.3 / 0.2 pM).

[0057] FIG. 33 A-D shows analytical linearity and limit of detection (LoD) of the duplex-qPCR assay. A, B) Standard curves indicating the linear dynamic range of detection for the 16S rRNA (A) and rpml (B) targets. C, D) Limit of detection (LoD95) determined by probit regression using 20 replicate reactions per dilution. The estimated LoD95 values were 2.15 copies / reaction for 16S rRNA (C) and 1.0 copies / reaction for rpml (D), with 95% confidence intervals (CI) shown as shaded regions.

[0058] FIG. 34 shows analytical specificity of the duplex Treponema qPCR assay. In the Cy5 channel (upper panel), the pan-Treponema target (16S rRNA) amplified the T. pallidum sub pallidum species and other pathogenic treponemes (T. phagedenis ATCC 27087, T. denticola ATCC 35404 and 35405), while non -pathogenic Treponema species (Treponema sp. OMZ 804, OMZ 838, and TSD-166) and no-template controls (NTC) remained negative. In contrast, the FAM channel (lower panel), targeting the rpml gene specific to T. pallidum subsp.Docket No. 15947WOO1 / 43913.601 pallidum, amplified only the Nichols strain, with no detectable signal for other Treponemes or NTCs.DETAILED DESCRIPTION

[0059] Aspects of the present disclosure relate to methods for detecting and distinguishing between Treponema pallidum subspecies in a sample and distinguishing between venereal syphilis and non-venereal disease (e.g., yaws or bejel). Compositions, assays, and kits for use in the methods are also disclosed.

[0060] The 16S RNA gene has only been considered as a target by one research group (Centurion-Lara et al. 1997) in a conventional RT-PCR assay. Thus, there is not a specific probe for signal detection and the targeted region of 366 bp is unrelated to the region selected for the current assay (See, Figure 1). The numerous drawbacks of current PCR-based diagnostics for Treponema pallidum subspecies pallidum have been described in detail by Luo et al. (Luo et al. 2020) and listed in the Table 1 above. The methods and assays (e.g., multiplex assay) of the present disclosure targets the 16S RNA gene in a combination with a target that is specific for one subspecies (e.g, the pallidum 50SL35 gene). The 50SL35 gene was selected based on the analysis of the pathogenicity islands in Treponema pallidum. Given its structural function within the ribosome and requirement for gene expression of T. pallidum subspecies pallidum (Jaiswal et al. 2020), it is believed that selective pressure on this gene is limited. Thus, the methods and assays of the present disclosure can detect all stages of disease and should be subject to low genetic variability among different strains.

[0061] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0063] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, orDocket No. 15947WOO1 / 43913.601 words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0064] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0065] As used herein, the terms “amplifying” or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, typically starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. Generation of multiple DNA copies from one or a few copies of a target or template DNA molecule during a polymerase chain reaction (PCR), isothermal reaction, or a ligase chain reaction (LCR) are forms of amplification. Amplification is not limited to the strict duplication of the starting molecule. For example, the generation of multiple cDNA molecules from a limited amount of RNA in a sample using reverse transcription (RT)-PCR is a form of amplification. Furthermore, the generation of multiple RNA molecules from a single DNA molecule during the process of transcription is also a form of amplification.

[0066] As used herein, the terms "characterization", "characterize", "characterizing" refer to describing or categorizing by sequence information in some cases.

[0067] The terms “bead,” “microparticle,” and “particle” used herein interchangeably herein, refer to a substantially spherical solid support. The bead may be a magnetic bead or a magnetic particle. Microparticles that can be used herein can be any type known in the art. For example, the bead or microparticle can be a magnetic bead or magnetic particle. Magnetic beads / particles may be ferromagnetic, ferrimagnetic, paramagnetic, superparamagnetic or ferrofluidic. Exemplary ferromagnetic materials include Fe, Co, Ni, Gd, Dy, CrO2, MnAs, MnBi, EuO, and NiO / Fe. Examples of ferrimagnetic materials include NiFe2O4, CoFe2O4, Fe3O4 (or FeO.Fe2O3). Beads can have a solid core portion that is magnetic and is surrounded by one or more non-magnetic layers. Alternately, the magnetic portion can be a layer around a nonmagnetic core. The size of the microparticles used in the methods, compositions and kits described herein can vary. Preferably, the microparticles have a substantially uniform diameterDocket No. 15947WOO1 / 43913.601 of less than about 0.10 pm, less than about 0.20 pm, less than about 0.30 pm, less than about 0.40 pm, less than about 0.50 pm, less than about 0.60 pm, less than about 0.70 pm, less than about 0.80 pm, less than about 0.9 pm, less than about 1 pm, less than about 2 pm, less than about 3 pm, less than about 4 pm, or less than about 5 pm.

[0068] As used herein, "coding sequence" or a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is used herein to refer to a nucleotide sequence which, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0069] The terms “complementary" or "complementarity" are used herein in reference to "polynucleotides" and "oligonucleotides" (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. These terms may also include mimics of or artificial bases that may not faithfully adhere to the base-pairing rules. For example, the sequence "C-A-G-T," is complementary to the sequence "G-T-C-A." Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases are not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

[0070] “Label” and “detectable label” as used interchangeably herein refer to a moiety attached to an probe of the present disclosure. A label can produce a signal that is detectable by visual or instrumental means. Various labels include signal -producing substances, such as chromagens, fluorescent compounds, chemiluminescent compounds, radioactive compounds, and the like. Representative examples of labels include moieties that produce light, e.g., acridinium compounds, and moieties that produce fluorescence, e.g., fluorescein. Other labels are described herein. In this regard, the moiety itself may not be detectable but may become detectable upon reaction with yet another moiety. Use of the term “detectably labeled” is intended to encompass such labeling.

[0071] Any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 14C, 32P, 33P, 35S, 90Y, 99Tc, 11 Un, 1251, 1311, 177Lu, 166Ho, and 153Sm), an enzymatic label (such as horseradish peroxidase,Docket No. 15947WOO1 / 43913.601 alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5- fluorescein, 6-carboxyfluorescein, 3’6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6- hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immuno-polymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oregon. In some aspects, the detectable label comprises a fluorescent dye. In some aspects, probes are labeled with a first fluorescent label and a second fluorescent label that is different from the first. In some aspects, the first and / or second probes comprise first and second fluorescent dyes selected from the group consisting of CY5 and FAM.

[0072] As used herein, the term “fragment" refers to portions of a nucleotide sequence. Fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

[0073] As used herein, the term “gene" refers to deoxyribonucleotide or ribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both amplified and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into heterogeneous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.Docket No. 15947WOO1 / 43913.601

[0074] "As used herein, the term “genome" refers to the entirety of an organism's hereditary information that is encoded in its primary DNA or RNA or nucleotide sequence (DNA or RNA as applicable). The genome includes both the genes and the non-coding sequences. For example, the genome may represent a microbial genome, such as an archaeal genome, a fungal genome (e.g., yeast), a protozoan genome, or a viral genome.

[0075] As used herein the phrase "hybridization product" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization product may be formed in solution or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., hybridization of a biotinylated probe used in target enrichment step (c) to a nucleic acid of interest and magnetic streptavidin beads).

[0076] As used herein, the terms "identification", "identify", and "identifying" refer to recognizing a specific microbe or microbes in a sample from a subject, for example, archaea, bacteria, fungi (e.g., yeast), protozoa, and / or viruses in sample from a subject.

[0077] As used herein, the term "identifier" refers to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating genome of a nucleic acid fragment. The identifier function can sometimes be combined with other functionalities such as adapters or primers and can be located at any convenient position.

[0078] As used herein, the term, "isolated" means that the referenced material, such as, for example, biological material (e.g., DNA, cDNA, RNA, mRNA, a nucleic acid, a protein, or a polypeptide) is free of components found in the natural environment in which the material is normally found. In particular, isolated biological material is free of cellular components. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, an isolated genomic DNA, or a restriction fragment. In some embodiments, an isolated nucleic acid is preferably excised from the chromosome in which it may be found. Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in some embodiments, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. In some embodiments, an isolated material can be purified.

[0079] As used herein, the phrases "nucleic acid", and "polynucleotide" and "nucleic acid sequence" and "nucleotide sequence" include a nucleic acid, an oligonucleotide, a nucleotide,Docket No. 15947WOO1 / 43913.601 a polynucleotide, and any fragment, variant, or derivative thereof. The nucleic acid or polynucleotide may be double- stranded, single- stranded, or triple-stranded DNA or RNA (including cDNA), or a DNA-RNA hybrid of genetic or synthetic origin, wherein the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides and any combination of bases, including, but not limited to, adenine, thymine, cytosine, guanine, uracil, inosine, and xanthine hypoxanthine. As further used herein, the term "cDNA" refers to an isolated DNA polynucleotide or nucleic acid molecule, or any fragment, derivative, or complement thereof. It may be double-stranded, single- stranded, or triple-stranded, it may have originated recombinantly or synthetically, and it may represent coding and / or noncoding 5' and / or 3' sequences.

[0080] As used herein, the phrases, "nucleic acid hybridization" or "hybridization" refer to antiparallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and / or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid).

[0081] As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, at least 15, at least 20 nucleotides, at least 30 nucleotides, at least 60 nucleotides, at least 80 nucleotides, at least 90 nucleotides, at least 100 nucleotides, at least 110 nucleotides, at least 120 nucleotides, at least 130 nucleotides, at least 140 nucleotides, at least 150 nucleotides, at least 160 nucleotides, or at least 180 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. The nucleic acids that comprise the oligonucleotides include but are not limited to DNA, RNA, linked nucleic acids (LNA), bridged nucleic acids (BNA) and peptide nucleic acids (PNA). Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.

[0082] As used herein, the phrases "percent (%) sequence similarity", "percent (%) sequence identity", refer to the degree of identity or correspondence between different nucleotideDocket No. 15947WOO1 / 43913.601 sequences of nucleic acid molecules or amino acid sequences of proteins that may or may not share a common evolutionary origin. Sequence identity can be determined using any publicly available sequence comparison algorithms, such as, for example, BLAST, FASTA, DNA Strider, and GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin).

[0083] To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

[0084] As used herein, the phrase “Polymerase chain reaction" ("PCR") refers to the method disclosed in U.S. Patent Nos. 4,683,195 and 4,683,202, herein incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified". With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin- enzyme conjugate detection; incorporation of 32P -labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplification s. With PCR, it is also possible to amplify a complex mixture (library) of linear DNA molecules, provided they carry suitable universal sequences on either end such that universal PCR primers bind outside of the DNA molecules that are to be amplified.Docket No. 15947WOO1 / 43913.601

[0085] Any suitable PCR methodology, combination of PCR methodologies, or combination of amplification techniques may be utilized in the methods (e.g. droplet-based detection, separation, and / or sequencing of target nucleic acids) disclosed herein, such as cdPCR, dPCR LATE, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, endpoint PCR, hot- start PCR, in situ PCR, intersequence-specific PCR, inverse PCR, linear after exponential PCR, ligation-mediated PCR, methylation-specific PCR, miniprimer PCR, multiplex ligationdependent probe amplification, multiplex PCR, nested PCR, overlap-extension PCR, polymerase cycling assembly, qualitative PCR, quantitative PCR, real-time PCR, RT-PCR, single-cell PCR, solid-phase PCR, thermal asymmetric interlaced PCR, touchdown PCR, or universal fast walking PCR, etc.

[0086] As used herein, the term “primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product that is complementary to a nucleic acid strand is induced (e.g., in the presence of nucleotides and an inducing agent such as a biocatalyst (e.g., a DNA polymerase or the like) and at a suitable temperature and pH). The primer is typically single stranded for maximum efficiency in amplification but may alternatively be double stranded. If double stranded, the primer is generally first treated to separate its strands before being used to prepare extension products. In some embodiments, the primer is an oligodeoxyribonucleotide. The primer is sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

[0087] As used herein, the term "sequencing" refers to any methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a molecule of DNA.

[0088] As used herein, the phrase “statistically significant” refers to the likelihood that a relationship between two or more variables is caused by something other than random chance. Statistical hypothesis testing is used to determine whether the result of a data set is statistically significant. In statistical hypothesis testing, a statistically significant result is attained whenever the observed p-value of a test statistic is less than the significance level defined of study. The p-value is the probability of obtaining results at least as extreme as those observed, given that the null hypothesis is true. Examples of statistical hypothesis analysis include Wilcoxon signed-rank test, t-test, Chi-Square or Fisher’ s exact test. As used herein, the term “significant” refers to a change that has not been determined to be statistically significant (e.g., it may not have been subject to statistical hypothesis testing).Docket No. 15947WOO1 / 43913.601

[0089] As used herein, the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. "Stringency" typically occurs in a range from about Tm to about 20°C to 25°C below Tm. A "stringent hybridization" can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. For example, when fragments are employed in hybridization reactions under stringent conditions the hybridization of fragments which contain unique sequences (i.e., regions which are either non-homologous to or which contain less than about 50% homology or complementarity) are favored. Alternatively, when conditions of "weak" or "low" stringency are used hybridization may occur with nucleic acids that are derived from organisms that are genetically diverse (i.e., for example, the frequency of complementary sequences is usually low between such organisms).

[0090] The terms “subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. In some embodiments, the subject is a human. The subject or patient may be undergoing other forms of treatment. The subject or patient may be undergoing monitoring of the course of a bacterial infection and / or treatment of a bacterial infection. The subject or patient may have or be suspected of having a bacterial infection and / or a symptom, disease, condition, or disorder associated with the bacterial infection. In some aspects, the subject or patient may have or be suspected of having a T. pallidum pallidum infection and one or more symptoms of a venereal disease. In other aspects, the subject or patient may have or be suspected of having a T. pallidum endemicum (TEN) infection and have one or more systems of bejel. In yet other aspects, the subject or patient may have or be suspected of having a T. pallidum pertenue (TPE) infection and have one or more systems of yaws.

[0091] As used herein, “melting temperature” or "Tm" as used herein refers to the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. As indicated by standard references, a simple estimate of the Tm value may be calculated by the equation: Tm=81.5+0.41 (% G+C), when a nucleic acid is in aqueous solution at IMNaCl. Anderson et al, "Quantitative Filter Hybridization" In: Nucleic Acid Hybridization (1985). More sophisticated computations take structural, as well as sequence characteristics, into account for the calculation of Tm.Docket No. 15947WOO1 / 43913.601

[0092] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those that are well known and commonly used in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.2. Methods for detecting and distinguishing between Treponema pallidum subspecies

[0093] Aspects of the present disclosure relate to methods for detecting and distinguishing between Treponema pallidum subspecies. Generally, the methods of the disclosure involve detecting within a sample a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies and a second target nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum (e.g., a gene that is either located within a pathogenicity island that is unique to the subspecies or is a unique gene that is located within a pathogenicity island that is shared among some, but not all of the subspecies).

[0094] The disclosure contemplates the use of any technique that is available to the skilled artisan for detecting nucleic acids within samples. Examples of such techniques include, without limitation, polymerase chain reaction (PCR), qualitative PCR, quantitative PCR (qPCR), isothermal amplification, next generation sequencing (NGS), DNA microarrays, CRISPR-based detection, fluorescence in situ hybridization (FISH), nucleic acid lateral flow assays (NALFA), digital PCR (dPCR), hybrid capture assays, surface plasmon resonance (SPR), peptide nucleic acid (PNA)-based assays, and nanopore sequencing. The examples described herein demonstrate excellent results using exemplary embodiments of qualitative, real-time PCR.

[0095] In an aspect, the present disclosure provides a method for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the method comprising: (a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises: (i) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample;Docket No. 15947WOO1 / 43913.601 and (ii) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; (b) detecting the Treponema pallidum bacteria in the sample if the nucleic acid amplification assay generates the first signal; and (c) distinguishing the subspecies of Treponema pallidum present in the sample if the nucleic acid amplification assay generates the second target second signal.

[0096] The disclosure contemplates distinguishing between any T. pallidum subspecies. In some aspects, the subspecies are selected from the group consisting of T. pallidum pallidum (TP A), T. pallidum endemicum (TEN), and T. pallidum pertenue (TPE). In some aspects, the nucleic acid amplification assay comprises PCR. In some embodiments, the nucleic acid amplification assay comprises quantitative PCR. In yet other embodiments, the nucleic acid amplification assay comprises qualitative PCR. In some cases, the nucleic acid assay comprises a multiplex assay (e.g., 96-plex).

[0097] The methods of the disclosure contemplate detecting any first target bacterial nucleic acid sequence that is conserved across the subspecies in combination with at least a second target bacterial nucleic acid that is unique in at least one or more subspecies but not conserved across the subspecies. In some aspects, the first target bacterial nucleic sequence comprises a region of the 16s rRNA gene that is conserved across the subspecies. In some embodiments, the first target bacterial nucleic acid sequence is conserved across the subspecies of T. pallidum pallidum (TP A), T. pallidum endemicum (TEN), and T. pallidum pertenue (TPE).

[0098] In some aspects, the conserved nucleic acid comprises a pathogenicity island for Treponema pallidum. Any nucleic acid sequence that is conserved across T. pallidum subspecies within a pathogenicity island can be used in the methods, assays, and kits of the disclosure.

[0099] In some embodiments, the nucleic acid that is conserved across the subspecies (e.g., T. pallidum pallidum (TP A), T. pallidum endemicum (TEN), and T. pallidum pertenue (TPE)) comprises a nucleic acid sequence within the 16s rRNA gene.

[0100] Aspects of the disclosure involve nucleic acid probes and / or primer pairs that are capable of hybridizing to and amplifying target nucleic acid sequences. In some aspect, the methods, assays and kits of the disclosure utilize a first probe and / or primer pair that hybridizes to a polynucleotide sequence within a pathogenicity island that is conserved across the subspecies. In other aspects, the methods, assays, and kits of the disclosure utilize a first probeDocket No. 15947WOO1 / 43913.601 and / or primer pair that hybridizes to a polynucleotide sequence within a gene that is conserved across the subspecies.

[0101] In some embodiments, the first probe hybridizes to a polynucleotide sequence corresponding to nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene. In other embodiments, the first probe hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene. For example, the first probe can be designed to hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides corresponding to nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene.

[0102] The skilled artisan will appreciate that a variety of probe and / or primer sequences can be used to hybridize to and amplify a target bacterial nucleic acid sequence in a sample.

[0103] In some embodiments, the first probe comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that differs from SEQ ID NO: 1 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In other embodiments, the first probe comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

[0104] In some embodiments, the first primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene. In other embodiments, the first forward and reverse primer respectively hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene. For example, the first primer pair can be designed to hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 nucleotides corresponding to nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene.

[0105] In yet other embodiments, the first primer pair comprises a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 or nucleotide sequences that differ from SEQ ID NO: 2 and SEQ ID NO: 3 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In still otherDocket No. 15947WOO1 / 43913.601 embodiments, the first primer pair respectively comprises the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 3.

[0106] The methods, assays, and kits of the present disclosure contemplate detecting at least a second target bacterial nucleic acid sequence that is unique for one subspecies of Treponema pallidum. For example, at least a second target bacterial nucleic acid sequence can be detected that is unique for the subspecies pallidum, endemicum. or pertenue. In some aspects, the second target bacterial nucleic acid comprises a region of a gene that is within a pathogenicity island that is unique for one subspecies of Treponema pallidum. In other aspects, the second target bacterial nucleic acid comprises a region of a gene that is unique for one subspecies but is located within a pathogenicity island that is shared across subspecies.

[0107] Distinguishing subspecies pallidum

[0108] In some aspects, the at least one second target bacterial nucleic acid sequence comprises a sequence that is unique for the subspecies T. pallidum pallidum. The disclosure contemplates targeting any gene that is unique for the subspecies T. pallidum pallidum. In some embodiments, the gene that is unique for the subspecies T. pallidum pallidum comprises a gene within a pathogenicity island for T. pallidum pallidum. Pathogenicity islands unique for T. pallidum pallidum are described by Jaiswal et al. (BMC Genomics. 2020; 21 :33, PMID: 31924165, which is incorporated herein by reference in its entirety). In some embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI1 of FIG. 6 of Jaiswal et al. In other embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI2 of FIG. 6 of Jaiswal et al. In still other embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI3 of FIG. 6 of Jaiswal et al. In yet other embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAM of FIG. 6 of Jaiswal et al.

[0109] In other aspects, the at least a second target bacterial nucleic acid sequence comprises a region of a gene that is present within PAI 2 of Jaiswal et al and which are not present in subspecies pertenue or endemicum. Exemplary such genes are listed in Table 2 below.

[0110] Table 2: List of genes related to PAI2 of subs t. palladiumDocket No. 15947WOO1 / 43913.601

[0111] In some aspects, the second target bacterial nucleic acid sequence comprises a region of the 50SL35 gene that is unique for one subspecies of Treponema pallidum. Any region of the 50SL35 gene that is unique for the subspecies Treponema pallidum pallidum (e.g., it is not present in the other subspecies, e.g., endemicum or pertenue) can be a suitable target bacterial nucleic acid sequence for detecting and distinguishing the subspecies.

[0112] In some embodiments, the second probe hybridizes to a polynucleotide sequence corresponding nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene. In other embodiments, the second probe hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene. For example, the second probe can be designed to hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, nucleotides corresponding to nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene.

[0113] In some embodiments, the second probe comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence that differs from SEQ ID NO: 4 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In other embodiments, the second probe comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4.

[0114] In some embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotidesDocket No. 15947WOO1 / 43913.601919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene. In other embodiments, the second forward and reverse primer respectively hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene. For example, the second primer pair can be designed to hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 nucleotides corresponding to nucleotides 919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene.

[0115] In yet other embodiments, the second primer pair comprises a forward primer and a reverse primer respectively comprises the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 or nucleotide sequences that differ from SEQ ID NO: 5 and SEQ ID NO: 6 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In still other embodiments, the second primer pair respectively comprises the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 or nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 5 or SEQ ID NO: 6.

[0116] The methods, assays, and kits can be used to distinguish between subspecies of T. pallidum in samples by detecting the first and second target nucleic acids in the sample (e.g., by amplification of first and second bacterial nucleic acid sequences that are respectively conserved across the subspecies and unique for one of the subspecies). In some aspects, the subspecies is identified as subspecies T. pallidum pallidum if the first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pallidum.

[0117] The skilled artisan will appreciate that detection of first and second target nucleic acid sequences within a sample obtained from a subject that are specific for a particular subspecies can be indicative of a disease, disorder, or condition associated with the subspecies. In some aspects, identification of the presence of the subspecies T. pallidum pallidum in the sample is indicative that the subject from whom the sample was obtained has venereal syphilis.

[0118] Distinguishing subspecies pertenue

[0119] In other aspects, the at least a second target bacterial nucleic acid sequence comprises a sequence that is unique for the subspecies T. pallidum pertenue. The disclosure contemplates targeting any gene that is unique for the subspecies T. pallidum pertenue.Docket No. 15947WOO1 / 43913.601

[0120] In some embodiments, the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum pertenue. In some embodiments, the gene that is unique for the subspecies T. pallidum pertenue comprises a gene within a pathogenicity island for T. pallidum pertenue. Pathogenicity islands unique for T. pallidum pertenue are described by Jaiswal et al. (BMC Genomics. 2020; 21 :33, PMID: 31924165, which is incorporated herein by reference in its entirety). In some embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI1 of FIG. 7 of Jaiswal et al. In other embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI2 of FIG. 7 of Jaiswal et al. In still other embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI3 of FIG. 7 of Jaiswal et al.

[0121] In some embodiments, the at least the second probe hybridizes to a polynucleotide sequence corresponding to a gene that is in PAI2 of T. pallidum pertenue but is unique to T. pallidum pertenue. In some embodiments, the gene comprises a gene located within a region of the T. pallidum pertenue genome that is from 80,000 bp to about 150,000 bp.

[0122] In other embodiments, the second probe hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 80,000 to 150,000 in the T. pallidum pertenue PAI2.

[0123] In some embodiments, the gene comprises a gene encoding RNA polymerase sigma factor and resides within a region of the genome between 80,000 bp and 150,000 bp of T. pallidum pertenue. In some embodiments, the second probe hybridizes to a polynucleotide sequence corresponding to a gene encoding a protein in Table 3 below or a polynucleotide sequence corresponding to a portion thereof.

[0124] In some embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides flanking the gene within PAI2 of T. pallidum pertenue. In other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence with the coordinates of a gene that encodes a protein listed in Table 3 below. In yet other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence flanking a portion of the coordinates of a gene that encodes a protein listed in Table 3 below.

[0125] In some aspects, the subspecies is identified as subspecies T. pallidum pertenue if the first signal is generated and at least second signal is generated due to the amplification of aDocket No. 15947WOO1 / 43913.601 second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pertenue.

[0126] In some aspects, identification of the presence of the subspecies T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained does not have venereal syphilis. In other aspects, identification of the presence of the subspecies T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained does have yaws.

[0127] Distinguishing subspecies endemicum

[0128] In yet other aspects, the at least a second target bacterial nucleic acid sequence comprises a sequence that is unique for the subspecies T. pallidum endemicum. The disclosure contemplates targeting any gene that is unique for the subspecies T. pallidum endemicum.

[0129] In some embodiments, the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum endemicum. In some embodiments, the gene that is unique for the subspecies T. pallidum endemicum comprises a gene within a pathogenicity island for T. pallidum endemicum. Pathogenicity islands unique for T. pallidum endemicum are described by Jaiswal et al. (BMC Genomics. 2020; 21 :33, PMID: 31924165, which is incorporated herein by reference in its entirety). In some embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI1 of FIG. 8 of Jaiswal et al. In other embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI2 of FIG. 8 of Jaiswal et al. In still other embodiments, the second target bacterial nucleic acid sequence comprises a sequence within PAI3 of FIG. 8 of Jaiswal et al.

[0130] In some embodiments, the second probe hybridizes to a polynucleotide sequence corresponding to a gene encoding a protein in Table 3 below or a polynucleotide sequence corresponding to a portion thereof. In other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence with the coordinates of a gene that encodes a protein listed in Table 3 below. In yet other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence flanking a portion of the coordinates of a gene that encodes a protein listed in Table 3 below.

[0131] In some aspects, the subspecies is identified as subspecies T. pallidum endemicum if the first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum endemicum.Docket No. 15947WOO1 / 43913.601

[0132] In some aspects, identification of the presence of the subspecies T. pallidum endemicum in the sample is indicative that the subject from whom the sample was obtained does not have venereal syphilis. In other aspects, identification of the presence of the subspecies T. pallidum endemicum in the sample is indicative that the subject from whom the sample was obtained has bejel.\ \ \ Distinguishing venereal subspecies from non-venereal subspecies

[0134] In yet other aspects, the methods, assays, and kits of the present disclosure involve detecting at least a second target bacterial nucleic acid sequence comprising a sequence that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum. The disclosure contemplates detecting any gene that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum. In some aspects, the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum. Examples of such genes are listed in Table 3. The skilled artisan can design suitable probes and / or primer pairs that can hybridize to and amplify target bacterial nucleic acid sequences within all or a portion of the coordinates of the subsp. pertenue and endemicum listed in Table 3 below.

[0135] Table 3 - List of genes related to PAI1 of subsp. pertenue and endemicumDocket No. 15947WOO1 / 43913.601

[0136] In some aspects, identification of the presence of the subspecies T. pallidum endemicum or T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained does not have venereal syphilis. In other aspects, identification of the presence of the subspecies T. pallidum endemicum and / or T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained from has a non-venereal disease. In yet other aspects, the detection of the first signal in combination with a second signal generated by detection of a target bacterial nucleic acid sequence located within the genomic coordinates listed in Table 3 is indicative that the subject from whom the sample was obtained from has yaws or bejel.3. Assays

[0137] Aspects of the disclosure relate to assays for use in the methods of the disclosure. In an aspect, the present disclosure provides a nucleic acid amplification assay for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the assay comprising: (a) a composition comprising a first probe and / or primer pair of the present disclosure; (b) a reagents for amplifying a first and at least a second target bacterial nucleic acid sequences, if present, in a sample obtained from a subject; and (c) a system for amplifying and detecting the first and at least the second target bacterial nucleic acid sequences using the composition of (a) and the reagents of (b).

[0138] In another aspect, the disclosure provides a nucleic acid amplification assay for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the assay comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of TreponemaDocket No. 15947WOO1 / 43913.601 pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample.

[0139] In yet another aspect, the present disclosure provides a nucleic acid amplification assay for identifying a subject as more likely than not having venereal syphilis, the assay comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, per tenue , and endemicum, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; and (c) a nucleic acid amplification system for amplifying the first and second target bacterial nucleic acid sequences, if present in the sample, using the first probe and / or primer pair and at least the second probe and / or primer pair.

[0140] In some aspects, the nucleic acid amplification assay comprises PCR. In some embodiments, the nucleic acid amplification assay comprises quantitative PCR. In yet other embodiments, the nucleic acid amplification assay comprises qualitative PCR. In some cases, the nucleic acid assay comprises a multiplex assay (e.g., 96-plex).4. Compositions

[0141] Aspects of the disclosure relate to compositions for use in the methods, assays, and kits of the disclosure. In some aspects, the disclosure provides a composition comprising a probe and / or primer pairs that hybridize to and amplify a first target bacterial nucleic acid sequence that is conserved across Treponema pallidum subspecies. In some embodiments, the composition comprises a first probe and / or primer pair that hybridizes to a polynucleotide sequence within a pathogenicity island that is conserved across the subspecies. In other embodiments, the composition comprises a first probe and / or primer pair that hybridizes to a polynucleotide sequence within a gene that is conserved across the subspecies.

[0142] In some embodiments, the composition comprises a first probe that hybridizes to a polynucleotide sequence corresponding to nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene. In other embodiments, the composition comprises a first probe hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene. For example, the first probeDocket No. 15947WOO1 / 43913.601 can be designed to hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides corresponding to nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene.

[0143] In some embodiments, the composition comprises a first probe comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that differs from SEQ ID NO: 1 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In other embodiments, the composition comprises a first probe comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1.

[0144] In some embodiments, the composition comprises a first primer pair comprising a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene. In other embodiments, the composition comprises a first forward and reverse primer that respectively hybridize to a polynucleotide sequence corresponding to at least a portion of nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene. For example, the first primer pair can be designed to hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or at least 18 nucleotides corresponding to nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene.

[0145] In yet other embodiments, the composition comprises a first primer pair comprising a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 or nucleotide sequences that differ from SEQ ID NO: 2 and SEQ ID NO: 3 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In still other embodiments, the composition comprises a first primer pair respectively comprising the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 3 or nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 or SEQ ID NO: 3.

[0146] In some aspects, the compositions comprise at least a second probe and / or primer pair for detecting at least a second target bacterial nucleic acid sequence that is unique for one subspecies of Treponema pallidum. For example, at least a second target bacterial nucleic acidDocket No. 15947WOO1 / 43913.601 sequence can be detected that is unique for the subspecies pallidum, endemicum, or pertenue. In some aspects, the composition comprises a probe and / or primer pair hybridizes to and amplifies a second target bacterial nucleic acid comprising a region of a gene that is within a pathogenicity island that is unique for one subspecies of Treponema pallidum. In other aspects, composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid comprising a region of a gene that is unique for one subspecies but is located within a pathogenicity island that is shared across subspecies.

[0147] In some aspects, the composition comprises a second probe and / or primer pair that hybridizes to at least a second target bacterial nucleic acid sequence comprising a sequence that is unique for the subspecies T. pallidum pallidum. In some embodiments, the composition comprises a second probe and / or primer that hybridizes to and amplifies a gene that is unique for the subspecies T. pallidum pallidum within a pathogenicity island for T. pallidum pallidum. In some embodiments, the second probe and / or primer hybridizes to and amplifies a target bacterial nucleic acid sequence within PAI1 of FIG. 6 of Jaiswal et al. In other embodiments, the second probe and / or primer hybridizes to and amplifies a second target bacterial nucleic acid sequence within PAI2 of FIG. 6 of Jaiswal et al. In still other embodiments, the second probe and / or primer hybridizes to and amplifies a second target bacterial nucleic acid sequence within PAI3 of FIG. 6 of Jaiswal et al. In yet other embodiments, the second probe and / or primer hybridizes to and amplifies a second target bacterial nucleic acid sequence within PAI4 of FIG. 6 of Jaiswal et al.

[0148] In other aspects, the second probe and / or primer hybridizes to and amplifies at least a second target bacterial nucleic acid sequence located within a region of a gene that is present within PAI 2 of Jaiswal et al and which is not present in subspecies pertenue or endemicum. Exemplary such genes are listed in Table 3 above.

[0149] In some aspects, the second probe and / or primer hybridizes to and amplifies a second target bacterial nucleic acid sequence comprising a region of the 50SL35 gene that is unique for one subspecies of Treponema pallidum. In some embodiments, the composition comprises a second probe that hybridizes to a polynucleotide sequence corresponding nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene. In other embodiments, the composition comprises a second probe that hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene. In some embodiments, the composition comprises a second probe comprising the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence that differs from SEQ ID NO: 4 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.Docket No. 15947WOO1 / 43913.601In other embodiments, the composition comprises a second probe comprising the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4.

[0150] In some embodiments, the composition comprises a second primer pair comprising a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene. In other embodiments, the composition comprises a second forward and reverse primer that respectively hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene.

[0151] In yet other embodiments, the composition comprises a second primer pair comprising a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 or nucleotide sequences that differ from SEQ ID NO: 5 and SEQ ID NO: 6 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides. In still other embodiments, the composition comprises a second primer pair respectively comprising the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6 or nucleotide sequences having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 5 or SEQ ID NO: 6.

[0152] In other aspects, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pertenue. In some embodiments, the composition comprises a second probe and / or primer pair comprising a second target bacterial nucleic acid sequence comprising a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum pertenue. Pathogenicity islands unique for T. pallidum pertenue are described by Jaiswal et al. (BMC Genomics. 2020; 21 :33, PMID: 31924165, which is incorporated herein by reference in its entirety). In some embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequence comprises a sequence within PAI1 of FIG. 7 of Jaiswal et al. In other embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequence within PA 12 of FIG. 7 of Jaiswal et al. In still other embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequenceDocket No. 15947WOO1 / 43913.601 within PAI3 of FIG. 7 of Jaiswal et al. In some embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a polynucleotide sequence corresponding to a gene that is in PAI2 of T. pallidum pertenue but is unique to T. pallidum pertenue. In some embodiments, the gene comprises a gene located within a region of the T. pallidum pertenue genome that is from 80,000 bp to about 150,000 bp.

[0153] In other embodiments, the composition comprises a second probe that hybridizes to a polynucleotide sequence corresponding to at least a portion of nucleotides 80,000 to 150,000 in the T. pallidum pertenue PAI2. In some embodiments, the gene comprises a gene encoding RNA polymerase sigma factor and resides within a region of the genome between 80,000 bp and 150,000 bp of T. pallidum pertenue. In some embodiments, the second probe hybridizes to a polynucleotide sequence corresponding to a gene encoding a protein in Table 3 above or a polynucleotide sequence corresponding to a portion thereof.

[0154] In some embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides flanking a gene or a portion of a gene within PAI2 of T. pallidum pertenue. In other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence with the coordinates of a gene that encodes a protein listed in Table 3 above. In yet other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence flanking a portion of the coordinates of a gene or portion of a gene that encodes a protein listed in Table 3 above.

[0155] In yet other aspects, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence comprises a sequence that is unique for the subspecies T. pallidum endemicum. In some embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum endemicum. In some embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequence within PAI1 of FIG. 8 of Jaiswal et al. In other embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequence within PAI2 of FIG. 8 of Jaiswal et al. In still other embodiments, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequence within PAI3 of FIG. 8 of Jaiswal et al.Docket No. 15947WOO1 / 43913.601

[0156] In some embodiments, the second probe hybridizes to a polynucleotide sequence corresponding to a gene encoding a protein in Table 2 above or a polynucleotide sequence corresponding to a portion thereof. In other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence with the coordinates of a gene or a portion thereof that encodes a protein listed in Table 2 above. In yet other embodiments, the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to a polynucleotide sequence flanking a portion of the coordinates of a gene or a portion thereof that encodes a protein listed in Table 2 above.

[0157] In yet other aspects, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence comprising a sequence that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum. In some aspects, the composition comprises a second probe and / or primer pair that hybridizes to and amplifies a second target bacterial nucleic acid sequence within a pathogenicity island that comprises a gene or a portion of a gene that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum. Examples of such genes are listed in Table 2.

[0158] In some aspects, the probes in the composition comprise a detectable label. In some aspects, the first probe comprises a detectable label. In some aspects, the second probe comprises a detectable label. In some aspects, the first probe and the at least the second probe comprise detectable label. In certain aspects, the first probe and the at least the second probe comprise detectable labels that generate different signals. The disclosure is not limited to any particular type of detectable label.

[0159] In some embodiments, the detectable label comprises a fluorescent dye. In some embodiments, first probe comprises a first fluorescent dye and the second probe comprises a second fluorescent dye that is produces a different signal than the first fluorescent dye.

[0160] In some embodiments, the first probe comprises a detectable label comprising a CY5 dye. In some embodiments, the at least a second probe comprises a detectable label comprising a FAM dye. In some embodiments, the first probe comprises a detectable label comprising a CY5 dye and the second probe comprises a detectable label comprising a FAM dye. In other embodiments, the first comprise comprises a detectable label comprising a FAM dye. In still other embodiments, the at least a second probe comprises a detectable label comprising a CY5 dye. In yet other embodiments, the first probe comprises a detectable label comprising a FAM dye and the at least a second probe comprises a detectable label comprising a CY5 dye.Docket No. 15947WOO1 / 43913.6015. Methods of Treatment and / or Monitoring

[0161] In an aspect, the present disclosure provides a method of treating a subject for venereal syphilis. In another aspect, the present disclosure provides a method of treating a subject for a T. pallidum pallidum infection, the method comprising detecting the presence of the first target nucleic acid that is conserved across T. pallidum subspecies and the presence of the second target nucleic acid that is unique for T. pallidum pallidum in a sample obtained from the subject using a composition, method, assay, and / or kit of the disclosure, and administering to the subject an effective amount of an antibiotic agent to the subject based on the target nucleic acids detected in the sample.

[0162] In another aspect, the disclosure provide a method of monitoring a subject undergoing treatment for a T. pallidum pallidum infection, the method comprising detecting the presence of the first and at least the second target nucleic acids that are indicative of a T. pallidum pallidum infection in a sample obtained from a subject undergoing treatment with an antibiotic agent at a first time point and a second time point, and determining a change in the amount of the first and / or second target nucleic acids detected from the first time point to the second time point, and adjusting the effective amount of the antibiotic agent administered to the subject based on the determined change in the amount.

[0163] In an aspect, the present disclosure provides a method of treating a subject for yaws. In another aspect, the present disclosure provides a method of treating a subject for a T. pallidum pertenue infection, the method comprising detecting the presence of the first target nucleic acid that is conserved across T. pallidum subspecies and the present of the second target nucleic acid that is unique for T. pallidum pertenue in a sample obtained from the subject using a composition, method, assay, and / or kit of the disclosure, and administering to the subject an effective amount of an antibiotic agent to the subject based on the target nucleic acids detected in the sample.

[0164] In another aspect, the disclosure provide a method of monitoring a subject undergoing treatment for a T. pallidum pertenue infection, the method comprising detecting the presence of the first and at least the second target nucleic acids that are indicative of a T. pallidum pertenue infection in a sample obtained from a subject undergoing treatment with an antibiotic agent at a first time point and a second time point, and determining a change in the amount of the first and / or second target nucleic acids detected from the first time point to the second time point, and adjusting the effective amount of the antibiotic agent administered to the subject based on the determined change in the amount.Docket No. 15947WOO1 / 43913.601

[0165] In an aspect, the present disclosure provides a method of treating a subject for yaws. In another aspect, the present disclosure provides a method of treating a subject for a T. pallidum endemicum infection, the method comprising detecting the presence of the first target nucleic acid that is conserved across T. pallidum subspecies and the presence of the second target nucleic acid that is unique for T. pallidum endemicum in a sample obtained from the subject using a composition, method, assay, and / or kit of the disclosure, and administering to the subject an effective amount of an antibiotic agent to the subject based on the target nucleic acids detected in the sample.

[0166] In another aspect, the disclosure provide a method of monitoring a subject undergoing treatment for a T. pallidum endemicum infection, the method comprising detecting the presence of the first and at least the second target nucleic acids that are indicative of a T. pallidum endemicum infection in a sample obtained from a subject undergoing treatment with an antibiotic agent at a first time point and a second time point, and determining a change in the amount of the first and / or second target nucleic acids detected from the first time point to the second time point, and adjusting the effective amount of the antibiotic agent administered to the subject based on the determined change in the amount.6. Kits

[0167] The compositions can be used in a kit of the present disclosure to implement a method or assay of the present disclosure.

[0168] In another aspect, the present disclosure provides a kit for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the kit comprising: (a) a composition of the present disclosure and (c) instructions for performing a nucleic acid amplification assay for detecting the Treponema pallidum bacteria in the sample and distinguishing the subspecies of Treponema pallidum present in the sample.

[0169] In another aspect, the present disclosure provides a kit for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the kit comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, ' and (c) instructions for performing a nucleic acid amplification assay for detecting the Treponema pallidum bacteria in the sample and distinguishing the subspecies of Treponema pallidum present in the sample.Docket No. 15947WOO1 / 43913.601

[0170] In yet another aspect, the present disclosure provides a kit for identifying a subject as more likely than not having venereal syphilis, the kit comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, per tenue, and endemicunr, and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum,' and (c) instructions for performing a nucleic acid amplification assay for identifying the subject has more likely than not having venereal syphilis if the nucleic acid amplification assay generates both a first and second signal due to the amplification of the first and second target bacterial nucleic acid sequences within a sample obtained from the subject.

[0171] In some aspects, the nucleic acid amplification assay comprises PCR. In some embodiments, the nucleic acid amplification assay comprises quantitative PCR. In yet other embodiments, the nucleic acid amplification assay comprises qualitative PCR. In some cases, the nucleic acid assay comprises a multiplex assay (e.g., 96-plex).

[0172] The kit can also include a control or reference standard and / or instructions for use thereof. In addition, the kit can include ancillary agents such as vessels for storing or transporting the detection agents and / or buffers or stabilizers. In some embodiments, the kit comprises reagents for performing PCR (e.g., multiplexed (e.g., 96-plex) quantitative or quantitative real-time PCR).

[0173] The kit may include instructions for performing the methods of the present disclosure. For example, the kit can comprise instructions for distinguishing between the T. pallidum subspecies in the sample. In another example the kit can comprise instructions for detecting a T. pallidum pallidum nucleic acid in a sample. In another example, the kit can comprise instructions for identifying the subject as more likely than not as having venereal syphilis. In yet another example, the kit can comprise instructions for detecting a T. pallidum pertenue nucleic acid in a sample. In a yet even another example, the kit can comprise instructions for identifying the subject as more likely than not as not having a venereal disease. In a further example, the kit can comprise instructions for identifying the subject as more likely than not as having yaws. In yet a further example, the kit can comprise instructions for detecting T. pallidum endemicum nucleic acid in a sample. In even a further example, the kit can comprise instructions for identifying the subject as more likely than not as having bejel.

[0174] Instructions included in kits can be affixed to packaging material or can be included as a package insert. While the instructions are typically written or printed materials, they are notDocket No. 15947WOO1 / 43913.601 limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.

[0175] Alternatively or additionally, the kit can comprise a calibrator or control, e.g., purified, and optionally lyophilized, and / or at least one container (e.g., tube, microtiter plates or strips) for conducting the assay, and / or a buffer, such as an assay buffer or a wash buffer, either one of which can be provided as a concentrated solution, a substrate solution for the detectable label, or a stop solution. Preferably, the kit comprises all components, i.e., reagents, standards, buffers, diluents, etc., which are necessary to perform the assay. The instructions also can include instructions for generating a standard curve.

[0176] The kit can also optionally include other reagents required to conduct a diagnostic assay or facilitate quality control evaluations, such as buffers, salts, enzymes, enzyme co-factors, substrates, detection reagents, and the like. Other components, such as buffers and solutions for the isolation and / or treatment of a test sample (e.g., pretreatment reagents), also can be included in the kit. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further comprise reagents suitable for the reconstitution of the lyophilized components.

[0177] The various components of the kit optionally are provided in suitable containers as necessary, e.g., a microtiter plate. The kit can further include containers for holding or storing a sample (e.g., a container or cartridge for a whole blood, plasma, serum sample, swab, tissue, or CSF sample). Where appropriate, the kit optionally also can contain reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or the test sample. The kit can also include one or more instruments for assisting with obtaining a test sample, such as a syringe, pipette, forceps, measured spoon, or the like.

[0178] If desired, the kit can contain a solid phase, such as a magnetic particle, bead, test tube, microtiter plate, cuvette, membrane, scaffolding molecule, film, filter paper, disc, or chip.

[0179] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects andDocket No. 15947WOO1 / 43913.601 embodiments of the present disclosure and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.7. Samples

[0180] Aspects of the disclosure involve detection of a first target bacterial nucleic acid and at least a second target bacterial nucleic acid in samples. The disclosure is not limited to the type of sample in which the first and / or at least a second target bacterial nucleic acids can be detected. In an embodiment, a T. pallidum pallidum subspecies is detected in at least one sample. In an embodiment, a T. pallidum pertenue subspecies is detected in at least one sample. In an embodiment, a T. pallidum endemicum subspecies is detected in at least one sample.

[0181] The sample may be a sample from a subject, the environment, a laboratory, or any sample in which has or is suspected of having a T. pallidum subspecies. In some embodiments, the at least one sample is a biological sample. In some embodiments, the at least one sample is selected from the group consisting of cerebral spinal fluid, plasma, serum, whole blood, a swab (e.g., from a skin lesion or ulcer), tissue (e.g., from a lesion), placental or amniotic fluid, and other bodily fluids. In an embodiment, the at least one sample is a cerebral spinal fluid sample. In an embodiment, the at least one sample is a plasma sample. In an embodiment, the at least one sample is a serum sample. In an embodiment, the at least one sample is a whole blood sample. In an embodiment, the at least one sample is a swab sample (e.g., e.g., from a skin lesion or ulcer). In an embodiment, the at least one sample is a tissue sample (e.g., from a lesion). The tissue sample may be a tissue biopsy. The biopsied tissue may be fixed, embedded in paraffin or plastic, and sectioned, or the biopsied tissue may be frozen and cryosectioned. Alternatively, the biopsied tissue may be processed into individual cells or an explant, or processed into a homogenate, a cell extract, a membranous fraction, or a protein extract.

[0182] The sample may be used “as is”, processed for cell lysis or disruption of bacterial cells, or the nucleic acid may be purified from the sample prior to sample preparation. Methods of isolating nucleic acid from a sample are known in the art. In certain embodiments, the isolated nucleic acid is reverse transcribed and amplified after isolation. This allows detection of both RNA and DNA bacterial nucleic acids. Specifically, RNA in the total nucleic acid may be reverse transcribed with reverse transcriptase. Primers may then be used for subsequent amplification. Second strand synthesis may then be carried out using DNA polymerase to generate cDNA for the RNA nucleic acid sequences.Docket No. 15947WOO1 / 43913.601

[0183] The DNA / cDNA mixture may then be amplified using DNA polymerase. In general, amplification is carried out using polymerase chain reaction (PCR). A PCR reaction may comprise nucleic acid, primers, polymerase, water, buffer, and deoxynucleotide triphosphates (dNTPs). PCR may be performed according to standard methods in the art. By way of nonlimiting example, the PCR reaction may comprise denaturation, followed by about 5-10 cycles of denaturation, annealing and extension, followed by a final extension. In an exemplary embodiment, the PCR reaction comprises denaturation at about 98° C. for about 30 seconds, followed by about 5 to about 10 cycles of (about 98° C. for about 10 seconds, about 62-72° C. for about 30 seconds, about 72° C. for about 30 seconds), followed by a final extension at about 72° C. for about 5 minutes. Optionally, the amplified nucleic acid is then purified, for example, via column purification. The nucleic acid in the sample may then be sheared via methods known in the art to generate fragments. The fragments may be about 100 to about 2000 bp. For example, the fragments may be about 200 to about 1500 bp, about 400 to about 1000 bp, about 400 to about 800 bp, or about 500 bp.

[0184] The at least one subject from which the sample is obtained has or is suspected of having a T. pallidum pallidum infection and / or a symptom, disease, condition, or disorder associated with the infection (e.g., venereal syphilis). In some embodiments, the at least one sample is obtained from a subject that has or is suspected of having a T. pallidum pertenue infection and / or a symptom, disease, condition, or disorder associated with the bacterial infection (e.g., yaws). In some embodiments, the at least one sample is obtained from a subject that has or is suspected of having a T. pallidum endemicum infection and / or a symptom, disease, condition, or disorder associated with the infection (e.g., bejel).

[0185] In some cases, the at least one subject has or is suspected of having a venereal disease (e.g., syphilis). In some cases, the at least one subject has or is suspected of having yaws. In some cases, the at least one subject has or is suspected of having a bejel.

[0186] In some embodiments, the sample comprises or is suspected to comprise at least a first target bacterial nucleic acid that is conserved across T. pallidum subspecies and at least a second target bacterial nucleic acid that is unique for one subspecies of T. pallidum. In some embodiments, the sample comprises or is suspected of comprising at least a first target bacterial nucleic acid that is conserved across T. pallidum subspecies and at least a second target bacterial nucleic acid that is unique for T. pallidum pallidum. In other embodiments, the sample comprises or is suspected of comprising at least a first target bacterial nucleic acid that is conserved across T. pallidum subspecies and at least a second target bacterial nucleic acid that is unique for T. pallidum pertenue. In yet other embodiments, the sample comprises or isDocket No. 15947WOO1 / 43913.601 suspected of comprising at least a first target bacterial nucleic acid that is conserved across T. pallidum subspecies and at least a second target bacterial nucleic acid that is unique for T. pallidum endemicum.

[0187] Preferably, the samples comprise nucleic acids (e.g., DNA, RNA, cDNAs, microRNA, mitochondrial DNA, etc.). Samples may be complex samples or mixed samples, which contain nucleic acids comprising multiple different nucleic acid sequences (e.g. host and pathogen nucleic acids; mutant and wild-type species; heterogeneous tumor). Samples may comprise nucleic acids from more than one source (e.g. different species, different subspecies, etc.), subject, and / or individual.

[0188] In some aspects, the amount of sample obtained from the subject is less than about 3.9 mL, about 3.8 mL, about 3.7 mL, about 3.6 mL, about 3.5 mL, about 3.4 mL, about 3.3 mL, about 3.2 mL, about 3.1 mL, about 3.0 mL, about 2.9 mL, about 2.8 mL, about 2.7 mL, about 2.6 mL, about 2.5 mL, about 2.4 mL, about 2.3 mL, about 2.2 mL, about 2.1 mL, about 2.0 ml, about 1.9 mL, about 1.8 mL, about 1.7 mL, about 1.6 mL, about 1.5 mL, about 1.4 mL, about 1.3 mL, about 1.2 mL, about 1.1 mL, about 1.0 mL, about 0.9 mL, about 0.8 mL, about 0.7 mL, about 0.6 mL, or about 0.5 mL.EXAMPLES

[0189] The present disclosure has multiple aspects, illustrated by the non-limiting examples as described herein.EXAMPLE 1

[0190] This example provides a non-limiting description of one exemplary embodiment of an assay of the present disclosure, for instance a qualitative, real-time PCR assay for detection and discrimination of Treponema pallidum subspecies. The exemplary embodiment of this assay involves a set of forward and reverse primers, two sets of each to amplify two different regions of the bacterial genome. To discriminate which regions are detected, exemplary probes comprising detectable labels (e.g., TaqMan probes labeled with different fluorescent dyes) hybridize to their respective amplicons. Thermocycling conditions and reagent compositions were optimized for running the assay on Abbott Laboratories’ m2000 and Alinity® m platforms. Examples 2 and 3 below describe expansive testing conducted to demonstrate linearity, analytical sensitivity, and specificity of the assay on both of those AbbottDocket No. 15947WOO1 / 43913.601Laboratories platforms, as well as its performance on clinical samples deemed positive by another molecular assay.

[0191] The purpose of the exemplary assay is two-fold: 1) to provide confirmation that Treponema pallidum is present and 2) to provide a discriminatory, molecular diagnosis to indicate which Treponema subspecies is present, and thereby determine whether someone has venereal syphilis, bejel, or yaws. Therefore, this exemplary embodiment of an assay of the present disclosure (e.g., qPCR) detects one target that is common to all Treponema subspecies, and another target that is specific to one species (e.g., subspecies pallidum that is the causative infectious agent of syphilis) and which differentiates venereal from non-venereal disease.

[0192] In the exemplary embodiments of the assays of the present disclosure described in the examples, the primary target to achieve purpose 1) is 16s ribosomal RNA (rRNA). The alignment shown in FIG. 1 indicates that all 3 subspecies possess this gene and are identical in the selected primer and probe regions. The exemplary first probe (e.g., TP_16S_676Probe22) sequence (SEQ ID NO: 1) has been labeled with the CY5 dye. TPA contains several genes found on ‘pathogenicity islands’ that are unique to this subspecies and thus absent in TEN and TPE (see FIG. 2A and FIG. 2B). In one exemplary embodiment, the methods and assays distinguish subspecies by targeting the 50s L35 rRNP, which is unique to TPA. The exemplary second probe (e.g., TP_35L70probel8A) sequence (SEQ ID NO: 2) has been labeled with the FAM dye. A second positive signal in the FAM channel therefore indicates a person has venereal syphilis, as illustrated in Table 4 below.

[0193] Table 4

[0194] The methods and assays of the present disclosure thus provide a single test with the ability to both confirm (1) the presence of Treponema pallidum and (2) differentiate the subspecies to diagnose a patient as having venereal syphilis, bejel, or yaws.

[0195] The methods and assays of the presence disclosure also detected venereal syphilis at two targets in the genome. TPA is the major public health concern, and the methods and assays of the present disclosure provide an inherent confirmation when both detectable label (e.g., FAM and CY5) channels are positive.Docket No. 15947WOO1 / 43913.601

[0196] The methods and assays of the present disclosure also contemplate the use of an internal control (in the VIC channel), which validates the extraction and amplification steps are perform as expected.

[0197] Finally, the analytical sensitivity of the methods and assays of the present disclosure exhibit superior analytic sensitivity. On the m2000, with 95% confidence the methods and assays can detect 22.1 cp / ml, or 5.6 cp / rxn. On the Alinity® m, with 95% confidence the methods and assays can detect 34.3 cp / ml, or 4.3 cp / rxn.EXAMPLE 2

[0198] This example demonstrates exemplary embodiments of methods, compositions, kits and an assay of the present disclosure implanted on the Abbott Laboratories m2000 platform for performing qualitative, real-time PCR.

[0199] Exemplary assay parameters used in this example include the following:• 400 uL assay for swab / serum / CSF• 650 uL sample input• Results reported as CN• 600 uL CT / NG internal control• DNA prep pack (70mL ethanol added to Wash2; 2x24 worth of prep pack can be used to run 96 samples or 192 samples per prep pack)• Max 96 samples per run

[0200] Differentiates venereal from non-venereal syphilis.

[0201] Reagents for performing the assay can be divided into separate containers as follows:

[0202] Table 5 - Bottle#!: Activator

[0203] Table 6 - Bottle#2: OligosDocket No. 15947WOO1 / 43913.601

[0204] Table 7 - Bottle#3: Enzyme

[0205] Plasmids containing the regions targeted in each channel were generated as positive controls and then serially diluted to demonstrate linearity and sensitivity and calculate efficiency (see Tables 8 and 9 below). Plasmid genetic construction is shown in FIG. 4A and FIG. 4B. The total length of the 16S rRNA plasmid shown in FIG. 4A is 4480 bp. The total length of the 35L plasmid shown in FIG. 4B is 3144 bp ~ 2 x 108copies / uL. Plasmid was digested so bp size is no longer the entire plasmid, but that length will be used in the calculations.Docket No. 15947WOO1 / 43913.601

[0206] Table 8

[0207] Table 9

[0208] Table 10 below includes the data used to construct a plasmid curve, which is shown in FIG. 5.

[0209] Tables 10 and 11 show 16S plasmid-CY5 probit analysis for limit of detection (LOD).

[0210] Table 10Docket No. 15947WOO1 / 43913.601

[0211] Table 11Docket No. 15947WOO1 / 43913.601

[0212] FIG. 6 shows a dose-response plot constructed using the data from Tables 10 and 11.

[0213] The LOD is a measure of analytical sensitivity, i.e., the lowest detectable number of virus copies in a sample at which a test will return a positive result 95% of the time.

[0214] The LOD 95%=2.15 cps / RXN=0.051532 cps / uL(sample)=8.461 cps / mL (sample)

[0215] The calculations used to derive the above data were as follows:

[0216] Cps / RXN - / 35pL=cps / pL-- *55pL total cps for eluate / 400pL for initial sample volume xl000=cps / mL of original sample

[0217] Tables 12 and 13 show 35L plasmid-FAM probit analysis for limit of detection (LOD).Docket No. 15947WOO1 / 43913.601

[0218] Table 12

[0219] Table 13Docket No. 15947WOO1 / 43913.601

[0220] FIG. 7 shows a dose-response plot constructed using the data from Tables 12 and 13.

[0221] LOD 95%=1.011 cps / RXN=0.02888 cps / pL=3.972 cps / mL (Sample)=0.599 LOG cps / mL (Sample)

[0222] Calculations for the above were derived as follows: Cps / RXN -> / 35pL=cps / pL- - *55pL total cps for eluate / 400pL for initial sample volume xl000=cps / mL of original sample.

[0223] The data in Table 14 below and FIG. 8, FIG. 9, and FIG. 10 illustrate the linearity of cultured syphilis.

[0224] Table 14

[0225] Table 15 below illustrates the limit of detection (LOD) using cultured syphilis. The culture stock was serially diluted in bulk UTM and then 35 replicates were tested at each concentration in order to do the probit analysis. For concentration LOG 3 and LOG5 an instrument related error resulted in a replicate being removed so the #Detected is 34 / 34 and not 34 / 35.Docket No. 15947WOO1 / 43913.601

[0226] Table 15

[0227] Tables 16 contains data used to construct the dose-response plots for FAM-35L- venereal and C5-16S-non-venereal depicted in FIG. 11.

[0228] Table 16Docket No. 15947WOO1 / 43913.601EXAMPLE 3

[0229] This example demonstrates exemplary embodiments of methods, compositions, kits and an assay of the present disclosure implanted on the Abbott Laboratories Alinity® m platform for performing qualitative, real-time PCR.

[0230] Reagents for performing the assay can be prepared in two separate plates as shown in Table 17 and Table 18:

[0231] Table 17 - AMP PlateDocket No. 15947WOO1 / 43913.601

[0232] Table 18 - ACT Plate (Calculated for adding 50 uL into each well)

[0233] Table 19 shows results of the Alinity® specificity study.

[0234] Table 19

[0235] The design of the Alinity® m requires transcripts or extracted material to be added at an early stage so only original patient plasma samples were run. Samples comprising other microbes not depicted in Table 19 were both run on the m2000 version and were negative.

[0236] Table 20 shows results of the Alinity® sensitivity study.

[0237] Table 20Docket No. 15947WOO1 / 43913.601

[0238] Serial dilutions were made from an 8 Log cps / mL cultured stock diluted in UTM with enough volume to test 35 replicates at each dilution. This is a total of 350 samples tested for the LOD Study.

[0239] Table 21 shows a summary of data for the Alinity® sensitivity study.

[0240] Table 21

[0241] FIG. 12 and FIG. 13 respectively show the linearity of data from the Alinity® sensitivity study, including all LOD data (FIG. 12) and average CT data (FIG. 13).

[0242] Table 22 shows results of interpretation of the Alinity® sensitivity study.Docket No. 15947WOO1 / 43913.601

[0243] Table 22

[0244] * When CTs are below 33.5 (LOD) interpretation is positive for general syphilis but underdetermined for differentiation because negatives in the FAM / Venereal channel could be due to sensitivity.

[0245] Tables 23 and 24 show data comparing the results of clinical specimen testing on samples procured from Trina BioReactives (Naenikon, Switzerland) using the optimized Alinity® m assay with Abbott chemistry and the original syphilis prototype assay with AgPath chemistry (Thermo Fisher).

[0246] Table 23Docket No. 15947WOO1 / 43913.601

[0247] Table 24 - shows the original Agpath data used to populate the prototype Ct columns in Table 3

[0248] N=17 Swab positive samples were originally procured from Trina BioReactives (Naenikon, Switzerland) and run on the prototype. N=l l had sufficient volume for retesting while several others were pooled to make a 12thsample. Cts look comparable with a single sample missed in FAM but using the prototype CT that would be beyond LOD for the assay.

[0249] Table 25 and FIG. 14 below show assay comparisons of samples obtained from Trina BioReactives (Naenikon, Switzerland) run on the m2000 and Alinity® m platforms.Docket No. 15947WOO1 / 43913.601

[0250] Table 25

[0251] N=17 Swab positive samples were originally procured from Trina BioReactives (Naenikon, Switzerland) and run on the prototype. N=11 had sufficient volume for retesting on the Alinity® m. Cts look comparable with a single sample missed in FAM but using the prototype CT that would be beyond LOD for the assay. This missed FAM sample is also listed from the vendor as low positivity and is possibly missed due to multiple freeze thaws and low volume.

[0252] Tables 26 and 27 show a comparison of assay results using the m2000 and Alinity® m platforms.

[0253] Table 26 - M2000 resultsDocket No. 15947WOO1 / 43913.601

[0254] Table 27- Alinity® m results

[0255] FIG. 15 and FIG. 16 respectively show a comparison of m2000 and Alinity® m assay results for FAM. The syphilis culture dilution was from E-5 down to E-0. The Alinity® m data has a mid-dilution control.

[0256] FIG. 17 and FIG. 18 respectively show a comparison of m2000 and Alinity® m assay results for VIC IC. The syphilis culture dilution was from E-5 down to E-0. The Alinity® m data has a mid-dilution control.

[0257] FIG. 19 and FIG. 20 respectively show a comparison of m2000 and Alinity® m assay results for CY5. The syphilis culture dilution was from E-5 down to E-0. The Alinity® m data has a mid-dilution control.

[0258] FIG. 21 shows a comparison of m2000 and Alinity® m assay results for linearity.

[0259] Patient samples (n=17) collected from BioReactives (Naenikon, Switzerland) and T. Pallidum pallidum reference material from the US CDC were evaluated with the assay.

[0260] FIG. 22 shows the evaluation of analytical sensitivity of the duplex-qPCR using the TPA strain Nichols in 10-fold dilution series. Values represent the number of replicates detected for each assay on each instrument (orange: m2000, purple: Alinity m) numbers between brackets represent the mean CT-values obtained for each assay.

[0261] FIG. 23 shows a comparison of the probit dose response for the m2000 and Alinity® m assays. The upper row shows that the m2000-LOD determined by 95% detectability is 22.1Docket No. 15947WOO1 / 43913.601 cps / mL, whereas the lower row shows that the Alinity® m-Lod determined by 95% detectability is 34.8 cps / mL.

[0262] The data in these examples demonstrates the superior performance of the exemplary multiplex assays for venereal and non-venereal syphilis performed on the m2000 and Alinity® m platforms.EXAMPLE 4

[0263] The reference strain of T. Pallidum sub. pallidum Nichols was provided by the CDC, Atlanta, Georgia, USA at a titer of 108units / mL. Serial dilutions of the strain corresponding to 105units / mL, 104units / mL, and 103units / mL were tested and yielded positive detection in both channels (FAM and CY5) for all cases. FIG. 24 shows results of assay performance on reference strain material TPP Nichols which was propagated in rabbit.

[0264] FIG. 25 and FIG. 26 show conditions for the Alinity® m. The exemplary first target bacterial nucleic acid (e.g., 50SL35 gene)-set yielded a linear range from 109-101copies / pL and amplification efficiency 97.28%. The exemplary second target bacterial nucleic acid (16S gene) set yielded a linear range from 109- 102copies / pL and amplification efficiency 101.78%.

[0265] Detailed experiments using plasmids, and the cultured Nichols reference strain were performed to demonstrate linearity, specificity, limit of detection, and analytical sensitivity on the m2000 and Alinity® m platforms.

[0266] FIG. 27A and FIG. 27B show linear range and sensitivity results for the exemplary first target bacterial nucleic acid (e.g., 16S rRNA), which yielded a linear range from 109-102copies / pL and amplification efficiency 101.78%.

[0267] FIG. 28 A and FIG. 28B show linear range and sensitivity results for the exemplary second target bacterial nucleic acid (e.g., 50SL35), which yielded a linear range from 109-l 01copies / pL and amplification efficiency 97.28%.

[0268] FIG. 29A and FIG. 29B shows analytical sensitivity results for the exemplary second target bacterial nucleic acid (e.g., 50SL35 gene)-set, which yielded a linear range from 109- 102copies / pL and amplification efficiency 101.78%.

[0269] In addition, the 17 clinical samples (swabs resuspended in viral transport media) collected from different stages of the disease having different demographics (age, sex, and location of the lesion) were tested and the assay provided 100% agreement with the results obtained by this supplier. Table 30 shows results of an evaluation of an exemplary multiplexDocket No. 15947WOO1 / 43913.601 qPCR for diagnostic of venereal syphilis on clinical swabs samples collected from Trina BioReactives (Naenikon, Switzerland).

[0270] Table 28

[0271] FIG. 30A and FIG. 30B show the results of an evaluation of the exemplary embodiment of an assay of the present disclosure (e.g., multiplex qPCR) for diagnostic of venereal syphilis on clinical swabs samples collected from Trina BioReactives (Naenikon, Switzerland). The multiplex assay detected all the positive samples at different degrees of bacterial load, showing a 100% agreement with the test performed by the provider.EXAMPLE 5Materials and methods

[0272] Treponema pallidum pallidum strains and Samples

[0273] Strains

[0274] The strain Nichols of T. pallidum pallidum was kindly provided by Dr Allan Pillay from the CDC repository. The pathogenic Treponeme strains T. denticola ATCC 35405, T. denticola ATCC 35404, T. phagedenis ATCC 27087, and the non-pathogenic strains Treponema sp. OMZ 804 ATCC 700766, Treponema sp. OMZ 838, and Treponema sp. TSD-166 were purchased from the American Type Culture Collection (ATCC).

[0275] SamplesDocket No. 15947WOO1 / 43913.601

[0276] A total of 17 swab samples were acquired from Trina Bioreactives AG (Nanikon, Switzerland) with confirmed T. pallidum pallidum infection. In addition, 540 samples collected from a clinical study for HSV infections were used to evaluate the performance of the novel duplex qPCR assay and compared with a previously described assay (Heymans et al. 2010).

[0277] Assay Design

[0278] Sequence Dataset Selection and Analysis

[0279] Three Treponema pallidum sequences, including T. pallidum pertenue (TPP), T. pallidum endemicum (TPE), and T. pallidum pallidum (TPA), available in the GenBank Database under the accession numbers CP032623, CP032303, and CP024088 were downloaded and analyzed for their 16S rRNA genes. In addition, other pathogenic Treponeme including T. dendicola (NR 036899), T. phagedenis (VOQA01000001) and non-pathogenic Treponemes such as Treponema sp. OMZ 838 (KP101523) and Treponema socranskii (CP054258) were part of the analysis. The pathogenic island unique to T. pallidum pallidum, the rpml gene, was selected as a differential target (Jaiswal et al. 2020) for venereal syphilis. Consequently, two groups of sequences were obtained, aligned, and subjected to a detailed examination to identify target regions.

[0280] Primer and Probe Design

[0281] Different sets of primers and probes were designed with two primary objectives: (i) the general targeting of pathogenic Treponema and (ii) the differential detection and discrimination of subspecies pallidum (TPA). All primers and probes were crafted using Oligo 7.6 software (Molecular Biology Insights, United States), following the methodology described in (Perez et al. 2020). Briefly, parameters were adjusted to optimize primer and probe selection for the TaqMan assay, focusing on the priming efficiency of each component. This process adhered to the conditions optimized by Rios et al. (Rios et al. 2018), and included a subsequent in silico evaluation for specificity using the GenBank nr database and BLASTn searches. To prevent inefficient amplification reactions, such as primer-dimer formation, all primers were also analyzed for their thermodynamic properties, secondary structures, and primer-primer interactions.

[0282] Venereal / non-venereal syphilis (pathogenic Treponeme) automated assays

[0283] The RealTime venereal / non-venereal syphilis Research Use Only (RUO) assays were designed for both the m2000 and Alinity m platforms. This test includes an internal control of an unrelated DNA sequence introduced into each specimen at the beginning of sample preparation during lysis and is simultaneously amplified by the assay to assess sample integrity and extraction efficiency, as described in Anderson et al. (Anderson et al. 2024). TheDocket No. 15947WOO1 / 43913.601 m2000sp / rt platform permits analysis of up to 96 samples (including controls) while the Alinity m platform enables continuous, random-access and can process 300 samples in approximately 8 hours, with the first result available in less than 115 minutes.

[0284] Linear Range, Efficiency, and Limit of Detection (LoD)

[0285] The linear ranges of each target for the assay were estimated by evaluating 10-fold serial dilutions of quantified DNA standards in nuclease-free water. Amplification efficiencies were determined following the methodology previously described (Perez et al. 2017) using Biogazelle's qbase C software, which incorporates a correction for efficiency estimation with the expression (1)

[0286] E = 101 / slope

[0287] The limit of detection (LoD) for each single-plex target and as a duplex-qPCR assay was assessed by 10-fold serial dilutions of quantified DNA standards in nuclease-free water. The evaluated dilutions ranged from 10'6DNA copies / mL to 10-2 DNA copies / pL for both 16S rRNA and rpml target genes. Each dilution was tested for a total of 35 replicates. The LoD was determined using Probit regression analysis included in MedCalc Statistical Software version 19.0.7 (MedCalc Software bvba, Ostend, Belgium, 2019). The LoD was defined as the concentration (copies of the gene / reaction) that produced 95% positive results, accompanied by a 95% confidence interval (CI), as proposed by Vaks et al. (Vaks et al. 2016).

[0288] Analytical Sensitivity

[0289] Analytical sensitivity (ASe) was assessed through dilution-to-extinction (DTE) experiments, employing ten-fold serial dilutions of cultures of T. pallidum sub. pallidum, strain Nichols, into two different matrices: universal transport medium (UTM) and negative human serum, the latter having been previously screened via Next Generation Sequencing to verify the absence of Treponemes. Additionally, the matrix effect was evaluated on both the efficiency and sensitivity of the assays. Similar to the determination of the Limit of Detection (LoD), the analytical sensitivity (ASe) for each clinical matrix was quantified using Probit regression analysis, conducted using MedCalc Statistical Software version 19.0.7 (MedCalc Software bvba, Ostend, Belgium, 2019). The ASe was defined as the concentration of gene copies per milliliter (copies / mL) that achieved 95% positive detection, with a corresponding 95% confidence interval (CI)(Vaks et al. 2016).

[0290] Intra- and Inter-Assay Variability

[0291] The repeatability and reproducibility of the duplex qPCR assay were evaluated following the approach described by Rios et al. (Rios et al. 2018). Briefly: three templateDocket No. 15947WOO1 / 43913.601 concentrations representing strong positive (105copies / mL), medium positive (103copies / mL), and weak positive (102copies / mL) reactions were tested using the Nichols strain of T. pallidum sub pallidum. Intra-assay variability was determined by analyzing 20 replicate reactions at each concentration within a single run. Inter-assay variability was assessed by testing the same concentration set in independent runs performed on different days by two operators. For each condition, the coefficient of variation (CV) was calculated using the formula CV = (SD of Ct values / mean Ct value) x 100, as previously described (Perez et al. 2012).

[0292] Interpretation of Results

[0293] Following the criteria previously described by Perez et al. (Perez et al. 2020), nonspecific amplifications resulting from primer-primer or primer-probe interactions were not observed after 55 amplification cycles. Based on this, a cut-off value for the duplex qPCR assay was established using expression (1) below to minimize the risk of spurious amplification signals or fluorescence artifacts. Accordingly, all samples with a cycle threshold (Ct) value below 40 (Ct < 40) were interpreted as positive. Samples with Ct values > 40 were interpreted as negative.

[0294] (l)Cut — off = Ct(LoD) X 3S£)(10x replicates LoD)

[0295] These parameters were also incorporated into the automated assay specification to ensure standardized and reproducible result interpretations across all runs.

[0296] Evaluation of Diagnostic performance

[0297] Study Populations

[0298] Two independent specimen sets were analyzed to provide evidence for sensitivity estimation against known positives. Population 1 is 17 Treponema-qPCR positive donor lesion swabs in VTM with clinical compatibility, supplied by a commercial vendor (Trina BioReactives). The vendor internal comparator assay is referred to hereafter as Test V. Population 2 is 540 specimens previously collected from a clinical HSV infection study, for which syphilis status was unknown. Each specimen was tested in parallel using the Treponema duplex qPCR (Test A) and a published comparator assay (Test B) (Heymans et al. 2010). The Heymans study reported a context-specific diagnostic performance of 73-87% sensitivity and 93-97% specificity across clinical settings; these data informed the priors used for Test B.

[0299] Latent-Class Modeling

[0300] There is no universally accepted gold standard for Treponema qPCR and the comparator in Population 1 (Test V) differs from the published assay used in Population 2 (Hess et al. 2012). Bayesian latent class models (LCM) were used to estimate diagnosticDocket No. 15947WOO1 / 43913.601 sensitivity (DSe) and specificity (DSp) as described in Perez et al. (Perez et al. 2020) with some modification.

[0301] Data Structure and “Overlap” Definition

[0302] For each population a 2 * 2 table of joint outcomes was constucted for Test A (duplex (2 qPCR) and the available comparator. In Population 2, the overlap denotes the number of samples testing positive by both assays (A+, B+). Given fixed marginals of A+ = 49, B+ = 30, (2 (2 (2 and n = 540, the feasible range was 0 < yxl< 30. The remaining cells were y10= A+ - yx,n - (y1('i')+y1('o')+yoi'))- InPopulation 1 (known positives), all 17 were A+, and Test V results (if available) were tabulated accordingly.

[0303] Bayesian Latent Class Model

[0304] A two-population, two-test LCM was fit under conditional independence as the primary specification. For each population p G { 1, 2}, the joint outcome probabilities were modeled as:with P(i4 = l | £) = l ) = DSeAand P(A = 0 I D = 0) = DSpA(analogously for Test B). Prevalence was fixed at nf1= 1 for Population 1 (all true positives) and estimated asfor Population 2. A conditional-dependence variant including covariance terms (yi, yo) was evaluated as a sensitivity analysis.

[0306] Priors

[0307] The assay (Test A): DSea~ Beta(2, 2); DSpa~ Beta(2, 2) (non-informative, symmetric). Comparator (Test B): Informative Beta priors derived from Heymans et al. (2010). For the selected STI-clinic scenario, mean DSe = 0.728 and DSp = 0.955 with effective prior sample sizeNo = 50, giving DSe_B ~ Beta(36.4, 13.6) and DSp_B ~ Beta(47.8, 2.2). Alternative priors for the general -practice and dermatovenereology settings (DSe ~ 0.75 / 0.87; DSp ~ 0.97 / 0.93) were examined in sensitivity analyses. Prevalence (Population 2): ?tA(2) ~ Beta(2, 2).

[0308] Computation

[0309] Models were implemented in R (v4.3.1) using JAGS (v4.3.1) via rjags. Three chains were run with 2 000 adaptation iterations, 5000 burn-in iterations, and 20000 post-burn-in iterations per chain (thinning = 10). Convergence was evaluated by visual inspection of traceplots, Gelman-Rubin R-hat (< 1.05), and effective sample sizes. Posterior medians and 95% equal-tail credible intervals (Crls) are reported. For each possible overlap value, posterior DSe and DSp were also summarized to assess sensitivity of estimates to the assumed overlap.Results

[0310] Dual color qPCR for detection and discrimination ofT pallidum subsp. pallidum.Docket No. 15947WOO1 / 43913.601

[0311] To meet the current need for sensitive and specific molecular diagnosis of Treponema infections (World Health Organization. 2025) the design prioritized inclusion of loci exhibiting high within-clade conservation and clear discrimination from non-target Treponemes and host / background microbiota. Multiple candidate regions were screened in silico against curated Treponema genomes (pathogenic T. pallidum subspecies (e.g., T. pallidum subsp. endemicum, subsp. pallidum, i subsp. pertenue)) and nearest non-target relatives (e.g., oral / commensal treponemes such as T. socranskii). Primer / probe design was then constrained by amplicon length suitable for duplex real-time PCR, as previously described by Rios et al. (2018). For the pan-pathogenic Treponema target (Target General; TG) labeled in Cy5, the selected primerprobe set mapped within the 16S rRNA gene, which is highly conserved across the pathogenic T. pallidum lineages. Notably, it possesses multiple mismatches relative to non-target treponemes and off-target bacterial taxa (Figure 31 A-B). An evaluation of the pathogenic panel demonstrated that the forward primer and probe binding sites have complete identity to the included species, for a value of 100% in silico inclusivity. (Figure 3 IB). To discriminate T. pallidum subsp. pallidum from other pathogenic Treponema, an additional primer-probe set labeled with FAM was designed against a pathogenicity-island region that includes the rplm gene, unique to subsp. pallidum and absent from the pertenue and endemicum subspecies (Jaiswal et al. 2020) (Figure 31). Once again, in silico analysis of a rplm alignment showed 100% identity within the intended subspecies, whereas non-target pathogenic treponemes and commensals lack this gene, consistent with high predicted exclusivity for the subgroup-specific target. Thus, the fitness-for-purpose defined for the assay was twofold: (i) clinical detection of pathogenic Treponema and (ii) epidemiologic discrimination of subsp . pallidum from the other pathogenic treponemes. This resulted in a duplex qPCR, complete with an internal control (VIC), which simplifies workflow and reduces cost and turnaround time (Rios et al. 2018). To summarize, all pathogenic Treponema subspecies are detected in Cy5 via the 16S rRNA target, but only venereal syphilis (TPP) is detected in FAM via the rplm target.

[0312] Optimization and Evaluation of Analytical Parameters

[0313] The assay parameters and components most likely to influence performance, including thermal profile, oligonucleotide and magnesium chloride concentrations, were optimized. PCR efficiency directly reflects target-doubling capacity and can be affected by non-specific interactions (e.g., primer-primer or primer-probe), therefore efficiency was used as the primary metric to refine the assay. Different concentration sets for primers, probes, and MgCh and combinations that consistently yielded efficiencies closest to 100% for all targeted regions were selected (Figure 32). Considering that the variation in primers / probe and MgCh concentrationsDocket No. 15947WOO1 / 43913.601 during titration exceeded ranges >10% without substantially affecting the efficiencies nor the linearity (e.g. efficiency and linearity were never lower than 80%), the assay is considered highly robust (Bustin et al. 2025).

[0314] Linear range and Limit of Detection

[0315] The dynamic range was defined as the concentration interval over which the standard curve remained linear, enabling accurate quantification. Consistent with recommendations for microbial qPCR (Kralik & Ricchi, 2017), the linear range extended from 109copies / |1L down to 10 copies / pL (Figure 33A-B). Limit of Detection (LoD) was defined here as the smallest nucleic acid concentration detected with > 95% probability without the action of interfering substances (Forootan et al., 2017). Thus, each target region was first evaluated in nuclease-free water, testing 10 replicates per dilution and estimating LoD by probit analysis. The resulting LoDs were comparable: the an-Treponema target and the subsp. / / i / ii-specific target were 2.15 copies / reaction and 1 copy / reaction, respectively (Figure 33C-D).

[0316] Matrix Effect and Analytical Sensitivity

[0317] Clinical matrices often contain PCR inhibitors such as heme, polysaccharides, and preservatives, which can impair nucleic acid amplification and must therefore be evaluated for the intended diagnostic context (Perez et al. 2012). To assess potential matrix effects, two clinically relevant specimen types were tested: (i) a swab transport medium representative of lesion exudates from primary syphilis, and (ii) human serum representative of secondary or early latent infection. The T. pallidum pallidum Nichols strain was serially diluted in each matrix at defined titers (105-10° copies / mL) and processed end-to-end in 15 replicates, from nucleic acid extraction through amplification, on both Abbott automated platforms (m2000 and Alinity m). Under the finalized duplex qPCR conditions, neither matrix caused a measurable loss of amplification efficiency or linearity (Table 29). Amplification efficiencies remained within the accepted range (90-110%), Ct values for all concentrations were < 40 cycles, and internal control (IC) signals were stable, indicating the absence of detectable inhibition. These results confirm that the duplex assay performs equivalently across both specimen types and is suitable for diagnostic workflows addressing primary and secondary syphilis.

[0318] Analytical sensitivity (ASe) is defined as the matrix-specific lowest concentration detected with > 95% probability (Forootan et al. 2017). Based on probit analysis of 30 replicates per dilution, the estimated L0D95 values were approximately 3 copies / reaction for swab transport medium and 4 copies / reaction for serum (Table 29). The difference between matrices was not statistically significant; therefore, a conservative LoD95 of 4 copies / reaction was adopted for the duplex assay irrespective of matrix. Both rpml (FAM) and 16S rRNADocket No. 15947WOO1 / 43913.601(Cy5) targets demonstrated near-perfect linearity (R2> 0.999) and optimal amplification efficiency, confirming minimal matrix interference and highly consistent duplex assay performance across the evaluated sample types.

[0319] Analytical Specificity Evaluation

[0320] Analytical specificity was assessed according to three components: inclusivity, exclusivity, and selectivity, to evaluate the assay’s ability to correctly discriminate Treponema pallidum subspecies pallidum without cross-reactivity to non-target organisms or interference from clinical matrices.

[0321] Inclusivity

[0322] The duplex, dual-color qPCR assay demonstrated complete inclusivity for the T. pallidum complex. The reference strain (Nichols) was consistently detected in the pathogenic Treponema channel targeting 16S rRNA (Cy5), confirming detection of the pathogenic-level signature. In parallel, the subspecies-specific channel targeting rpml (FAM) accurately discriminated T. pallidum subsp. pallidum, the etiologic agent of venereal syphilis, without loss of sensitivity relative to the pan-target. Other pathogenic treponemes, including T. denticola ATCC 35405 (oral spirochete associated with periodontal disease), T. denticola ATCC 35404, and T. phagedenis ATCC 27087 (a nonvenereal Treponema species isolated from a patient with syphilis-like lesions), produced amplification exclusively in the 16S rRNA Cy5-channel but not in the rpml FAM-channel (Figure 34; Table 31), yielding 100% concordance with expected results and confirming assay inclusivity and accurate subspecies discrimination.

[0323] Exclusivity

[0324] The assay was further challenged against a comprehensive exclusivity panel comprised of non-pathogenic treponemes, and a broad selection of unrelated bacterial and viral human pathogens (Table 33). Non-pathogenic Treponema species including T. sp. OMZ 804 (ATCC 700766, isolated from human dental plaque), T. sp. OMZ 838, and T. sp. TSD-166 yielded no amplification in either assay channel (Figure 34, Table 30). Likewise, no amplification was detected across all non-Treponema bacteria, viral isolates, or matrix controls. All no-template controls (NTCs) remained negative across experiments, confirming the absence of background signal and non-specific amplification.

[0325] Selectivity (matrix interference)

[0326] To assess potential inhibition from clinical sample matrices alone, nucleic acid extracts from T. pallidum-negative swab transport medium (UTM) and serum were tested in 1 Oxreplicates. Neither matrix produced false-positive amplification in the pan-target or discriminatory channels. Internal control (IC) amplification occurred consistently within theDocket No. 15947WOO1 / 43913.601 expected Ct range, confirming that assay performance was unaffected by matrix-derived inhibitors. Collectively, these findings demonstrate that the duplex qPCR assay exhibits high analytical specificity, with no evidence of cross-reactivity or matrix interference.

[0327] Precision (repeatability and intermediate precision)

[0328] Assay precision was evaluated in accordance with ISO / CLSI recommendations for quantitative molecular methods and MIQE reporting standards (Bustin et al. 2025). Repeatability (within-run precision) and intermediate precision (between-run / operators) were assessed using 10 replicate reactions per target and per concentration across two independent runs. The duplex qPCR demonstrated excellent consistency under identical run conditions. For the rpml target (FAM channel), repeatability coefficients of variation (CVs, based on Ct) ranged from 0.78% to 1.37%, and for the 16S rRNA target (Cy5 channel), CVs ranged from 0.47% to 1.52% (Table 31). These low CVs confirm minimal variability among replicate measurements, reflecting robust assay optimization and stable fluorescence detection across concentrations. Intermediate precision, comparing results from two operators on separate days, was also tight between experiments. Combined data yielded reproducibility CVs ranging from 0.95% to 1.42% for rpml and 1.20% to 1.90% for 16S rRNA. All CVs were below 3%, indicating negligible inter-run variability and meeting typical acceptance criteria for clinical molecular assays (Perez et al. 2020). These findings support the assay’s suitability for routine diagnostic use and compliance with MIQE guidelines for precision reporting (Bustin et al. 2025).

[0329] Diagnostic performance assessed by Bayesian latent-class analysis

[0330] Two cohorts of patient samples were evaluated. Population 1 (n = 17) consists of vendor-confirmed TPP positive swabs (genital, penis, anus, urethral, lesions) in UTM from individuals in France. All 17 samples were detected in both the Cy5 and FAM channels, with nearly identical Cts. Notably, three samples flagged by the vendor as having Tow positivity level’ all yielded Cts ranging from 34-38 with the duplex assay. Population 2 (n = 540) specimens were obtained initially to screen patients for HSV-1 and HSV-2 and had never been evaluated previously for syphilis. Here, they were tested with the duplex qPCR (Test A) and compared to results when using primers and probes from a published comparator assay (Test B) (Heymans et al. 2010). Test A detected 49 positives.

[0331] Diagnostic sensitivity (DSe) and specificity (DSp) of the duplex Treponema qPCR were estimated using a two-population, two-test Bayesian latent class model (LCM) under conditional independence. Population 1 positive Treponema status anchored assay sensitivity, while Population 2 (unknown Treponema status) contributed to joint estimation of both DSeDocket No. 15947WOO1 / 43913.601 and DSp, using informative Beta priors derived from the published comparator assay of Heymans et al. (2010, J Clin Microbiol 48:4605-4609). Posterior estimates were explored as a function of the assumed overlap of joint positives in Population 2 (A+Cl B+), spanning the full feasible range (0-30). Across this range, median posterior estimates for the duplex qPCR remained high, with DSe = 0.905-0.966 and DSp = 0.907-0.972. Credible intervals narrowed as the overlap increased, reflecting reduced posterior uncertainty at higher joint-positive counts. For example, when overlap = 0, DSe = 0.711-0.985 and DSp = 0.882-0.929; when overlap = 30, DSe = 0.890-0.995 and DSp = 0.951-0.990 (95% CrI). At the current working overlap (yn2= 30), posterior medians (95% CrI) were DSe = 0.966 (0.892-0.995) and DSp = 0.973 (0.952-0.989), with an estimated prevalence for Population 2 of 0.071 (0.049-0.099). These findings indicate consistently high diagnostic accuracy of the duplex assay across all feasible joint-positive scenarios, with results comparable to or exceeding those of the published reference assay. In summary, the Bayesian LCM analysis supports robust diagnostic performance of the duplex qPCR, with both DSe and DSp exceeding 95% under plausible data structures. This performance profile meets expectations for molecular diagnostic validation in the absence of a perfect reference standard ((FDA) 2010).

[0332] Table 29. Matrix Effect on the duplex-qPCR assay performance across both targets.Target 1: rpml Target 1: rpml Target 2: 16S rRNA Target 2: 16S rRNAParameter _ (UTM) (Serum) (UTM) (Serum)Amplification efficiency101.4 104.6 101.3 100.3(%)Linearity (R2) 0.999 0.9993 0.999 0.9997Analytical sensitivity 11.29 16.57 8.97 2.13(copies / mL)Analytical sensitivity 2.87 4.22 2.28 0.54(copies / reaction)

[0333] UTM: universal transport medium; Serum: human serum matrix. Analytical sensitivity reflects the 95% limit of detection (LoD) estimated from the probit function as described in Material and Methods section obtained from the extrapolation across the independent dilution series.

[0334] Table 30. Evaluation of analytical specificity.Species Target (rplm) FAM Result Target (16 S rRNA) Cy5 ResultParechovirus A Negative NegativeAdeno-associated dependovirus A Negative NegativeRotavirus A Negative NegativeDocket No. 15947WOO1 / 43913.601Human alphaherpesvirus 3 Negative NegativeHuman mastadenovirus C Negative NegativeHuman polyomavirus 1 Negative NegativeCardiovirus A Negative NegativeHuman mastadenovirus E Negative NegativeHuman immunodeficiency vims 1 Negative NegativeChlamydia trachomatis Negative NegativeHepatitis B Vims Negative NegativeHepatitis C Vims Negative NegativeHepatitis Delta Virus Negative NegativeHuman Immunodeficiency Virus Negative NegativeSARS-CoV-2 Negative NegativeTreponema pallidum subsp. pallidum Positive PositiveTreponema denticola Negative PositiveTreponema denticola Negative PositiveTreponema phagedenis Negative PositiveTreponema sp. OMZ 804 Negative NegativeTreponema sp. OMZ 838 Negative NegativeTreponema sp. TSD-166 Negative Negative

[0335] Table 31. Precision Summary for duplex-qPCR assay showing repeatability and reproducibility across low, medium, and high analyte (template) concentrations.

[0336] Notes: n = 20 replicates per concentration per run. Repeatability = within-run precision.Reproducibility = combined within- and between-run precision (pooled across Run 1 and Run 2). CV is reported as the range across concentrations for each row; per-concentration CVs are computed on Ct as 100><SD / Mean Ct.

[0337] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.Docket No. 15947WOO1 / 43913.601

[0338] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.

[0339] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:

[0340] Clause 1. A composition comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the first probe comprises a first detectable label that generates a first signal when the first probe and / or primer pair hybridize to and amplify the first target bacterial nucleic acid sequence; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the at least the second probe comprises a second detectable label that generates a second signal when the at least the second probe and / or primer pair hybridize to and amplify the at least the second target bacterial nucleic acid sequence.

[0341] Clause 2. A method for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the method comprising: (a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises the composition of clause 1; (b) detecting the Treponema pallidum bacteria in the sample if the nucleic acid amplification assay generates the first signal; and (c) distinguishing the subspecies of Treponema pallidum present in the sample if the nucleic acid amplification assay generates the second target second signal.

[0342] Clause 3. A nucleic acid amplification assay for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the assay comprising: (a) the composition of clause 1; (b) a reagents for amplifying the first and at least the second target bacterial nucleic acid sequences, if present, in a sample obtained from a subject; and (c) a system for amplifying and detecting the first and at least the second target bacterial nucleic acid sequences using the composition of (a) and the reagents of (b).

[0343] Clause 4. A kit for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the kit comprising: (a) the composition of clause 1; and (c) instructions for performing a nucleic acid amplification assayDocket No. 15947WOO1 / 43913.601 for detecting the Treponema pallidum bacteria in the sample and distinguishing the subspecies of Treponema pallidum present in the sample.

[0344] Clause 5. A method for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the method comprising: (a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises: (i) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (ii) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; (b) detecting the Treponema pallidum bacteria in the sample if the nucleic acid amplification assay generates the first signal; and (c) distinguishing the subspecies of Treponema pallidum present in the sample if the nucleic acid amplification assay generates the second target second signal.

[0345] Clause 6. A nucleic acid amplification assay for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the assay comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample.

[0346] Clause 7. A kit for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the kit comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum,' and (c) instructions for performing a nucleic acid amplification assay for detecting the TreponemaDocket No. 15947WOO1 / 43913.601 pallidum bacteria in the sample and distinguishing the subspecies of Treponema pallidum present in the sample.

[0347] Clause 8. A method for identifying a subject as more likely than not having venereal syphilis, the method comprising: (a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises: (i) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across Treponema pallidum subspecies pallidum, pertenue, and endemicum, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (ii) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; (b) identifying the subject has more likely than not having venereal syphilis if the nucleic acid amplification assay generates both the first signal and the second signal.

[0348] Clause 9. A nucleic acid amplification assay for identifying a subject as more likely than not having venereal syphilis, the assay comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, pertenue, and endemicum, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; and (c) a nucleic acid amplification system for amplifying the first and second target bacterial nucleic acid sequences, if present in the sample, using the first probe and / or primer pair and at least the second probe and / or primer pair.

[0349] Clause 10. A kit for identifying a subject as more likely than not having venereal syphilis, the kit comprising: (a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, pertenue, and endemicum,' and (b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum,' and (c) instructions for performing a nucleic acid amplification assay for identifying the subjectDocket No. 15947WOO1 / 43913.601 has more likely than not having venereal syphilis if the nucleic acid amplification assay generates both a first and second signal due to the amplification of the first and second target bacterial nucleic acid sequences within a sample obtained from the subject.

[0350] Clause 11. The composition of clause 1, method of any one of clauses 2, 5 and 8, assay of any one of clauses 3, 6, and 9, or kit of any one of clauses 3, 6, and 9, further comprising: (i) recommending a treatment for venereal syphilis if the subject is indicated as having or identified as more likely than not having venereal syphilis; (ii) recommending a treatment for yaws if the subject is indicated as having or identified as more likely than not as having yaws; or (iii) recommending a treatment for bejel if the subject is indicated as having or more likely than not has bejel.

[0351] Clause 12. The composition of clause 1 or 11, method of any one of clauses 2, 5, 8 and 11, assay of any one of clauses 3, 6, 9 and 11, or kit of any one of clauses 4, 7 and 10-11, wherein the subspecies are selected from the group consisting of T. pallidum pallidum (TP A), T. pallidum endemicum (TEN), and T. pallidum pertenue (TPE).

[0352] Clause 13. The composition of any one of clauses 1 and 11-12, method of any one of clauses 2, 5, 8 and 11-12, assay of any one of clauses 3, 6, 9 and 11-12, or kit of any one of clauses 4, 7 and 10-12, wherein the first target bacterial nucleic sequence comprises a region of the 16s rRNA gene that is conserved across the subspecies.

[0353] Clause 14. The composition of any one of clauses 1 and 11-13, method of any one of clauses 2, 5, 8 and 11-13, assay of any one of clauses 3, 6, 9 and 11-13, or kit of any one of clauses 4, 7 and 10-13, wherein the conserved nucleic acid comprises a pathogenicity island for Treponema pallidum.

[0354] Clause 15. The composition of any one of clauses 1 and 11-14, method of any one of clauses 2, 5, 8 and 11-14, assay of any one of clauses 3, 6, 9 and 11-14, or kit of any one of clauses 4, 7 and 10-14, wherein the first probe hybridizes to a polynucleotide sequencing corresponding to nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene.

[0355] Clause 16. The composition of any one of clauses 1 and 11-15, method of any one of clauses 2, 5, 8 and 11-15, assay of any one of clauses 3, 6, 9 and 11-15, or kit of any one of clauses 4, 7 and 10-15, wherein the first probe comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that differs from SEQ ID NO: 1 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

[0356] Clause 17. The composition of any one of clauses 1 and 11-16, method of any one of clauses 2, 5, 8 and 11-16, assay of any one of clauses 3, 6, 9 and 11-16, or kit of any one of clauses 4, 7 and 10-16, wherein the first primer pair comprises a forward primer and a reverseDocket No. 15947WOO1 / 43913.601 primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene.

[0357] Clause 18. The composition of any one of clauses 1 and 11-17, method of any one of clauses 2, 5, 8 and 11-17, assay of any one of clauses 3, 6, 9 and 11-17, or kit of any of one of clauses 4, 7 and 10-17, wherein the first primer pair comprises a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 or nucleotide sequences that differ from SEQ ID NO: 2 and SEQ ID NO: 3 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

[0358] Clause 19. The composition of any one of clauses 1 and 11-18, method of any one of clauses 2, 5, 8 and 11-18, assay of any one of clauses 3, 6, 9 and 11-18, or kit of any of one of clauses 4, 7 and 10-18, wherein the second target bacterial nucleic acid sequence comprises a region of the 50SL35 gene that is unique for one subspecies of Treponema pallidum.

[0359] Clause 20. The composition of any one of clauses 1 and 11-19, method of any one of clauses 2, 5, 8 and 11-19, assay of any one of clauses 3, 6, 9 and 11-19, or kit of any of one of clauses 4, 7 and 10-19, wherein the second probe hybridizes to a polynucleotide sequence corresponding nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene.

[0360] Clause 21. The composition of any one of clauses 1 and 11-20, method of any one of clauses 2, 5, 8 and 11-20, assay of any one of clauses 3, 6, 9 and 11-20, or kit of any of one of clauses 4, 7 and 10-20, wherein the second probe comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence that differs from SEQ ID NO: 4 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

[0361] Clause 22. The composition of any one of clauses 1 and 11-21, method of any one of clauses 2, 5, 8 and 11-21, assay of any one of clauses 3, 6, 9 and 11-21, or kit of any of one of clauses 4, 7 and 10-21, wherein the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene.

[0362] Clause 23. The composition of any one of clauses 1 and 11-22, method of any one of clauses 2, 5, 8 and 11-22, assay of any one of clauses 3, 6, 9 and 11-22, or kit of any of one of clauses 4, 7 and 10-22, wherein the second primer pair comprises a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 or nucleotide sequences that differ from SEQ ID NO: 5 and SEQ ID NO: 6 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.Docket No. 15947WOO1 / 43913.601

[0363] Clause 24. The composition of any one of clauses 1 and 11-23, method of any one of clauses 2, 5, 8 and 11-23, assay of any one of clauses 3, 6, 9 and 11-23, or kit of any of one of clauses 4, 7 and 10-23, wherein the subspecies is identified as subspecies T. pallidum pallidum if the first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pallidum.

[0364] Clause 25. The composition, method, assay, or kit of clause 24, wherein identification of the presence of the subspecies T. pallidum pallidum in the sample is indicative that the subject from whom the sample was obtained has venereal syphilis.

[0365] Clause 26. The composition of any one of clauses 1 and 11-18, method of any one of clauses 2, 5, 8 and 11-18, assay of any one of clauses 3, 6, 9 and 11-18, or kit of any of one of clauses 4, 7 and 10-18, wherein the at least the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum pertenue.

[0366] Clause 27. The composition, method, assay, or kit of any clause 26, wherein the subspecies is identified as subspecies T. pallidum pertenue if the first signal is generated and at least a second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pertenue.

[0367] Clause 28. The composition of any one of clauses 1 and 11-18, method of any one of clauses 2, 5, 8 and 11-18, assay of any one of clauses 3, 6, 9 and 11-18, or kit of any of one of clauses 4, 7 and 10-18, wherein the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum endemicum.

[0368] Clause 29. The composition, method, assay, or kit clause 28, wherein the subspecies is identified as subspecies T. pallidum endemicum if the first signal is generated and at least a second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum endemicum.

[0369] Clause 30. The composition of any one of clauses 1 and 11-18, method of any one of clauses 2, 5, 8 and 11-18, assay of any one of clauses 3, 6, 9 and 11-18, or kit of any of one of clauses 4, 7 and 10-18, wherein the at least the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum.Docket No. 15947WOO1 / 43913.601

[0370] Clause 31. The composition, method, assay, or kit of clause 30, wherein the subspecies is identified as subspecies T. pallidum endemicum or T. pallidum pertenue if the first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in T. pallidum pallidum.

[0371] Clause 32. The composition, method, assay, or kit of any one of clauses 26-31, wherein identification of the presence of the subspecies T. pallidum endemicum in the sample is indicative that the subject from whom the sample was obtained has bejel.

[0372] Clause 33. The composition, method, assay, or kit of any one of clauses 26-31, wherein identification of the presence of the subspecies T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained has yaws.

[0373] Clause 34. The composition, method, assay, or kit of any one of clauses 26-33, wherein identification of the presence of the subspecies T. pallidum endemicum or T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained does not have venereal syphilis.

[0374] Clause 35. The composition, method, assay, or kit of any one of the preceding clauses, wherein the first probe comprises a first detectable label.

[0375] Clause 36. The composition, method, assay, or kit of any one of the preceding clauses, wherein the second probe comprises a second detectable label.

[0376] Clause 37. The composition, method, assay, or kit of any one of the preceding clauses, wherein the first signal is generated by a first detectable label.

[0377] Clause 38. The composition, method, assay, or kit of any one of the preceding clauses, wherein the second signal is generated by a second detectable label.

[0378] Clause 39. The composition, method, assay, or kit of any one clauses 35-38, wherein the first and second detectable label respectively comprise a first fluorescent dye and a second fluorescent dye that is different from the first fluorescent dye.

[0379] Clause 40. The composition, method, assay, or kit of any one of the clauses 35-39, wherein the first and second fluorescent dye are selected from the group consisting of CY5 and FAM.

[0380] Clause 41. The composition, method, assay, or kit of any one of the preceding clauses, wherein amplifying or amplification comprises qualitative PCR (qPCR).Docket No. 15947WOO1 / 43913.601REFERENCES Aho, J., et al. (2022), 'Rising syphilis rates in Canada, 2011-2020', Can Commun Dis Rep, 48 (23), 52-60. Anderson, M., et al. (2024), 'Development and Validation of Three Automated High- Throughput Molecular Tests to Detect Monkeypox Virus Infections', J Infect Dis, 229 ( Suppl ement_2), S137-s43. Arnold, S. R. and Ford-Jones, E. L. (2000), 'Congenital syphilis: A guide to diagnosis and management', Paediatr Child Health, 5 (8), 463-9. Branscum, A. J., Gardner, I. A., and Johnson, W. O. (2005), 'Estimation of diagnostictest sensitivity and specificity through Bayesian modeling', Prev Vet Med, 68 (2-4), 145-63. Bustin, S. A., et al. (2025), 'MIQE 2.0: Revision of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments Guidelines', Clin Chem. 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(2020), 'The pan-genome of Treponema pallidum reveals differences in genome plasticity between subspecies related to venereal and non- venereal syphilis', BMC Genomics, 21 (1), 33. Kielaite, D., Januskiene, S., and Paliulyte, V. (2025), 'Syphilis Infection During Pregnancy: The Possible Effect on the Course of Pregnancy and Fetal Outcomes-A Case Report and Literature Review', Biomedicines, 13 (1). Kralik, P. and Ricchi, M. (2017), 'A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything', Front Microbiol, 8, 108. Kriesel, John D., et al. (2015), 'Multiplex PCR testing for nine different sexually transmitted infections', International Journal of STD & AIDS, 27 (14), 1275-82. LEI, Z-S;., et al. (2013), 'Evaluation of nPCR for detection of Treponema pallidum in primary syphilis', Progress in Modem Biomedicine, 13 (23), 3. LaFond Rebecca, E. and Lukehart Sheila, A. 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(2017), 'Validation of optimal reference genes for quantitative real time PCR in muscle and adipose tissue for obesity and diabetes research', Sci Rep, 7 (1), 3612.Docket No. 15947WOO1 / 43913.601 Perez, L. J., et al. (2020), 'A Validated Multiplex Real-Time PCR Assay for the Diagnosis of Infectious Leptospira spp.: A Novel Assay for the Detection and Differentiation of Strains From Both Pathogenic Groups I and If, Front Microbiol, 11, 457. Perez, L. J., et al. (2012), 'An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus', Mol Cell Probes, 26 (3), 137-45. Rios, L., et al. (2018), 'Multi-Target Strategy for Pan / Foot-and-Mouth Disease Virus (FMDV) Detection: A Combination of Sequences Analysis, in Silico Predictions and Laboratory Diagnostic Evaluation', Front Vet Sci, 5, 160. Robayo-Amortegui, H., et al. (2024), 'Endocarditis and ascending aortic aneurysm with aortic valve insufficiency secondary to late syphilis: Case report', Sci Prog, 107 (4), 368504241308956. Rosset, F., et al. (2025), 'The Epidemiology of Syphilis Worldwide in the Last Decade', J Clin Med, 14 (15). Salazar, Juan C., Hazlett, Karsten R. O., and Radolf, Justin D. (2002), 'The immune response to infection with Treponema pallidum, the stealth pathogen', Microbes and Infection, 4 (11), 1133-40. Salle, R., et al. (2022), 'Specific detection of Treponema pallidum in clinical samples: validation of a qPCR assay combining two genomic targets', Sex Transm Infect. Salome, S., et al. (2024), 'Congenital Syphilis: A Re-Emerging but Preventable Infection', Pathogens, 13 (6). Smith, J. L., et al. (1971), 'Neuro-ophthalmological study of late yaws and pinta. II. The Caracas project', Br J Vener Dis, 47 (4), 226-51. Scott, L. J., et al. (2010), 'A new multiplex real-time PCR test for HSV1 / 2 and syphilis: an evaluation of its impact in the laboratory and clinical setting', Sex Transm Infect, 86 (7), 537-9. Shields, M., et al. (2012), 'A longitudinal evaluation of Treponema pallidum PCR testing in early syphilis', BMC Infect Dis, 12, 353. Tipple, C., et al. (2011), 'Getting the measure of syphilis: qPCR to better understand early infection', Sex Transm Infect, 87 (6), 479-85.Docket No. 15947WOO1 / 43913.601 Vaks, J. E., et al. (2016), 'Verification of Claimed Limit of Detection in Molecular Diagnostics', J Appl Lab Med, 1 (3), 260-70. Vanhaecke, C., et al. (2016), 'Clinical and Biological Characteristics of 40 Patients With Neurosyphilis and Evaluation of Treponema pallidum Nested Polymerase Chain Reaction in Cerebrospinal Fluid Samples', Clin Infect Dis, 63 (9), 1180-86. Wang, C., et al. 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Claims

Docket No. 15947WOO1 / 43913.601ClaimsWhat is claimed is:

1. A method for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the method comprising:(a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises:(i) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and(ii) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample;(b) detecting the Treponema pallidum bacteria in the sample if the nucleic acid amplification assay generates the first signal; and(c) distinguishing the subspecies of Treponema pallidum present in the sample if the nucleic acid amplification assay generates the second target second signal.

2. A nucleic acid amplification assay for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the assay comprising:(a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and(b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample.

3. A kit for detecting and distinguishing between Treponema pallidum subspecies in a sample obtained from a subject in need thereof, the kit comprising:Docket No. 15947WOO1 / 43913.601(a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies,; and(b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, ' and(c) instructions for performing a nucleic acid amplification assay for detecting the Treponema pallidum bacteria in the sample and distinguishing the subspecies of Treponema pallidum present in the sample.

4. The method of claim 1, assay of claim 2, or kit of claim 3, wherein the subspecies are selected from the group consisting of T. pallidum pallidum (TP A), T. pallidum endemicum (TEN), and T. pallidum pertenue (TPE).

5. The method of claim 1 or 4, assay of claim 2 or 4, or kit of claim 3 or 4, wherein the first target bacterial nucleic sequence comprises a region of the 16s rRNA gene that is conserved across the subspecies.

6. The method of any one of claims 1, 4 and 5, assay of any one of claims 2, 4 and5, or kit of any one of claims 3-5, wherein the conserved nucleic acid comprises a pathogenicity island for Treponema pallidum.

7. The method of any one of claims 1 and 4-6, assay of any one of claims 2 and 4-6, or kit of any one of claims 3-6, wherein the first probe hybridizes to a polynucleotide sequencing corresponding to nucleotides 103892 to 1038945 in the T. pallidum pallidum 16s rRNA gene.

8. The method, assay or kit of claim 7, wherein the first probe comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence that differs from SEQ ID NO: 1 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

9. The method, assay or kit of claim 7, wherein the first primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequencesDocket No. 15947WOO1 / 43913.601 corresponding to nucleotides 1038895 to 1038914 and nucleotides 1038949 to 1038971 in the T. pallidum pallidum 16s rRNA gene.

10. The method, assay or kit of claim 8 or 9, wherein the first primer pair comprises a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 2 and SEQ ID NO: 3 or nucleotide sequences that differ from SEQ ID NO: 2 and SEQ ID NO: 3 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

11. The method, assay or kit of any one of claims 1-10, wherein the second target bacterial nucleic acid sequence comprises a region of the 50SL35 gene that is unique for one subspecies of Treponema pallidum.

12. The method, assay or kit of claim 11, wherein the second probe hybridizes to a polynucleotide sequence corresponding nucleotides 919564 to 919581 in the T. pallidum pallidum 50SL35 gene.

13. The method, assay or kit of claim 11 or 12, wherein the second probe comprises the nucleotide sequence of SEQ ID NO: 4 or a nucleotide sequence that differs from SEQ ID NO: 4 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.

14. The method, assay or kit of any one of claims 11-13, wherein the second primer pair comprises a forward primer and a reverse primer that respectively hybridize to polynucleotide sequences corresponding to nucleotides 919532 to 919551 and nucleotides 919594 to 919613 in the T. pallidum pallidum 50SL35 gene.

15. The method, assay or kit of any one of claims 12-14, wherein the second primer pair comprises a forward primer and a reverse primer respectively comprising the nucleotide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 or nucleotide sequences that differ from SEQ ID NO: 5 and SEQ ID NO: 6 by no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 nucleotides.Docket No. 15947WOO1 / 43913.60116. The method, assay, or kit of any one of claims 1-15, wherein the subspecies is identified as subspecies T. pallidum pallidum if the first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pallidum.

17. The method, assay, or kit of claim 16, wherein identification of the presence of the subspecies T. pallidum pallidum in the sample is indicative that the subject from whom the sample was obtained has venereal syphilis.

18. The method, assay, or kit of any one of claims 1-10, wherein the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum pertenue.

19. The method, assay, or kit of any one of claims 1-10 and 18, wherein the subspecies is identified as subspecies T. pallidum pertenue if the first signal is generated and at least a second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum pertenue.

20. The method, assay, or kit of any one of claims 1-10, wherein the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is unique for the subspecies T. pallidum endemicum.

21. The method, assay, or kit of any one of claims 1-10 and 20, wherein the subspecies is identified as subspecies T. pallidum endemicum if the first signal is generated and at least a second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is unique for the subspecies T. pallidum endemicum.

22. The method, assay, or kit of any one of claims 1-10 and claims 18-21, wherein the second target bacterial nucleic acid sequence comprises a sequence within a pathogenicity island that comprises a gene that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in the subspecies T. pallidum pallidum.

23. The method, assay, or kit of any one of claims 1-10 and 18-22, wherein the subspecies is identified as subspecies T. pallidum endemicum or T. pallidum pertenue if theDocket No. 15947WOO1 / 43913.601 first signal is generated and at least second signal is generated due to the amplification of a second target bacterial nucleic acid sequence that is shared between the subspecies T. pallidum endemicum and T. pallidum pertenue but absent in T. pallidum pallidum.

24. The method, assay, or kit of any one of claims 1-10 and 18-23, wherein identification of the presence of the subspecies T. pallidum endemicum in the sample is indicative that the subject from whom the sample was obtained has bejel.

25. The method, assay, or kit of any one of claims 1-10 and 18-24, wherein identification of the presence of the subspecies T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained has yaws.

26. The method, assay, or kit of any one of claims 1-10 and 18-25, wherein identification of the presence of the subspecies T. pallidum endemicum or T. pallidum pertenue in the sample is indicative that the subject from whom the sample was obtained does not have venereal syphilis.

27. A method for identifying a subject as more likely than not having venereal syphilis, the method comprising:(a) performing a nucleic acid amplification assay in a sample obtained from the subject, wherein the nucleic acid amplification assay comprises:(i) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across Treponema pallidum subspecies pallidum, pertenue , and endemicum, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample; and(ii) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; and(b) identifying the subject has more likely than not having venereal syphilis if the nucleic acid amplification assay generates both the first signal and the second signal.Docket No. 15947WOO1 / 43913.60128. A nucleic acid amplification assay for identifying a subject as more likely than not having venereal syphilis, the assay comprising:(a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, pertenue, and endemicum, wherein the nucleic acid amplification assay generates a first signal if the first target bacterial nucleic acid is present in the sample;(b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum, wherein the nucleic acid amplification assay generates at least a second signal if the second target bacterial nucleic acid is present in the sample; and(c) a nucleic acid amplification system for amplifying the first and second target bacterial nucleic acid sequences, if present in the sample, using the first probe and / or primer pair and at least the second probe and / or primer pair.

29. A kit for identifying a subject as more likely than not having venereal syphilis, the kit comprising:(a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies pallidum, pertenue, and endemicunr,(b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that comprises a gene within a pathogenicity island that is present in Treponema pallidum pallidum,' and(c) instructions for performing a nucleic acid amplification assay for identifying the subject has more likely than not having venereal syphilis if the nucleic acid amplification assay generates both a first and second signal due to the amplification of the first and second target bacterial nucleic acid sequences within a sample obtained from the subject.

30. The method, assay, or kit of any one of claims 1-29, further comprising:(i) recommending a treatment for venereal syphilis if the subject is indicated as having or identified as more likely than not having venereal syphilis;(ii) recommending a treatment for yaws if the subject is indicated as having yaws; or(iii) recommending a treatment for bejel if the subject is indicated as having bejel.Docket No. 15947WOO1 / 43913.60131. A composition comprising:(a) a first probe and / or primer pair that hybridizes to and amplifies a first target bacterial nucleic acid sequence that is conserved across the Treponema pallidum subspecies, wherein the first probe comprises a first detectable label that generates a first signal when the first probe and / or primer pair hybridize to and amplify the first target bacterial nucleic acid sequence; and(b) at least a second probe and / or primer pair that hybridizes to and amplifies at least a second target bacterial nucleic acid sequence that is unique for at least one subspecies of Treponema pallidum, wherein the at least the second probe comprises a second detectable label that generates a second signal when the at least the second probe and / or primer pair hybridize to and amplify the at least the second target bacterial nucleic acid sequence.

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