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WO 2016/188962 Al 1 December 2016 (01.12.2016) P O P C T

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WO 2016/188962 Al 1 December 2016 (01.12.2016) P O P C T (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2016/188962 Al 1 December 2016 (01.12.2016) P O P C T (51) International Patent Classification: (74) Agents: GOODFELLOW, Hugh Robin et al; Carpmaels C12Q 1/68 (2006.01) & Ransford LLP, One Southampton Row, London WC1B 5HA (GB). (21) International Application Number: PCT/EP2016/061599 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) Date: International Filing AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, 23 May 20 16 (23.05.2016) BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (25) Filing Language: English DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, (26) Publication Language: English KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, (30) Priority Data: MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, 1508860.2 22 May 2015 (22.05.2015) GB PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, (71) Applicant: NATIONAL UNIVERSITY OF IRELAND, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. GALWAY [IE/IE]; University Road, Galway (IE). (84) Designated States (unless otherwise indicated, for every (72) Inventors: REDDINGTON, Kate Mary; Deerpack East, kind of regional protection available): ARIPO (BW, GH, Newport Road, Westport, Co. Mayo (IE). TUITE, Nina; GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, 43 Ardilaun Road, Newcastle, Co. Galway (IE). TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, MINOGUE, Elizabeth; Ballinagough, Whitegate, Co. TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, Clare (IE). BARRY, Thomas Gerard; Dunguaire East, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, Kinvara, Co. Galway (IE). LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, [Continued on nextpage] (54) Title: DIAGNOSTIC METHOD FOR BACTERIAL ORGANISMS USING THE SMPB GENE (57) Abstract: A method for determining the presence or ab sence of a member of a group of bacterial organisms in a sample, wherein the method comprises determining whether a target region of the smpB gene is present in said sample is provided. Primers, probes and kits for use in these methods also form part of the invention, as does the use of an smpB gene target region to detect the presence or absence of a member of a group of bacterial organisms in a sample, in a range of clinical and non- clinical applications. TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, ,. - - „ KM ML MR NE SN TD TG) with sequence listing partt of description (Rule 5.2(a)) Published: DIAGNOSTIC METHOD FOR BACTERIAL ORGANISMS USING THE SMPB GENE FIELD OF THE INVENTION The present invention relates to methods for determining the presence or absence of a member of a group of bacterial organisms in a sample. More specifically, the invention relates to methods for determining the presence or absence of a member of a group of bacterial organisms by determining whether a target region of the smpB gene is present in a sample. Primers, probes and kits for use in these methods also form part of the invention, as does the use of an smpB gene target region to detect the presence or absence of a member of a group of bacterial organisms in a sample, in a range of clinical and non-clinical applications. BACKGROUND TO THE INVENTION Bacterial infections are a major cause of disease worldwide. A wide range of bacterial organisms are responsible for infection and there is a need for assays to detect and to identify bacterial organisms. The need extends to detecting and identifying bacterial organisms in a clinical context (e.g. in a sample from a subject or patient) and also to detecting and identifying bacterial organisms in other samples, such as clinical products, food products (including drinking water) and environmental samples. Detecting and identifying bacterial organisms in a clinical context is useful for example to diagnose infection in a subject or patient, whereas identifying bacterial organisms in other samples is also useful to detect and identify bacterial organisms as a source of contamination and as potential causes of disease. The presence of undesirable bacteria in food has the potential to cause serious illness or even death as a consequence of its ingestion by the consumer. Likewise, bacterial contamination of clinical products (e.g. blood products) can have harmful consequences if infection is caused as a result of the use of such products. Further, there is the need to monitor the environment for bacterial contamination. This is of particular concerns in locations such as hospitals where there are people who are vulnerable to infection, or in recreational facilities such as swimming pools and lakes, which may be contaminated with high levels of bacteria. Accordingly, there is a need for detecting and identifying bacterial contamination in numerous products that are consumed or used by humans, as well as in the environment and in detecting and identifying bacterial organisms causing infection in patients/subjects. Traditional assays to detect and identify bacterial organisms commonly rely on microbiological culture methods and biochemical methods to identify the cause of microbial infections. Such methods are often time-consuming, requiring several days to culture the infectious agent before definitive identification can take place. Furthermore, such methods can have poor sensitivity. In a clinical setting this can not only lead to delays in providing treatment, but also cause the wrong treatment to be administered. Nucleic acid based assays to detect and identify bacterial organisms are desirable as not only are they quick to carry out, but also they have the potential to afford a high level of specificity and sensitivity. Nucleic acid based assays to detect and identify bacterial organisms require suitable nucleic acid targets to be present in the bacterial organism of interest. Accordingly, there is a need for the identification of novel molecular targets that can be used for rapid, selective and specific detection and identification of bacteria. A number of nucleic acid targets have been identified for use in bacterial diagnostics assays including the ssrA [1], lepA [2] and 16sRNA genes [3]. Whilst these targets have been useful in the development of assays for certain bacterial organisms, there are limitations associated with these targets owing to the high degree of nucleotide sequence homology across the bacterial kingdom. In other instances, sequence heterogeneity can also pose a problem. For example, the use of 16S rRNA as a diagnostics target has been complicated by the fact that bacteria harbour multiple heterogeneous copies of this gene (ranging from 1-15). Sequence heterogeneity within single bacterial genomes can create a significant problem for diagnostic development. It can also lead to an overestimation of microbial populations present in samples [4,5]. It is an aim of the present invention to identify further nucleic acid targets that can be used in nucleic acid based assays to detect and identify bacterial organisms. Furthermore, the identification of further targets will facilitate the development of multi-target assays that can further increase specificity of assays. For reliable detection and identification of a bacterial organism, the target will preferably be highly specific. An assay is specific if there is low or no cross-reactivity with nucleic acids from other organisms, i.e., the assay does not generate false positive results, or does so with low frequency, and if most or all organisms of interest are identified, i.e. the assay does not generate false negative results or does so with low frequency. The former is defined with respect to "exclusivity" panels, and the latter with respect to "inclusivity" panels. Haemophilus influenza is a Gram-negative, coccobacillary, facultatively anaerobic bacterium which frequently colonises the Upper Respiratory Tract (URT) and is a significant causative agent of Respiratory Tract Infections (RTI) and pneumonia worldwide. There is a requirement for the development of a rapid H. influenzae diagnostics assay which would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients. There is currently no single nucleic acid diagnostics target described in the literature that can unambiguously identify H. influenzae. Accordingly, there is a need for the identification of novel molecular targets that can be used for rapid and specific detection of H. influenzae. Traditional culture and phenotypic based methodologies for the identification of H. influenzae are slow and in many cases cannot differentiate H. influenzae from the closely related H. haemolyticus or other Haemophilus species [6, 7, 8]. In recent years a number of novel molecular based approaches have been described for the detection of H. influenzae including MALDI-TOF mass spectrometry and nucleic acid based diagnostics assays [9, 10, 11, 12]. As mass spectrometers are still relatively expensive and require specialist training they are not available in all diagnostic and clinical laboratories. More importantly, the requirement for bacterial culture prior to analysis by MALDI-TOF MS takes a minimum of one day, often longer. Furthermore, false negative reporting of results can occur if antimicrobial therapy has been administered prior to collection of the clinical samples. Several real-time PCR (RT-PCR) diagnostic assays have been described for H. influenzae. These include those that target the fucK, hpdA, bexA, ompP2, P6 genes, respectively [8,12,13,14].
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