WO2020161651A1

WO2020161651A1 - Early detection of multiple resistances to anti-bacterial treatment

Info

Publication number: WO2020161651A1
Application number: PCT/IB2020/050938
Authority: WO
Inventors: Tsachi TSADOK PERETS
Original assignee: Mor Research Applications Ltd.
Priority date: 2019-02-05
Filing date: 2020-02-05
Publication date: 2020-08-13

Abstract

Method and kits for non-invasive diagnosis of possible multiple mutated DNA-based resistances of H. pylori to distinct anti-H. pylori treatment modalities are provided. The methods and kits are based on the use of molecular multiplex means for profiling H. pylori DNA extracted form stool. Diagnosis methods disclosed herein enable the design of personalized treatment regimens that would be effective in eradicating H. pylori of any genetic profile.

Description

EARLY DETECTION OF MULTIPLE RESISTANCES TO ANTI¬

BACTERIAL TREATMENT

FIELD OF THE INVENTION

The present disclosure relates to diagnosis of multiple resistances of Helicobacter pylori to various, distinct treatment modalities, particularly, but not exclusively, to multiple antibiotic resistances.

BACKGROUND

Helicobacter pylori (H. pylori ) is a spiral-shaped, microaerophilic bacterium that colonizes the mucous layer of the human stomach and is an etiologic agent for peptic and duodenal ulcers, with an estimation of 50% prevalence worldwide. Once established, the pathogen may reside in the majority of carriers for years or decades in the absence of symptoms. Prevalence of infection with H. pylori depends on geography and socioeconomic status. It is estimated that prevalence in Europe ranges from 11% to 60%, while in Asia the numbers are higher, reaching almost 90%. In North America, H. pylori prevalence is around 30%, but in distinct groups, such as the Aboriginal populations living in Canada, the prevalence is estimated as high as 95%.

H. pylori has been identified as a Group 1 carcinogen by the World Health Organization and International Agency for Research on Cancer (WHO/IARC), as it is associated with the development of gastric cancer. The successful eradication of H. pylori infection leads to healing of the peptic ulcer disease and to a long-term relief of dyspeptic symptoms even in some patients without ulcers. Eradication therapy of H. pylori infection is also recommended as the hrst-line treatment for low-grade mucosa-associated lymphoid tissue (MALT) lymphoma and, furthermore, successful H. pylori eradication therapy is an effective strategy for preventing gastric cancer.

Diagnostic methods for the detection of H. pylori infection are divided into invasive (gastroscopy is required) and non-invasive methods. Although several methods give highly accurate results, there is no single gold standard for the diagnosis of H. pylori infection, rather, the selection of diagnosis method depends on the clinical situation and whether there is otherwise a need for gastroscopy. According to the test-and-treat strategy, patients with a low risk for gastric cancer can be tested for H. pylori with non-invasive methods and treated if H. pylori is detected.

Although non-invasive diagnostic methods, such as urea breath tests and stool antigen tests, in general, show highly accurate detection rates, these methods do not give any information on the antimicrobial susceptibility of the infecting isolate of H pylori. Furthermore, even if gastric biopsies are taken in gastroscopy and sent for cell culturing, antimicrobial susceptibility testing results are usually not available in all positive cases due to the low sensitivity of the cell culture. However, the need for antimicrobial susceptibility testing of H. pylori is increasing: in Europe, H. pylori has been demonstrating resistance rates to clarithromycin, metronidazole and levofloxacin of approximately 18%, 35% and 15%, respectively. In China, H. pylori resistance rates to clarithromycin, metronidazole and levofloxacin are approximately 60%, 70% and 55%, respectively. These numbers show a distinct trend of increasing antibiotic resistance of H. pylori. Existence of H. pylori strains resistant to two or more antibiotic in an antibiotic-based regimen may result in eradication failure in most cases.

There is an ongoing need for identifying resistances of H. pylori to specific antibiotic treatments prior to administration of therapy so as to decrease treatment and eradication failure and aid in preventing future complications.

SUMMARY

The present disclosure relates to multiplex diagnostic methods for identification of multiple resistances to distinct anti-// pylori treatment modalities stemming from mutated H. pylori genome. Multiple resistances detectable include, but are not limited to, resistances to the common first-, second-, and/or third-line antibiotic treatments. The methods are non- invasive and based on identifying H. pylori DNA extracted and purified form a stool sample obtained from a symptomatic or a-symptomatic patient.

Diagnosis methods disclosed herein enable the design of specific treatment regimens that would be effective in eradicating H. pylori of any genetic profile. Early assessment of putative resistances to known treatment modalities will prevent the administration of ineffective treatment and, moreover, will enable the tailoring of a subject- specific, i.e., personalized treatment protocol and surveillance that would fit an individual’s particular H. pylori genetic makeup, so as to provide a successful eradication of the pathogen.

The present disclosure relates to a method of diagnosing multiple mutated DNA- based resistances of H. pylori to distinct anti -H. pylori treatment modalities in a subject infected with H. pylori, the method comprising the steps of:

(i) obtaining a stool sample from the subject;

(ii) extracting and purifying bacterial DNA from the stool sample;

(iii) optionally, amplifying the purified bacterial DNA to obtain amplicons of genes of interest;

(iv) subjecting the purified bacterial DNA or the amplicons to gene profiling by the use of one or more multiplex molecular means; and

(v) diagnosing multiple mutated DNA-based resistances of H. pylori to distinct anti-// pylori treatment modalities if multiple mutated genes associated with distinct anti- H. pylori treatment resistances are detected.

A contemplated method for diagnosing multiple anti -H. pylori treatment resistances is useful for diagnosing H. pylori infection combined with early diagnosis of possible multiple mutated DNA-based resistances to various, distinct anti -H. pylori treatment modalities in a subject suspected of being infected with H. pylori. Early diagnosis herein is diagnosing putative resistances to anti-bacterial treatment at the time of first diagnosing H. pylori infection.

The diagnosis, for example, early diagnosis, of anti-bacterial treatment resistances in a subject provides the advantage of decreasing H. pylori infection eradication failure, or, alternatively, increasing H. pylori infection eradication success in a subject, whereby the subject is provided with a treatment protocol for which no mutated DNA-based resistance has been detected.

In some embodiments, a contemplated method provides the identification or diagnosis of mutated DNA-based resistances to at least two distinct anti -H. pylori treatment modalities. In some embodiments, at least one of two or more mutated DNA-based resistances is resistance to treatment with a first-line, second-line, and/or third-line state of the art antibiotics such as, but not limited to, clarithromycin, metronidazole, levofloxacin, tetracycline, and/or amoxicillin.

Non-limiting examples of mutated H. pylori genes that may confer antibiotic resistances, detectable by a contemplated method, include the genes 235 rRNA, gyrA,frxA, rdxA or rpsU. It is noted that a contemplated method is suitable for detection of any anti bacterial treatment resistances originating from any mutations yet to be discovered.

For example, a contemplated method provides the detection of simultaneous resistances to treatment with clarithromycin, metronidazole and levofloxacin; simultaneous resistances to treatment with clarithromycin, and metronidazole; simultaneous resistances to treatment with clarithromycin and levofloxacin; simultaneous resistances to treatment with metronidazole, tetracycline and levofloxacin; or resistances to treatment with clarithromycin, amoxicillin and levofloxacin.

The multiplex molecular means used in accordance with a contemplated method may be, for example, a DNA microarray or multiplex PCR. Non-limiting examples of DNA microarray systems include printed microarrays, in .v/Yu-synthesized microarrays, high- density bead arrays, electronic microarrays and suspension bead microarrays. In some embodiments, an electronic microarray is utilized for DNA profiling.

It is noted that any of the multiplex genome profiling means used in accordance with a contemplated method is specifically designed to suit the prevalence of mutations conferring resistances to anti-// pylori treatment modalities in specific geographic regions or ethnic populations.

The present disclosure relates to a kit for diagnosis of multiple mutated DNA-based resistances of H. pylori to distinct anti -H. pylori treatment modalities, comprising: (a) means and/or reagents to conduct multiplex genome profiling; (b) detection means; (c) optionally, means and reagents to extract and purify bacterial DNA from a stool sample; and (d) optionally, written instructions.

In exemplary embodiments, a contemplated kit is designed for utilization of an electronic microarray system for DNA profiling and in accordance with these embodiments, the kit comprises: (a) an activated microelectronic cartridge comprising capture probes designed to promote specific hybridization of target H. pylori genes of interest; and (b) fluorescent reporter oligonucleotides. Further embodiments and the full scope of applicability of the present disclosure will become apparent from the detailed description given hereinafter. It should be understood that the detailed description and specific examples are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION

The present disclosure relates, at least in part, to the diagnosis of multiple resistances of Helicobacter pylori to various and distinct treatment modalities, particularly, but not exclusively, to multiple resistances to antibiotic treatment.

Helicobacter pylori (H. pylori ) infects (colonizes) the stomach of over half of the world’s population (e.g., about 1/3 of the U.S. population). H. pylori is responsible for disease outcomes ranging from asymptomatic gastritis (inflammation of the stomach) to peptic and duodenal ulcers and gastric cancers. Both human and bacterial genetic variability appear to contribute to differences in disease outcome. H. pylori exhibits extensive inter-strain genetic diversity as well as intra-strain genetic diversification during infection.

A subject is classified as having an H. pylori infection if H. pylori whole organisms, H. pylori genes, H. pylori proteins, H. pylori protein activity (urease activity) or human antibodies specific for H. pylori proteins are detected in the subject’s tissues (tissue biopsies, blood, stool, saliva, etc.).

Infection with H. pylori can be effectively treated with proton pump inhibitors and various antibiotics. First-line therapies for treating H. pylori infection comprise administration of the antibiotic drug clarithromycin. Since clarithromycin is a widely used antimicrobial drug, the prevalence of clarithromycin resistant H. pylori strains is increasing continuously.

Methods for diagnosis of H. pylori currently available include both invasive and non-invasive tests. The invasive tests (pathological evaluation of biopsies obtained via endoscopy) offer high sensitivity and specificity as well as the option of genotyping the H. pylori strain. Invasive methods include rapid urease testing, culture, histology and molecular diagnostics. However, these procedures include risk of esophageal and/or gastric perforation and bleeding, and risks from the medications used for patient sedation, as well as substantial costs associated with the endoscopic procedure and pathology review of the biopsy specimens. A further important disadvantage of biopsy-based evaluation is that due to non-homogeneous colonization of the pathogen in the infected tissue, biopsies may be taken from parts of the tissue that may not contain the bacterium or contain very low levels thereof, which are undetectable. Furthermore, for most gastric biopsies taken in gastroscopy and sent for culturing, antimicrobial susceptibility testing results are usually not available in all positive cases due to low sensitivity of the cell cultures.

Non-invasive tests for diagnosis of H. pylori include the serology test, urea breath test, stool antigen test and molecular diagnostics. Further, optional, non-invasive tests not yet fully developed so as to be assimilated in clinical practice include a real-time polymerase chain reaction (PCR) assay for detection of H. pylori infection and, optionally, simultaneous clarithromycin susceptibility testing in stool samples (see, for example, International Publication No. WO 2016/61398; U.S. Patent No. 9,868,995; Schabereiter- Gurtner et ah, 2004, J. Clinical Microbiology, 42(10):4512-4518; Scaletsky et ah, 2011, Helicobacter, 16: 311-315; Noguchi et ah, 2007, Journal of Microbiological Methods. 45:89-94; Dewhirst et ah, 2005, J. Bacteriology, 187(17): 6106-6118). These tests have varying levels of sensitivity and specificity, and most of them are unable to provide assessment of H. pylori antibiotic resistance altogether or able to provide limited indication of resistance to only one type of antibiotic drug, mostly resistance to clarithromycin.

Disclosed herein are non-invasive methods and kits useful for diagnosis of mutated DNA-based resistance of H. pylori to multiple anti -H. pylori treatment modalities. The contemplated methods and kits are based on bacterial DNA profiling, wherein the profiled bacterial DNA is extracted from a stool sample obtained from a subject infected or suspected of being infected with H. pylori.

The methods and kits described herein improve sensitivity over currently available tests such as conventional PCR or real-time PCR, may further provide, e.g., quantification of H. pylori load and ratio of specific genotypes. In designing and optimizing the described methods and kits, at least two major hurdles to non-invasive detection of anti -H. pylori treatment resistances are addressed: the low abundance of H. pylori in stool, and the nucleotide variability among H. pylori strains.

The diagnosis methods and kits disclosed herein can be used for detection of resistances in a clinical setting, to inform treatment strategies and to afford surveillance of antibiotic resistances in a population.

Specifically, disclosed herein is a method of diagnosing H. pylori infection combined with early diagnosis of putative multiple mutated DNA-based resistances to various, distinct anti -H. pylori treatment modalities in a subject suspected of being infected with H. pylori, the method comprising the steps of:

(i) obtaining a stool sample from the subject;

(ii) extracting and purifying bacterial DNA from the stool sample;

(iv) subjecting the purified bacterial DNA or the amplicons to multiplex gene profiling by the use of one or more multiplex molecular means; and

(v) diagnosing H. pylori infection in the subject and further diagnosing mutated DNA-based resistances to multiple distinct anti-// pylori treatment modalities, if multiple mutated H. pylori genes associated with distinct anti -H. pylori treatment resistances are detected.

Further disclosed herein is a method of diagnosing multiple mutated DNA-based resistances of H. pylori to anti -H. pylori distinct treatment modalities in a subject inflicted with H. pylori, the method comprising the steps of:

(i) obtaining a stool sample from the subject;

(ii) extracting and purifying bacterial DNA from the stool sample;

(iv) subjecting the bacterial DNA or the amplicons to gene profiling by the use of one or more multiplex molecular means; and

(v) diagnosing multiple mutated DNA-based resistances of H. pylori to distinct anti -H. pylori treatment modalities, if multiple mutated H. pylori genes associated with specific distinct anti -H. pylori treatment resistances are detected. The disclosed methods provide the benefit of early detection and characterization of multiple anti-// pylori treatment resistances in a subject, wherein“early detection” herein refers to identification of specific anti -H. pylori treatment resistances already at the time of first diagnosing H. pylori infection in the subject. According to a disclosed method, early detection of multiple anti -H. pylori treatment resistances in the subject is enabled before the subject is being subjected to a routine, e.g., a first-line or second-line, anti -H. pylori treatment protocol and, therefore, provides the benefit of tailoring the specific and efficient treatment modality which will result in successful H. pylori eradication in the subject. Early identification of specific anti -H. pylori treatment resistances affords personalized medicine (PM) whereby a personal, subject-specific treatment modality may be designed, focusing on the exact relevant treatment that will effectively eradicate H. pylori, while avoiding subjecting the patient to ineffective and unnecessary treatment modalities. Such therapeutic approach has the potential of tailoring therapy with the best response and highest safety margin to ensure better patient care. By enabling earlier diagnosis, a disclosed method enables risk assessments and, therefore, optimal treatments, thereby affording an improved and cost-effective health care.

In a further aspect, disclosed herein is a method for decreasing H. pylori infection eradication failure or, alternatively, increasing H. pylori infection eradication success, in a subject infected with H. pylori, the method comprising the steps of:

(i) extracting and purifying bacterial DNA from a stool sample obtained from the subject;

(ii) optionally, amplifying the purified bacterial DNA to obtain amplicons of genes of interest;

(iii) subjecting the bacterial DNA or the amplicons to gene profiling by the use of one or more multiplex molecular means;

(iv) detecting mutated H. pylori genes that confer resistances to multiple distinct anti -H. pylori treatment modalities; and

(v) providing to the subject a treatment modality for which no mutated DNA-based resistance has been detected, thereby decreasing H. pylori infection eradication failure in the subject or, alternatively, increasing H. pylori infection eradication success. In the context of embodiments described herein, the term“multiple” is to be interpreted as“at least two” or“two or more”, for example, two, three, four, five, seven or more. Thus, it is noted that a contemplated method provides the diagnosis of at least two, e.g., two, three, four, five or more resistances to various and, more importantly, distinct anti-// pylori treatment modalities. For example, two or more different antibiotic protocols or regimens such as first-line and second-line antibiotic regimens, are referred to herein as “distinct treatment modalities”.

Anti-//, pylori treatment modalities

The term“eradication”, as used herein in the context of treating H. pylori infection, is the destruction or extermination of the pathogen H. pylori in a subject. A successful eradication means inability to detect H. pylori in post treatment tests. Essentially, at least 98%, but preferably 100% of the bacterium infecting or residing i.e., colonizing, in the gastrointestinal tract of a subject is exterminated in a successful eradication, whereas destruction of lower levels of bacterium, e.g., 70-90%, is considered as eradication failure.

Preventing a disease, as used herein, refers to inhibiting the full development of a disease or condition caused by, induced by, propagated by, exacerbated due to, or resulting from H. pylori infection, for example inhibiting the progression of peptic ulcers and/or preventing the development of a gastric cancer in a subject infected with H. pylori. Treating a disease, as referred to herein, means ameliorating, inhibiting the progression of, delaying worsening of, and even completely preventing the development of a disease or pathological condition caused by, induced by, propagated by, exacerbated due to, or resulting from H. pylori infection. Treatment herein refers to a therapeutic intervention directly aimed at colonizing H. pylori for the purpose of eradicating the bacterium. Treatment may be similar to prevention, except that instead of complete inhibition, the development or progression of a disease is inhibited or slowed. In some embodiments, an anti -H. pylori treatment will decrease the probability that a condition, for example, a gastric cancer, will develop. In some embodiment, an anti -H. pylori treatment will prevent the commence and/or development of a condition or disease, for example, a gastric cancer.

The term“treatment modality”, as referred to herein, is a therapeutic method, an agent or a means designed for, used by, or applied to, e.g., administered to, a subject who displays symptoms or signs of a particular condition or disease, or to a patient known to be inflicted with a particular condition or disease, for the purpose of preventing or treating the particular condition or disease. For example, the disease may be H. pylori infection, and a treatment modality may be an agent and/or means provided to the subject under a protocol or regimen designed for eradicating the H. pylori infection. Herein treatment modality is interchangeable with the terms“treatment protocol”,“treatment approach”, "therapeutic treatment" and“treatment type”.

The terms“first-line therapy”, as used herein, is the initial, preferred, or the one accepted as the best treatment for a disease. It is often the therapy that combines the best efficacy with the best safety profile and/or the lowest cost. This term is synonym with “first-line treatment” and is exchangeable with terms such as“induction therapy”,“primary therapy”,“first therapeutic approach” and“primary treatment”. If the first-line therapy does not cure the disease or it causes severe side effects, other treatment may be added or used instead. For example, a second-line therapy is a treatment that is given when first-line therapy doesn’t work or stops working. A third-line therapy usually substitutes a first-line and/or a second-line therapies which have failed.

Often enough, the goal of achieving a cure in all treated H. pylori patients at the first therapeutic intervention, as generally occurring in most common infective diseases, is not accomplished. The initial susceptibility of H. pylori to both clarithromycin and imidazoles, key drugs for triple first-line therapies, has progressively been undergoing a marked reduction, and the eradication rate following therapy regimens including these antibiotics is decreasing. Similarly, the low H. pylori resistance towards quinolones, mainly used in second-line therapy, observed in the past has increased during the last decades, whilst both amoxicillin and tetracycline resistances still remain low. Antibiotic resistances play a crucial role in the management of H. pylori infection. Therefore, knowledge of the resistance mechanisms may contribute to designing more rational antibiotic combinations that will increase treatment success.

Only a few antibiotics are active against H. pylori in the acidic environment of the stomach. The main antibiotics employed in H. pylori eradication include, but are not limited to, macrolides such as clarithromycin and azithromycin, imidazoles such as metronidazole, quinolones such as levofloxacin, amoxicillin and tetracycline. Alternative molecules, such as furazolidone, bismuth salts and rifabutin are also employed although to a lesser extent due to low availability and significant side-effects.

Macrolides are a class of antibiotics derived from Saccharopolyspora erythraea, a type of soil-borne bacteria. These antibiotics have a broad spectrum of activity against many gram-positive bacteria, inhibiting protein synthesis by reversibly binding to the P site of the 50S unit of the bacterial ribosome. Five macrolide antibiotics are currently available for use: erythromycin, clarithromycin, azithromycin, fidaxomicin and telithromycin. The five macrolide antibiotics have a similar range of activities, with clarithromycin and azithromycin being more active than erythromycin against H. pylori. Clarithromycin presents minimal inhibitor concentrations (MICs) being the lowest as compared to the other molecules. For example, MIC values as low as 0.016-0.5 mg/L are generally reported. Antibiotic resistance is being recognized as MIC values > 1.0 mg/L (broad range of: 2-256 mg/L).

Metronidazole, marketed under the brand name Flagyl among others, is an antibiotic and antiprotozoal medication. It is used either alone or with other antibiotics. Resistance to metronidazole is high. In H. pylori strains, MIC values of 0.5-2 mg/L are reported, with antibiotic resistance being recognized as MIC values > 8 mg/L (range: 16- 128 mg/L). Bactericide activity of metronidazole depends on the reduction of its nitro- groups to produce anionic radicals which are able to damage the DNA-helicoidal structure.

The use of fluoroquinolones such as levofloxacin for H. pylori eradication is increasing worldwide because of its role in‘rescue therapy’ regimens following the failure of clarithromycin-based treatments. MIC values of 0.25-0.50 mg/L are generally reported, with antibiotic resistance being recognized as MIC values > 1 mg/L (range: 4-32 mg/L). Levofloxacin exerts a dose-dependent bactericide effect by binding the sub-unit A of DNA gyrase (topoisomerase II), an essential enzyme for the maintenance of DNA helicoidal structure. In susceptible strains, levofloxacin stops DNA and, at high doses, even RNA synthesis.

Amoxicillin is a b-lactam antibiotic included in all current therapeutic regimens for H. pylori eradication. MIC values ranging from 0.06 to 0.25 mg/L are generally reported in susceptible strains, and antibiotic resistance being recognized as MIC values > 1 mg/L (range: 1-8 mg/L). Amoxicillin acts by interfering with the peptidoglycan synthesis, especially by blocking transporters named penicillin binding proteins (PBP). This drug has been the first antibiotic used in H. pylori therapy because of a presumed absence of resistance, but evidence of stable amoxicillin resistant strains with a MIC of 8 mg/L has been reported.

Tetracycline is a fundamental antibiotic in quadruple regimens for H. pylori eradication. MIC values 0.25-2 mg/L are generally reported with antibiotic resistance being recognized as MIC values > 4 mg/L. Bacterial resistance towards tetracycline appears to be increasing. Tetracycline acts as a bacteriostatic against either Gram positive or Gram negative species by inhibiting codon-anticodon link at level of 30S ribosomal subunit and blocking the attachment of aminoacyl-tRNA to the acceptor site.

Other antibiotics pertaining to H. pylori eradication treatment include, but are not limited to, rifabutin, rifaximin, sitafloxacin, moxifloxacin and nitroimidazole.

Proton-pump inhibitors (PPIs) are a group of drugs whose main action is a pronounced and long-lasting reduction of stomach acid production. The combination of a PPI with at least two of amoxicillin, clarithromycin or metronidazole, achieves a high eradication rate. The rationale of PPI-based triple or quadruple therapy has been established by many clinical trials and a large body of experimental evidence. PPIs have pivotal direct as well as indirect effects in the eradication of H. pylori infection, the most potent of which are stabilizing and raising the antibacterial effects of combined antibiotics, especially clarithromycin and amoxicillin in the hostile gastric environment. Non-limiting examples of PPIs used in anti-// pylori therapy include esomeprazole, lansoprazole, omeprazole, pantoprazole and rabeprazole.

Standard triple therapy (TT), also referred to as clarithromycin-containing triple therapy, which consists of proton-pump inhibitors (PPIs), amoxicillin, clarithromycin or metronidazole, was the first-line H. pylori eradication therapy tailored. In recent years, progressively declining eradication rate for TT are indicated.

Second-line therapies featuring alternative combination regimens have been proposed, including bismuth-containing quadruple therapy (BQT), non-bismuth quadruple therapy (also called concomitant therapy, CT), sequential therapy (ST), hybrid therapy (HY) and quinolone -based triple therapy. Their efficiencies have been tested in different countries, including countries with high or low clarithromycin resistance, however, the optimal regimen for 77. pylori eradication remains elusive. Second-line treatment after failure of first-line clarithromycin-based triple therapy is exemplified by: a 7-day BQT: bismuth sub-citrate potassium, metronidazole, tetracycline and PPI); PPI, amoxicillin, metronidazole triple therapy; BQT: PPI, bismuth, amoxicillin, metronidazole; BQT: PPI, bismuth, tetracycline, amoxicillin; BQT:PPI, bismuth, tetracycline, levofloxacin; PPI, amoxicillin and levofloxacin; PPI, levofloxacin, metronidazole; sequential therapy: PPI and amoxicillin for 5 days, followed by PPI, levofloxacin, nitroimidazole for 5 days; and moxifloxacin, PPI, amoxicillin.

Third-line therapies are exemplified by: 10 days sequential therapy: PPI, amoxicillin for 5 days, then PPI, levofloxacin and tetracycline for 5 days; Rifabutin- containing therapies such as rifabutin, amoxicillin or PPI; rifaximin, levofloxacin and PPI; and sitafloxacin, rabeprazole, amoxicillin.

The guidelines for first-line, second-line and third-line therapies are different in each country. In addition, the guidelines defer from one physician to another, and while some physicians may start treatment with clarithromycin triple therapy (along with PPI and amoxicillin), others start with concomitant therapy (clarithromycin, amoxicillin, nitroimidazole and PPI), with no prior assessment of putative resistances to any of the anti- 77. pylori treatments prescribed.

Mutated-DNA based resistance

Bacterial species develop resistances to a variety of drugs by several mechanisms such as natural selection of bacteria that are inherently resistant to one or more drugs such as antibiotics, and/or changes that occur randomly within the organism's genome, collectively referred to herein as“mutations”. Altered bacterial DNA that may confer resistance can be created when entirely new genes are incorporated to the bacterium by processes such as (a) transformation, during which naked DNA from the ambient environment binds specifically to a receptor on the surface of the bacterium; the DNA is internalized and incorporated into the recipient genome via homologous recombination; (b) conjugation, which is the direct transfer of one or more genes from a donor to a recipient, either by way of a specialized "conjugation" tube or through direct cell-to-cell contact; and/or (c) transduction, whereby a bacteriophage (a virus capable of infecting a bacterium) incorporates fragments of bacterial DNA into its own genome, which then are transferred to new bacterial strains by newly synthesized infectious bacteriophage particles. Each of these mechanisms has been shown to operate in the movement of clinically relevant DNA (e.g., antibiotic resistance genes and virulence genes) from one bacterium to another.

Mutation may occur at a fixed rate of 1 in 10⁹ to 1 in 10¹⁰ replicated base pairs and are characterized either as one -base (nucleotide) substitution (a point mutation), or frameshift mutations that results from either the insertion or deletion of one or more nucleotides.

The term“mutated-DNA based resistance”, as used herein, embraces resistance of the bacteria to any anti-bacterial treatment, resulting from any of the genome alterations and mutations described above.

In bacteria that become resistant to antibiotics, rapid proliferation of a clonal line descended from a mutated, or otherwise altered, progenitor bacterial cell occurs. Microbial resistance to antibiotics is widely accepted today as one of the major problems of public health at the world’s level. Resistance of a microbial population is determined when a bacterial strain can grow in the presence of an antibiotic concentration higher than the concentration that inhibits most of the strains belonging to the same species. Genes encoding the mechanisms of resistance to antibiotics may encode, for example, inactivated enzymes and/or pumps, and/or modified antibiotic action target. Resistant bacteria, e.g., mutated DNA-based resistant bacteria, may also develop mechanisms that can chemically transform the antibiotic, inactivate or remove it completely from the cell.

Numerous cases of resistance of the bacterial pathogen to macrolides are caused by one or more point mutations in the 23S ribosomal RNA gene (235 rRNA ) coding the 2906 nucleotides long 23S rRNA (specifically in the peptidyl transferase region of 23S rRNA), which is a component of the large subunit (50S) of bacterial ribosome. These mutations are able to inhibit the binding between clarithromycin and the ribosomal subunit. The more frequent mutations associated with clarithromycin resistance include substitution of adenine with cytosine in position 2143 (herein designated“A2143C” mutation) and in the 2142 position (A2142C mutation) of 23S rRNA. These mutational events are responsible for more than 90% of clarithromycin resistance in developed countries. The mutation at position 2143 seems to be associated with different resistance levels rather than an on/off behavior, with minimal inhibitor concentrations (MICs) values widely ranging from 0.016 to 256 mg/L. Conversely, the mutation at position 2142 is associated with more restricted MIC values, close to 64 mg/L. The A2143G point mutation, markedly reduces H. pylori eradication rate.

Mutations in positions A2058 or A2059 of the 235 rRNA, as common to most bacteria, confer macrolide resistance also in H. pylori. Adenosine 2058 is the essential nucleotide for interaction of the macrolide with the ribosome. Replacement of adenosine at position 2058 with guanine (A2058G mutation) confers resistance to erythromycin. Several other point mutations in 235 rRNA have been identified in antibiotic resistant H. pylori such as A2115G, G2141A, T2117C, T2182C, T2289C, G224A, C2245T, and C2611A. It is noted that new mutations conferring resistance to macrolides are continuously occurring and being detected.

Point mutations in quinolones resistance-determining region (QRDR) of gyrA gene prevent binding of the levofloxacin antibiotic to the enzyme DNA gyrase, thereby conferring antibiotic bacterial resistance. Point mutation conferring resistance to levofloxacin include C261A, C261G, G271A, G271T, and A272G in the gene gyrA, and certain mutations in gyrB gene.

Multiple point mutations in pbpl gene are the major mechanism of amoxicillin resistance. The penicillin binding proteins (PBPs) are enzymes involved in the synthesis of the peptidoglycan layer of the bacterial wall, and mutated pbpl gene leads to a loss of affinity between amoxicillin and PBP-transpeptidase.

Simultaneous triple point mutations from the 965 to 967 position in loop of helix 31 is recognized as the major mechanism of tetracycline resistance. The main point mutation is a substitution of an AGA with a TTC triplet. Mutations conferring resistance to metronidazole include but are not limited to, G3A, C46T, G47A, G238A, G352A, C589A and G610A in the gene rdxA, and G37T in the gene rspU.

Table 1 summarizes some exemplary mutated DNA-based antibiotic resistances in H. pylori. Table 1. Antibiotic resistances due to mutated H. pylori DNA

A = adenine; G = guanine; C = cytosine; U = uracil.

Resistance patterns vary significantly between regions due, e.g., to differences in disease prevalence and the differing patterns of antibiotics usage. It is highly advisable that clinicians be informed about the primary antimicrobial resistance patterns for their local treatment population before deciding on empirical treatment.

A contemplated diagnosis method may provide resistances profiles based on a single analysis. In some embodiments, a disclosed method provides the simultaneous identification of resistances to both clarithromycin and levofloxacin based on a single stool sample.

In some embodiments, a disclosed method provides the simultaneous identification of resistances to both clarithromycin and metronidazole based on a single stool sample.

In some embodiments, a disclosed method provides the simultaneous identification of resistances to clarithromycin, metronidazole and levofloxacin based on a single stool sample.

In some embodiments, a disclosed method provides the simultaneous identification of resistances to both metronidazole and levofloxacin based on a single stool sample.

In some embodiments, a disclosed method provides the simultaneous identification of resistances to both tetracycline and levofloxacin based on a single stool sample.

In some embodiments, a disclosed method provides the simultaneous identification of resistances to clarithromycin, metronidazole, tetracycline and levofloxacin based on a single stool sample.

In some embodiments, a disclosed method provides the identification of resistances to clarithromycin, metronidazole and amoxicillin based on a single stool sample.

When a subject is classified, based on DNA profiling obtained by a diagnostic method described herein, as having two or more antibiotic resistant strains of H. pylori, or an H. pylori strain having multiple resistances, e.g., to two, three or more antibiotics, the subject can be given a therapeutic treatment for which susceptibility has not been negated, for example, a treatment modality that comprises provision of other antibiotics. For example, if the subject is classified as having clarithromycin resistant and metronidazole resistant strain(s) of H. pylori, in some instances the subject can be treated with levofloxacin and/or metronidazole. If the subject is classified as having an azithromycin and levofloxacin resistant strain(s) of H. pylori, in some instances the subject can be treated with tetracycline or metronidazole. Prior knowledge of the subject being non-responsive to certain antibiotic treatments affords tailoring of an immediately effective anti-// pylori treatment protocol that will successfully eradicate the pathogen and prevent development of, e.g., gastric cancer. Multiplex molecular means for DNA profiling

DNA profiling (also called DNA fingerprinting) is the process where a specific DNA pattern, i.e., profile, is obtained from a person or sample of bodily tissue. This process determines an individual's DNA characteristics, which are as unique as fingerprints. In the context of the present disclosure, the term“DNA profiling” is used for denoting DNA profiles of human gut infecting bacteria, wherein microbial DNA is analyzed, after being isolated, for example, from stool samples of individuals. Embodiments described herein mainly concern the profiling of H. pylori DNA colonizing in human gut.

“Multiplex molecular means”, as used herein, refers to a means which affords the handling of multiple or multitude molecules simultaneously in a single assay or in a single experiment cycle, for example, simultaneously amplifying, tagging, identifying, hybridizing, or detecting multiple genes, multiples mRNAs, multiple DNAs or fragments thereof, multiple antibodies or multiple proteins from the same specimen in a single analysis, assay or test. As used herein,“multifold”,“multiplex”,“multiple”,“multitude” are interchangeable and denote at least two, for example, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty and even more. Non-limiting examples of multiplex molecular means includes DNA microarrays and multiplex real time PCR.

Microarrays and gene chips

Simply dehned, a microarray for genome profiling is a collection of multiple microscopic spots also termed“features” or“tests spots” on a solid support, each feature accommodating one or more specific single strands DNA or RNA. DNA microarrays are based on the ability of single strands of DNA probes to hybridize to complementary (matching) sequences of DNA (cDNA) or RNA. The term“complementary”, as used herein, means that the sequence of the probe is exactly hybridizing to the sequence of the target. As used herein, the terms “probe”, “capture oligonucleotide” and “capture sequence” are interchangeable and refer to a specific DNA sequence, or to a universal oligonucleotide sequence flanking a specific primer, immobilized onto discrete features or test spots, whereas the term“target” refers to the“unknown” cDNA sequence being assayed. Each feature may contain millions of identical probes. The target may optionally be fluorescently labeled and then hybridized to the probes. In such cases, a successful hybridization event between the labeled target and the immobilized probe results in an increase of fluorescence intensity over a background level and can be measured using a fluorescent scanner. The fluorescence data can then be analyzed by a variety of methods for both quantitative and qualitative measurements.

Microarray platforms include two-dimensional hybridization microarrays and three-dimensional arrays or suspension bead arrays. Microarrays are characterized based on the nature of the probes, probes length and synthesis, number of possible features (i.e., density of the microarray), the solid-surface support used, and the specific method used for probe addressing and/or target detection. Multiplex microarray systems suitable for the purpose of embodiments described herein include, but are not limited to, printed and in .gz/_zz-synthesized microarrays, high-density bead arrays, electronic microarrays and suspension bead microarrays.

In printed microarrays, the probes are“printed” or spotted onto the microarray surface, which is most commonly a glass slide, using noncontact or contact printing technologies as well known in the art.“Printing”, in the context of printed microarrays, is the application of a few nanoliters of probe solution per spot to create an array of 100 to 150 pm features. Printed arrays are classified, based upon the nature of the probes, into double-stranded DNA (dsDNA) and oligonucleotide microarrays. In dsDNA microarrays, the probes consist of double-stranded DNA of 200 to 800 base pair (bp) which are denatured either in print buffer or after immobilization and become available for hybridization, and the oligonucleotide microarrays consist of short, chemically synthesized sequences of 25 to 80 bp. Printed microarrays are relatively simple and inexpensive. In addition, these microarrays enable to quickly adjust spotted probes based upon updated annotations or the discovery of new, emerging pathogens or resistance mechanisms.

In gz/_zz-synthesized arrays are extremely high-density microarrays comprising oligonucleotide probes that are synthesized directly on the surface of the microarray, which is typically a 1-2 cm² quartz wafer. These microarrays, are termed herein“DNA chips”, “gene chips”, or simply“chips” because they are characterized by orderly arranged short single stranded DNA (20 to 25 bp) in a grid, spotted at precise intervals, and are made by photolithographic techniques similar to those used for fabricating computer chips. The probes are spotted on the wafer as probe sets, each set comprising perfect-match probes and mismatch probes, wherein a typical mismatch probes contains a 1-bp difference in the center of the probe (i.e., position 13 of a 25-bp probe). Perfect-match probes and mismatch probes of a set are located in separate features, thus allowing the mismatch probes to act as a negative control to identify possible nonspecihc cross-hybridization events. Each set comprises multiple probes to improve sensitivity, specihcity, and statistical accuracy.

Gene chips are commercially available and some of them may be utilized for H. pylori DNA profiling in accordance with a disclosed method. Non-limiting examples include GeneChip® Microarray of Affymetrix (Santa Clara, CA), having >10⁶ features per microarray, NimbleGen® microarray manufactured by Roche (Madison, WI), and Agilent Technologies’ gene chips (Palo Alto, CA). Any of these platforms can be easily customized with unique oligonucleotide sequence content.

Bead arrays are based on 3-micron silica beads, randomly self-assembled in a uniform spacing of ~5.7 microns on a substrate such as fiber optic bundles (such as Sentrix® array matrix (SAM)) or a planar silica slides (such as Sentrix® BeadChip). Each bead is covered with hundreds of thousands (-700,000) of copies of a specific capture oligonucleotide (probes). For example, a SAM substrate may contain 96 1.4 mm hber optic bundles, each bundle being an individual array consisting of 50,000 5 pm light-conducting hbers, wherein each fiber is chemically etched to create a microwell for a single bead. Each SAM allows the analysis of 96 independent samples. The BeadChip can be used to assay 1 to 16 samples at a time on a silicon slide that has been processed to provide micro wells for individual beads.

Commercially available bead arrays are exemplified by BeadArrays system produced by Illumina® (San Diego, CA), comprising fiber optic bundles (SAM) as substrate.

Electronic microarrays utilize active hybridization via electric helds to control target oligonucleotides transport. Electronic microarrays are also, interchangeably, referred herein as“gene chips” and“microelectronic cartridge”. Usually, features (test sites) on the solid surface of a chip contain streptavidin. In the process of preparing the chip for multiplex assay, DNA or RNA capture probes attached to biotin (biotinylated probes) are first electronically directed to desired features on the chip (by applying a positive current at the features), and allowed to form streptavidin-biotin bonds, thereby forming active features. The strength of the streptavidin-biotin bond allows the capture probes to remain attached to the activated features while excess target DNA is washed away to prevent cross contamination. The positive current is then removed from the active features, and new features can be activated by the targeted application of a positive current and direction of other biotinylated oligonucleotide probes. Once the probes have been anchored at discrete features, the microarray is ready for hybridization. DNA and RNA, which are normally negatively charged nucleic acids, are transported to specihc activated features on the microarray when a positive current is applied to one or more of these features. Typically, target DNA passively hybridizes with the immobilized probes on the microarray but can also be concentrated electronically. Usually, DNA and RNA targets are fluorescently labeled, e.g., by sandwich hybridization assays.

Electronic microarrays are commercially available, and at least some of them may be utilized for H. pylori DNA profiling in accordance with a disclosed method. A non limiting example of a microelectronic cartridge is the NanoChip® 400 gene chip manufactured by Savyon Diagnostics (Ashdod, Israel). This tiny, bio-compatible silicon chip utilizes active hybridization via electric helds to control nucleic acid transport. The chip surface is covered with a permeable gel layer that contains streptavidin, which is utilized to attach biotinylated DNA capture probes to specific features. Sandwich hybridization assays at each feature with fluorescently labeled DNA fragments are enabled once the probes at discrete features have been hybridized to specific target DNA. Automatic processing and reading by a built-in image sensor are then performed, and the results are analyzed and presented.

Commercially available electronic cartridges are universal blank chips, and the features of the microarrays are specified directly by the user, which allows more flexibility in assay design and decreased costs associated with microarray manufacturing. Furthermore, the electronic gene chip assay currently available may be compatible with various automated DNA extraction systems thus enabling efficient assimilation as part of a fully automated process. The procedure is compatible with the laboratory needs in the community settings in terms of time-to-result and workflow.

Suspension bead arrays are three-dimensional arrays based on the use of microscopic polystyrene spheres (or hollow beads) as the solid support, and flow cytometry for bead and target detection. In suspension arrays, beads remain suspended in the hybridization solution. Robust multiplexing is accomplished using different microsphere sets based on color. Red (658 nm emission) and infrared (712 nm emission) fluorochromes at various concentrations are used for hlling 5.6 pm microspheres. Each microsphere in a 100-microsphere set has a distinct red-to-infrared ratio, and therefore, each bead has a unique spectral address. Microspheres with a specihc spectral address are coupled to specihc capture probes and are equivalent to features in a planar microarray. A fluorescent reporter is utilized for indicating probe-target DNA hybridization. In an DNA profiling assay, a single-file microsphere suspension passes by two lasers. A 635 nm laser excites the red and infrared fluorochromes impregnated in the microspheres and allows the classification of the beads and therefore the identity of the probe-target being analyzed. A 532 nm laser excites reporter fluorochromes such as R-phycoerythrin or Alexa® Fluor 532 to quantify any hybridization that occurs on the microsphere.

Although the feature density of suspension bead arrays is the lowest of all the platforms described herein, this platform, nevertheless, has important advantages: the availability of universal bead sets and their inherent flexibility make the development of user-defined applications feasible and relatively inexpensive.

Depending on the number of target sequences under examination, costs and customization requirements, it is appreciated that a skilled person can design and manufacture, or alternatively, customize a suitable microarray system to meet the genome profiling and diagnosis requirements of a contemplated method.

In some embodiments, DNA profiling is obtained using a gene chip. In exemplary embodiments, the gene chip is a one-color electronic microarray detection means. In accordance which such embodiments, a microelectronic cartridge loaded with probing oligonucleotides complementary to wild-type DNA and/or probes complementary to at least two mutated genes of this bacterial species is used. DNA is extracted, for example, form a stool sample and, optionally, PCR is utilized for amplifying the DNA. Optionally, amplification is conducted in the presence of biotinylated nucleotides to thereby produce biotinylated amplicons. Depending on the gene chip used, extracted or PCR amplified double-stranded DNA may optionally be denatured at this stage, and/or optionally fragmented. Target DNA is washed over the gene chip, and the loaded cartridge is inserted to a hybridization oven at 45°C for, e.g., 16 hours so as to allow complementary strands to stick together. Any unhybridized DNA pieces are gently washed off. When biotinylated amplicons are tested, one fluorescent stain that attaches to biotin is washed over the chip after termination of hybridization. Then, a means such as laser is used to detect the fluorescent dye and create a visual image of the pattern of the dye. If a target DNA/amplicon has anchored on the chip, it will glow. Lastly, a relevant computer program is applied for indicating which are the probes the labelled DNA has hybridized to, thereby identifying which species and/or mutant are present in the sample and to what extent. The higher the fluorescence intensity of a given feature is, the higher is the amount of the specific gene probed by that feature.

In some embodiments, DNA profiling is obtained using a gene chip, for example, an electronic microarray, as a two-color microarray detection means. In accordance with these embodiments, DNA is first extracted, for example, form a stool sample of a subject suspected of being infected with H. pylori. A control sample comprising, e.g., wild type H. pylori DNA, or mutated H. pylori genome bearing known mutated genes, is employed. The target and control DNA are denatured and, optionally, the long strands of DNA are cut into smaller, more manageable fragments. Each fragment is labelled, for example, by attaching a fluorescent dye thereto. Fluorescent dyes commonly used for DNA labelling include cyanine dye3 (Cy3) or its spectrally equivalent Hy3, which has a fluorescence emission wavelength of 570 nm (corresponding to the green part of the light spectrum), and cyanine dye5 (Cy5) or its spectrally equivalent Hy5 with a fluorescence emission wavelength of 670 nm (corresponding to the red part of the light spectrum). For example, when H. pylori genome suspected of bearing mutated genes that confer antibiotic resistances is assayed, the subject's target DNA is labeled with a green fluorescence dye (e.g., Cy3 or Hy3) and the control DNA, being wild type or“normal” H. pylori DNA, is labeled with a red fluorescence dye (e.g., Cy5 or Hy5). Both sets of labeled DNA are then loaded onto the chip and allowed to hybridize to the probes on the chip. If the subject does not have mutations in the gene(s) tested, both the red and green samples will bind to the probes on the chip that represent the sequences without the mutations (the "normal" sequences). However, if the subject does possess two or more mutations, the subject's DNA will bind to corresponding two or more probes on the chip that represent the mutated genes. Multiplex PCR

Some embodiments described herein pertain to the use of multiplex polymerase chain reaction (PCR) as a means for profiling bacterial genome.

PCR, in all of its forms, is an amplification technique which enables to increase the number of copies of a nucleic acid molecule in a sample or specimen. Basically, a biological sample collected from a subject is contacted with a pair of oligonucleotide primers under conditions that allow for the hybridization of the primers to nucleic acid template in the sample. The primers are extended under suitable conditions with a specific polymerase enzyme, dissociated from the template, and then re-annealed, extended, and dissociated so as to amplify the number of copies of the nucleic acid.

Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture.

The term“primer”, as used herein and in the art, refers to a short nucleic acid sequence that provides a starting point for DNA synthesis, namely, it serves to prime and lay a foundation for DNA polymerases that synthesize DNA and can only attach new DNA nucleotides to an existing strand of nucleotides in the presence of a primer. In in vitro studies, the primer is designed specifically for the DNA region of interest such that it matches the beginning of the DNA target to be amplified. Preferred DNA polymerases are thermostable DNA polymerase such as the Taq polymerase (isolated from the heat-tolerant bacterium Thermus aquaticus ). A pair of primers are used for amplification of each target DNA, having sequences that will hybridize to opposite strands of the template DNA. These primers are also to herein as“forward and reverse primers”.

Primers suitable for multiplex PCR assays are designed so as to feature the appropriate balance between length, melting temperature (Tm) and specificity. While specificity of a particular primer increases with its length, the length of a primer is limited by the temperature required to melt it. Usually, primers of short length in the range of 18- 22 bases are preferred. Primers with similar Tm, preferably between 55 °C to 65 °C are preferred. A Tm variation of between 3°C to 5°C is acceptable for primers used in a pool.

PCR primer pairs are available commercially or can be synthesized in the laboratory. Design of multiplex PCR primers may be facilitated by the use of commercially available software such as PrimerPlex that analyzes millions of possible multiplex sets in an a few seconds and presents a list of alternate sets.

Multiplex PCR reactions can be broadly divided in two categories: (1) single template PCR reaction, a technique that uses a single template such as a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within the template; and (2) multiple template PCR reaction, which uses multiple templates and several primer sets in the same reaction tube or in separate reaction tubes.

Multiplex PCR methods for detecting H. pylori infection as well as antibiotic resistance are known, but these tests provide assessment of only one type of mutated-DNA based antibiotic resistance, usually resistance to clarithromycin. At the time of the present disclosure, there are no known multiplex real-time PCR kits that test for more than one antibiotic -resistance. Commercially available means, such as the H. pylori ClariRes Assay of lngenetix, are designed for stool-based real-time PCR test and afford the detection of H. pylori in combination with clarithromycin susceptibility.

When a disclosed diagnostic method comprises the use of multiplex PCR as a means to profile the genome, the method may further provide quantitative information on the number of genomes per sample and the number of bacteria of specific genotypes, and thus provides information on the load/density of H. pylori infection.

Because H. pylori strains are highly diverse at a genetic level and individuals can be infected with more than one strain, it is important to design probes and primers based upon conserved or consensus fragments found in various strains. Alternatively, if the same gene is to be amplified from different genotypes/strains, degenerate probes or primers may be used, namely, mixtures of similar, but not identical, probes or primers.

As used herein, “oligonucleotide” refers to a plurality of joined nucleotides, between about 6 and about 300 nucleotides in length. Nucleotides include ribonucleotides, deoxyribonucleotides and modified nucleotides, referred to herein as“oligonucleotide analogs”. Oligonucleotide analogs contain moieties that function similarly to oligonucleotides but have non-naturally occurring portions, such as sugar moieties or inter sugar linkages, such as a phosphorothioate, alkylphosphorothioates or peptide nucleic acids. Oligonucleotide analogs further include biotinylated oligonucleotides, and/or fluorescence oligonucleotides i.e., oligonucleotides bearing biotin moieties and/or fluorescent moieties, respectively. The introduction of one or more of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, and the like.

Kits

An aspect of the present disclosure relates to a kit for diagnosis of H. pylori infection combined with early diagnosis of possible multiple mutated DNA-based resistances to various, distinct anti-// pylori treatment modalities in a subject suspected of being infected with H. pylori. A contemplated kit comprises one or more of the following components: (a) means and reagents to extract and purify bacterial DNA from a stool sample; (b) means and/or reagents to conduct genome profiling; (c) detection means; and, optionally, (d) written instructions, e.g., a user manual.

The means and reagents to extract and purify bacterial DNA from a stool sample contained in a contemplated kit, depend on the extraction/purification method of choice. For example, a kit may comprise reagents suitable for DNA extraction and purification conducted using an automated system such as KingFisher™ Flex purification system (Thermo Scientific, Vantaa, Finland), or using manual means such as the AllPrep PowerFecal DNA/RNA Kit or QIAamp Fast DNA Stool Mini Kit (both by Qiagen®).

The means and/or reagents to conduct multiplex genome detection and genome profiling depend on the multiplexing technology of choice, which may be, for example, multiplex PCR or multiplex microarray systems such as electronic microarrays, printed and in .v/ -synthesized microarrays, high-density bead arrays, and suspension bead microarrays.

In some of any of the diagnosis methods described herein, DNA extracted from a specimen, e.g., stool, may be amplified before being subjected to genome profiling. In such embodiments, a contemplated kit may comprise an amplification mixture that includes any combination of multiple primer pairs (e.g., two, three, four, five or more primer pairs), designed to anneal to complementary DNA templates comprising genes related to, or involved in, H. pylori susceptibility to two or more antibiotic drugs. Additionally, the kit may comprise at least one polymerase such as Taq polymerase, and deoxynucleotide (and/or nucleotide) triphosphates (dNTPs and/or NTPs) or analogs thereof.

Detection means, also referred to herein as labelling means, include a detectable compound that is conjugated directly or indirectly to a probed target DNA sequence to facilitate detection thereof. In some embodiments, labelling is performed using one or more fluorescent dyes such as Cy3, Hy3, Cy5 and Hy5 and/or fluorescent reporter oligonucleotides, i.e., a fluorescent dye attached to oligonucleotides.

Any of the kits described herein can be used in the context of a suitable hybridization and detection system, which may contain, for example, a hybridization oven, fluidics stations, scanners, flow cytometry and software for data analysis.

It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination or as suitable in any other described embodiment of the disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

As used herein the term“about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including",“having” and their conjugates mean "including but not limited to".

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments described may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above description illustrate some embodiments of the disclosure in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in embodiments of the present disclosure include molecular, chemical, biochemical, microbiological and/or recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See for example, Guide to Research Techniques in Neuroscience (Second Edition), Matt 2015; Elsevier's Integrated Review Biochemistry (Second Edition), 2012. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.

Materials and Methods

H. Pylori DNA purification

H. Pylori DNA is extracted from stool sample and purified using an automatic, high throughput system or a manual kit. Sample collection is relatively easy, non-invasive, and amenable for large sample sizes. Stool, however, is a complex sample source due to the presence of many compounds that can degrade DNA and inhibit downstream enzymatic reactions such as polyphenols, humic acid, lipids, and other PCR inhibiting compounds. High quality DNA extracted from frozen, fresh, or preserved stool, free of PCR inhibitors is obtained by the use of an automated, high throughput extracting format. In some embodiments, a magnetic bead-based approach for DNA purification is attempted, using commercially available extraction kits such as the Thermo Fisher Scientific’s KingFisher™ Flex automated purification system (Thermo Scientific, Vantaa, Finland), a magnetic bead based automated purification system, which affords obtaining pure and intact DNA with minimal hands on time (see, for example, h ttp : // www . ther o .com/kingfi slier) . In some exemplary embodiments, extraction and purification of H. pylori DNA from stool is conducted manually, for example by using a DNA stool kit, for example, the QIAamp Fast DNA Stool Mini Kit or the AllPrep PowerFecal DNA/RNA Kit (both by Qiagen®). Such kits provide rapid purification of high-quality genomic DNA from fresh or frozen stool samples. DNA is eluted in low-salt buffer and is free of protein, nucleases, and other impurities or inhibitors. The purified DNA is ready for use in PCR and other enzymatic reactions.

Optionally, the suitability of the purified DNA for downstream applications is determined by running a real-time PCR analysis. For example, a real-time PCR reaction is set up to a total volume of 20-25 pi using, e.g., Agilent’s Brilliant III 2X SYBR® as the master mix, and 2 pi of purified DNA at appropriate dilution as template with suitable primers, and following a standard amplification protocol, e.g., on the ABI 7900.

The DNA quantity is measured by reading the absorbance at 260 nm and the quality by analyzing the 260/280 nm ratio. The quality of DNA is also tested by the end-point PCR to control the presence of PCR-inhibitors in the eluate. A ratio A260/A280 close to 1.8 is indicative of high quality.

H. Pylori DNA profiling

Purified DNA extracted from various stool samples (the“target DNA” or“target sequences”), is loaded onto a microarray such as any of the microarray systems described herein. Optionally, the target genes are amplified by PCR before being subjected to a multiplex profiling. In exemplary embodiments, an electronic gene chip is employed, for example, the NanoCHIP® microelectronic cartridge (Savyon Diagnostics, Ashdod, Israel). The cartilage is loaded with target sequences and administered into a NC400 instrument (Savyon Diagnostics, Ashdod, Israel). The target sequences are electronically addressed to discrete loci on the NanoCHIP® cartridge, pre-activated with specific capture oligonucleotides. Detection is achieved through specific fluorescent reporter oligonucleotides. Optionally, the output analysis of each sample is compared to the characterization of the respective original stool sample, as characterized by real-time PCR.

Detection and reporting are conducted by a sandwich hybridization assay. The method is based on the detection of hybridization events between two specific oligonucleotide probes and the target sequence. The first oligonucleotide probe is the capture probe, optionally labeled with a detectable marker. Usually, the capture probe is shorter than the target sequence such that following hybridization, a short segment of each target sequence is left unpaired. The second oligonucleotide probe is the fluorophore labeled probe, a short oligonucleotide complementary to the unhybridized segment of the target sequence and conjugated to a fluorescence dye. When two or more target sequences are assayed, for example one wild type H. pylori gene and three mutated H. pylori genes, the reporting step of the multiplex analysis may comprise one or more report-washing cycles. For this purpose, two fluorophore labeled probes are used, one labelled with, e.g., a red fluorescence dye and one with, e.g., a green fluorescence dye. In the first reporting stage, a red fluorophore labeled prob complementary to wild-type gene (herein designated, for example, as“Target A”) and a green fluorophore labeled prob complementary to a first mutated gene (herein designated“Target B”) are contacted with corresponding target sequences hybridized to their respective capture probes, and allowed to hybridize. In reporting, red-labeled reporter is a perfect match for Target A and green-labeled reporter is a perfect match for Target B. Then, a first wash cycle is effected, wherein a thermal stripping step removes the reporters but leaves the target sequences bound to the capture probes ready for next reporting.

In the second reporting stage, a red fluorophore labeled prob complementary to a second mutated gene (“Target C”) and a green fluorophore labeled prob complementary to a third mutated gene (“Target D”) are contacted with target sequences anchored onto the chip via their corresponding capture probes, and allowed to hybridize. In reporting, red- labeled reporter is a perfect match for Target C and green-labeled reporter is a perfect match for Target D. Additionally or alternatively, more than two fluorophore labeled probs, for example, three, four, five or more probs may be used in a contemplated sandwich hybridization assay, each probes labelled with a distinct fluorophore, so as to enable simultaneous detection of more than two target sequences, e.g., more than two mutated genes, in one report-washing cycle

At the end of a gene profiling step in a contemplated method, two observations are provided: (1) presence of H. pylori ; and (2) presence of resistances to multiple anti-//. pylori treatment modalities, wherein the resistances are specified by the detection of specific mutations in the bacterial DNA.

EXAMPLE 1

Multiplex microarray-based diagnosis of multiple H. pylori mutated-DNA based antibiotic resistances

A patient suspected of being infected by H. pylori provides a stool sample. No special preparation (e.g., fasting, withholding drug consumption and the like) are needed. Stool is diluted 10% w/v in PBS and mixed thoroughly. DNA from, e.g., 250 pi of the stool sample is extracted and purified either using an automatic means such as Fisher Scientific’s KingFisher™ purification system, or a manual kit such as Qiagen® kit described in Materials and Methods.

The purified DNA is evaluated by reading the absorbance at 260 nm and the quality is assessed by analyzing the 260/280 nm ratio using a spectrophotometer (NanoDrop™ 2000, Thermo Scientific, Vantaa, Finland). If the DNA is found to be clean of contaminants it is amplificated by specific PCR reaction using specific primers for gene of interest. However, if the DNA is contaminated, a further extraction from the stool sample is performed.

Specific amplified DNA (amplicons) are cleaned post PCR from all leftovers such as DNTPs, buffers, polymerase residues and the like, and the purified DNA is then analyzed once more for quality and quantity using a spectrophotometer as described above.

The purified amplicons are profiled by using the electronic gene chip NanoCHIP® cartridge (Savyon Diagnostics, Ashdod, Israel) as described in Materials and Methods above. For this purpose, the gene chip is loaded with specific capture probes that hybridize to wild type H. pylori or to mutated H. pylori genome, particularly to mutated genes that confer antibiotic resistances to at least two distinct antibiotics. The NC400 instrument provides analysis of the hybridization profile of the target DNA, and a detailed report provides specific resistances-conferring mutations of the tested strain.

To be noted, a gene chip used in accordance with a contemplated multiplex diagnosis is specifically designed to suit the prevalence of mutations conferring resistance to antibiotics in specific geographic regions or ethnic populations. EXAMPLE 2

Multiplex real time PCR-based diagnosis of multiple H. pylori mutated-DNA based antibiotic resistances

A stool sample is collected from a patient suspected of being infected by H. pylori, and bacterial DNA is extracted, purified, assessed for quality and quantity, and amplified as described in Example 1.

The purified amplicons are profiled by performing real time PCR using a dedicated real time PCR machine such as Applied Biosystems™ QuantStudio™ 6 Flex Real-Time PCR System (Thermo Fisher Scientific).

For multiplex real time PCR, a set of 5 tubes is used. The software in a dedicated real time PCR automated system analyzes the amplification in each tube. Any signal of amplification with a specific probe indicates the existence of a specific antibiotic resistance-conferring mutation linked to the probe. The 5 tubes are:

Tube 1, containing a multiplex of specific probes for 23S rRNA mutations that confer resistance to clarithromycin, and a set of primers for the 23S rRNA gene;

Tube 2, containing a multiplex of specific probes for gyrA mutations that confer resistance to levofloxacin, and a set of primers for the gyrA gene;

Tube 3, containing a multiplex of specific probes for frxA mutations that confer resistance to levofloxacin, and a set of primers for the frxA gene;

Tube 4, containing a multiplex of specific probes for rdxA mutations that confer resistance to metronidazole, and a set of primers for the rdxA gene; and

Tube 5, containing a multiplex of specific probes for rpsU mutations that confer resistance to metronidazole, and a set of primers for the rpsU gene.

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing multiple mutated DNA-based resistances of Helicobacter pylori ( H . pylori ) to distinct anti -H. pylori treatment modalities in a subject infected with H. pylori, the method comprising the steps of:

(i) obtaining a stool sample from the subject;

(ii) extracting and purifying bacterial DNA from the stool sample;

(v) diagnosing multiple mutated DNA-based resistances of H. pylori to distinct anti -H. pylori treatment modalities if multiple mutated genes associated with distinct anti -H. pylori treatment resistances are detected.

2. A method of diagnosing Helicobacter pylori (H. pylori ) infection combined with early diagnosis of possible multiple mutated DNA-based resistances to various, distinct anti -H. pylori treatment modalities in a subject suspected of being infected with H. pylori, the method comprising the steps of:

(i) obtaining a stool sample of the subject;

(ii) extracting and purifying bacterial DNA from the stool sample;

(v) diagnosing H pylori infection in the subject and further diagnosing mutated DNA-based resistances to multiple distinct anti -H. pylori treatment modalities, if multiple mutated H. pylori genes associated with distinct anti -H. pylori treatment resistances are detected.

3. A method for decreasing Helicobacter pylori (H. pylori ) infection eradication failure in a subject infected with H. pylori, the method comprising the steps of:

(i) obtaining a stool sample of the subject;

(ii) extracting and purifying bacterial DNA from the stool sample; (iii) optionally, amplifying the purified bacterial DNA to obtain amplicons of genes of interest;

(iv) subjecting the bacterial DNA or the amplicons to gene profiling by the use of one or more multiplex molecular means;

(v) detecting mutated H. pylori genes that confer resistances to multiple distinct anti -H. pylori treatment modalities; and

(vi) providing to the subject a treatment modality for which no mutated DNA- based resistance has been detected, thereby decreasing H. pylori infection eradication failure in the subject.

4. A method for increasing Helicobacter pylori (H. pylori ) infection eradication success in a subject infected with H. pylori, the method comprising the steps of:

(i) obtaining a stool sample of the subject;

(ii) extracting and purifying bacterial DNA from the stool sample;

(vi) providing to the subject a treatment modality for which no mutated DNA- based resistance has been detected, thereby increasing H. pylori infection eradication success in the subject.

5. The method of claim 1 or 2, wherein the multiple mutated DNA-based resistances is two, three, four, five, six, seven or more mutated DNA-based resistances to distinct anti -H. pylori treatment modalities.

6. The method of claim 5, wherein at least one of the multiple distinct anti -H. pylori treatment modalities is antibiotic treatment.

7. The method of claim 5, wherein two or more of the distinct anti -H. pylori treatment modalities are distinct antibiotic treatments.

8. The method of claim 6 or 7, wherein the antibiotic treatment is treatment with at least one of clarithromycin, metronidazole, levofloxacin, tetracycline, or amoxicillin.

9. The method of any one of claims 1 to 8, wherein mutated H. pylori DNA is at least one mutation in at least one of the genes 23 S rRNA, gyrA,frxA, rdxA or rpsU.

10. The method of claim 9, wherein the gene mutations are: in gene 23S rRNA, point mutations A2142G, A2142C, A2143C and A2143G; in gene gyrA, point mutations C261A, C261G, G271A, G271T and A272G; in gen e frxA, the mutation -571TA; in gene rdxA, point mutations G3A, C46T, G238A, G47A, G352A, C589A, and G610A; and in gene rpsU, point mutation G37T.

11. The method of any one of claims 1 to 10, wherein the multiple mutated DNA- based resistances are selected from:

(i) resistances to treatment with clarithromycin, metronidazole and levofloxacin;

(ii) resistances to treatment with clarithromycin, and metronidazole;

(iii) resistances to treatment with clarithromycin and levofloxacin; or

(iv) resistances to treatment with metronidazole and levofloxacin.

12. The method of any one of claims 1 to 11, wherein the multiplex molecular means is a DNA microarray or multiplex PCR.

13. The method of claim 12, wherein the DNA microarray is selected from a printed microarray, in .s/m-synthesized microarray, high-density bead microarray, electronic microarray or suspension bead microarray.

14. A kit for diagnosis of multiple mutated DNA-based resistances of H. pylori to distinct anti -H. pylori treatment modalities, comprising: (a) means and reagents to conduct multiplex genome profiling; (b) detection means; (c) optionally, means and reagents to extract and purify bacterial DNA from a stool sample; and (d) optionally, written instructions.

15. The kit of claim 14, wherein the means to conduct multiplex genome detection and genome profiling is at least one of a multiplex PCR, printed microarray, in situ- synthesized microarray, high-density bead microarray, electronic microarray or suspension bead microarray.