Success of susceptibility-guided eradication of Helicobacter pylori in a region with high secondary clarithromycin and levofloxacin resistance rates

BACKGROUND Resistance to clarithromycin(CLA)and levofloxacin(LFX)of Helicobacter pylori(H.pylori)is increasing in severity,and successful eradication is essential.Presently,the eradication success rate has greatly declined,leaving a large number of patients with previous treatment histories.AIM To investigate secondary resistance rates,explore risk factors for antibiotic resistance,and assess the efficacy of susceptibility-guided therapy.METHODS We recruited 154 subjects positive for Urea Breath Test who attended The First Affiliated Hospital of China Medical University between July 2022 and April 2023.Participants underwent a string test after an overnight fast.The gastric juice was obtained and transferred to vials containing storage solution.Subsequently,DNA extraction and the specific DNA amplification were performed using quantitative polymerase chain reaction(qPCR).Demographic information was also analyzed as part of the study.Based on these results,the participants were administered susceptibility-guided treatment.Efficacy was compared with that of the empiric treatment group.RESULTS A total of 132 individuals tested positive for the H.pylori ureA gene by qPCR technique.CLA resistance rate reached a high level of 82.6%(n=109),LFX resistance rate was 69.7%(n=92)and dual resistance was 62.1%(n=82).Gastric symptoms[odds ratio(OR)=2.782;95%confidence interval(95%CI):1.076-7.194;P=0.035]and rural residence(OR=5.152;95%CI:1.407-18.861;P=0.013)were independent risk factors for secondary resistance to CLA and LFX,respectively.A total of 102 and 100 individuals received susceptibility-guided therapies and empiric treatment,respectively.The antibiotic susceptibility-guided treatment and empiric treatment groups achieved successful eradication rates of 75.5%(77/102)and 59.0%(59/411)by the intention-to-treat(ITT)analysis and 90.6%(77/85)and 70.2%(59/84)by the per-protocol(PP)analysis,respectively.The eradication rates of these two treatment strategies were significantly different in both ITT(P=0.001)and PP(P=0.012)analyses.CONCLUSION H.pylori presented high secondary resistance rates to CLA and LFX.For patients with previous treatment failures,treatments should be guided by antibiotic susceptibility tests or regional antibiotic resistance profile.

Clarithromycin的体内抗菌作用研究

新的１４元大环内酯ｃｌａｒｉｔｈｒｏｍｙｃｉｎ（ＣＲＭ）与红霉素（ＥＭ）对金葡球菌、化脓链球菌、肺炎链球菌、嗜血流感杆菌感染小鼠体内保护作用结果显示：口服ＣＲＭ对金葡球菌、化脓链球菌及肺炎链球菌的体内抗菌作用优于ＥＭ６．３～８．６倍。尤其对金葡球菌耐药菌及嗜血流感杆菌感染小鼠的半数保护剂量作用有效于ＥＭ的保护作用１７及１８倍，ＣＲＭ在体内具有良好的抗菌作用。

Clarithromycin的体外抗菌作用研究

评价国产ｃｌａｒｉｔｈｒｏｍｙｃｉｎ（ＣＲＭ）对临床分离到的４６４株致病菌的体外抗菌作用，并与红霉素（ＥＭ）、麦迪霉素（ＭＤＭ）、麦白霉素（ＭＬＭ）、柱晶白霉素（吉他霉素，ＬＵＭ）进行比较。结果表明ＣＲＭ对ＥＭ敏感的金葡球菌、表葡球菌、肺炎链球菌和化脓链球菌的抗菌作用优于ＥＭ，明显优于其它３种同类对照药，ＭＩＣ（９０）为０．０６２～２ｍｇ／Ｌ；对部分ＥＭ耐药的表葡球茵，化脓链球菌和粪链球菌也有一定的抗菌作用；对流感嗜血杆菌和对ＥＭ敏感的粪链球菌的抗菌作用同ＥＭ，优于其它３种同类对照药，ＭＩＣ_（９０）为０．５～２ｍｇ／Ｌ；对部分厌氧菌也有一定的抗菌作用，细菌接种量对ＣＲＭ的抗菌作用有一定的影响，但仍在敏感范围之中；ｐＨ值的变化影响ＣＲＭ抗菌作用，其抗菌作用在碱性条件下较强，尤以ｐＨ８．５时为显著；少量的人血清不影响ＣＲＭ的抗菌作用，但浓度加至７５％，ＣＲＭ的抗菌作用减弱。

国产clarithromycin片治疗急性细菌性感染临床研究

采用非盲随机对照临床验证设计，治疗药物为国产ｃｌａｒｉｔｈｒｏｍｙｃｉｎ（ＣＲＭ）片，对照药物为国产琥珀酸乙酰红霉素片，共治疗急性细菌性感染１７５例，其中随机对照１３４例（治疗组与对照组各６７例），开放治疗组４１例。结果显示ＣＲＭ片对１０８例患者总的临床痊愈率６４．８％，临床有效率９６．３％，细菌阴转率９６．９％，细菌清除率９６．３％，不良反应发生率７．４％。随机对照研究显示，治疗药对下呼吸道革兰氏阳性需氧菌感染以及口腔间隙厌氧菌感染的疗效与对照药比较均无显著性差异（Ｐ＞０．０５）。本验证细菌培养阳性１５３例，阳性率８７．４％，药敏试验显示了ＣＲＭ良好的体外抗菌作用。疗程结束时，治疗组中的９株耐药菌有８株被清除，反映ＣＲＭ的体内疗效更为显著。

国产clarithromycin治疗急性细菌性感染多中心临床研究

本项多中心临床研究旨在评价国产ｃｌａｒｉｔｈｒｏｍｙｃｉｎ（ＣＲＭ）的安全性和有效性。１２４例被确诊为急性呼吸道感染、皮肤软组织感染的成年患者入选随机对照试验。进口ＣＲＭ作对照药。患者被随机地分入国产或进口ＣＲＭ组。两药均按２５０ｍｇ，ｑ１２ｈ给药，疗程７～１４ｄ。国产ＣＲＭ组临床总有效率为９１．９％，细菌清除率９２．６％，不良反应发生率７．５％；对照组进口ＣＲＭ分别为９１．９％、９１．１％和７．６９％。以上结果经统计学处理，无显著性差异（Ｐ＞０．０５）。开放试验包括４５例急性细菌性感染病例，临床有效率为８６．７％，细菌清除率８７．８％，不良反应发生率６．７％。由以上结果可见，国产与进口ＣＲＭ治疗急性细菌性感染同样安全。

clarithromycin的研究进展及临床应用前景

clarithromycin(CAM)是新的大环内酯类抗生素,是红霉素(EM)的6位羟基被氧甲基取代的衍生物.该药抗菌机制与EM相似,但与EM相比,CAM有更广的抗菌谱、更高的血浓度和组织浓度、更广泛的细胞渗透性及更长的清除半衰期.加上它具有独特的药动学性质,使之成为临床治疗各类感染很有前途的药物.本文就CAM的基础和临床的研究进展综述如下.

Prevalence of primary Helicobacter pylori resistance to metronidazole and clarithromycin in Singapore

INTRODUCTIONEradication of Helicobacter pylori,a bacteriumresiding in stomach and causing peptic ulcer disease,can be achieved by using combination therapiesconsisting of one or two antibiotics with a protonpump inhibitor (PPI).The major antibiotics widelyused in the regimens to eradicate H.pylori aremetronidazole and clarithromycin.However,resistance to these antibiotics by H.pylori

Optimizing clarithromycin-containing therapy for Helicobacter pylori in the era of antibiotic resistance

The efficacy of triple therapy for Helicobacter pylori infection has dramatically declined over the last decade,largely related to increasing clarithromycin resistance rates.From a microbiological standpoint,bismuth quadruple therapy is the ideal replacement since it combines drugs for which resistance does not impair its efficacy.Nonetheless,several obstacles such as availability,complexity or tolerance prevent a general implementation of bismuth quadruple therapy,so nonbismuth quadruple regimens remain the best firstline treatment in clinical practice in many geographical areas.We review the rationale and efficacy of several optimization tools(increasing the length of duration,high-dose acid suppression,probiotics),which have been largely evaluated over the last 5 years to increase the effectiveness of standard triple therapy.Then,we update available evidence on the effectiveness of several non-bismuth quadruple therapies(sequential,concomitant,hybrid,miscellaneous therapy),which have gained interest lately.We also revise evidence on the efficacy of the aforementioned optimization tools for non-bismuth quadruples schemes and,finally we provide a novel regionalized therapeutic algorithm,based on novel formulas recently developed for predicting the outcome of non-bismuth quadruple regimens,upon local antibiotic resistance rates.

Punctual mutations in 23S rRNA gene of clarithromycinresistant Helicobacter pylori in Colombian populations

AIM To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori(H. pylori) and determine their association with therapeutic failure.METHODS PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin(29 from a low-risk gastric cancer(GC) population: TumacoColombia, and 5 from a high-risk population: TuquerresColombia) and 40 from a susceptible population(28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and Mc Nemar's test was performed to determine the relationship between H. pylori mutationsand clarithromycin resistance.RESULTS23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin(20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates(13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593 T1, A1653 G2, C1770 T, C1954 T1, and G1827 C in isolates from Tumaco, and A2144 G from Túquerres. The mutations T2183 C, A2144 G and C2196 T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori(κ = 0.71).CONCLUSION The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycinresistant H. pylori.

Primary clarithromycin resistance to Helicobacter pylori : Is this the main reason for triple therapy failure?

Conventional triple therapies for Helicobacter pylori (H. pylori ) eradication have recently shown a disappointing reduction in effectiveness in many countries. The main reason for failure was found to be bacterial resistance to one of the most commonly used antibiotics, clarithromycin. An additional problem for conventional triple therapy is the high rate of resistance to metronidazole found in Europe, America and Asia. In Italy, in the last 15 years a 2-fold increase in resistance has occurred. A recent study of the whole of Italy included about 20 patients from each region at the first endoscopic diagnosis of H. pylori infection. The most surprising result was the patchy distribution of resistance, which was almost absent in two regions (one northern and one southern), although the highest prevalence was found in some regions of the South. In the paediatricpopulation we found a 25% prevalence of resistance in a sample of H. pylori positive children observed between 2002 and 2007, mirroring data obtained in southern European countries. Clarithromycin resistance assessment is currently based on phenotypic detection performed after culture the agar dilution method or E-test, and genotypic methods based on polymerase chain reaction (PCR). In a recent comparative study we found a 71.2% agreement between the two methods. Culture-free techniques are highly accurate in finding even minimal traces of genotypically resistant strains. Moreover, PCR-based tools are accurate in detecting a heteroresistant status, defined as the co-existence of some strains that are susceptible and some resistant to the same antibiotic in an individual patient. Three point mutations, namely A2143G , A2142G and A2142C , are responsible for 90% of cases of primary clarithromycin resistance in H. pylori strains isolated in Western countries, although we previously demonstrated that the presence of the A2143G mutation, but not A2142G or A2142C , significantly lowered the H. pylori eradication rate. Treatment failure has considerable cost/benefit implications because of 'waste' of National Health System and patient resources, in terms of drugs, further diagnostic tests and medical examination expenses. Therefore, in future it would be very useful to be able to test for clarithromycin resistance before starting conventional triple therapy. Hopefully, fast, effective noninvasive tests may soon be devised to determine this condition.

Resistance to clarithromycin and gastroenterologist's persistence roles in nomination for Helicobacter pylori as high priority pathogen by World Health Organization

Due to the increasing prevalence of clarithromycin resistance, future of management of Helicobacter pylori(H. pylori) infections need to be recognized. To now, clarithromycin was the best effective, well-tolerated and safe antibiotic used in treatment of the bacterium, but, increasing trend of resistance reduced efficacy of recommended regimens. Indeed, gastroenterologists are mostly unable to start appropriate therapyaccording to the sensitivity profile-due to the certain difficulties in routine H. pylori culture procedure and being time consuming method. This announcement by World Health Organization(WHO) was an onset to reconsider current challenging dilemma about H. pylori clarithromycin resistant isolates. Therefore, investigating of various factors affecting this nomination by WHO is highly welcomed. In fact, WHO enumerated more than 16 pathogens which seriously threats human life and public health, thus better management or effective guidelines are necessary. Here for the first time, we nominated this phenomenon as ‘‘gastroenterologist's persistence'' which should be equally investigated as antibiotic resistance. The ability of gastroenterologists to win the game against H. pylori infections is highly influenced by their collaboration with diagnostic laboratories to apply susceptibility patterns before any prescription. In conclusion, closer collaboration between two important partners(gastroenterologists and microbiologists) in management of H. pylori infection may hopefully trigger an era to remedy current crisis in clarithromycin resistance, a later gastric cancer can be practically preventable.

Clinical role and importance of fluorescence in situ hybridization method in diagnosis of H pylori infection and determination of clarithromycin resistance in H pylori eradication therapy

H pylori is etiologically associated with gastritis, gas-tric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Eradicating H pylori may convert rapidly the outcome of related diseases with the use of more accurate diagnostic molecular tests. Indeed some of the tests cannot give the evidence of current infection; H pylori can be detected by noninvasive and invasive methods, the latter requiring an endoscopy. Eradication failure is a big problem in H pylori infection. Recently, clarithromycin resistance in H pylori strains is increasing and eradicati-on therapy of this bacterium is becoming more difficult. Molecular methods have frequently been applied besides phenotypic methods for susceptibility testing to detect clarithromycin resistance due to mutations in the 2143 and 2144 positions of 23S rRNA gene. Fluorescence in situ hybridization (FISH) method on paraffin embedded tissue is a rapid, accurate and cost-effective method for the detection of H pylori infection and to determine clarithromycin resistance within three hours according to the gold standards as a non-culture method. This method can also be applied to fresh biopsy samples and the isolated colonies from a culture of H pylori, detecting both the culturable bacillary forms and the coccoid forms of H pylori, besides the paraffin embedded tissue secti-ons. This technique is helpful for determining the bac-terial density and the results of treatment where clarith-romycin has been widely used in populations to increase the efficacy of the treatment and to clarify the treatment failure in vitro.

He/icobacter py/ori infection in hemodialysis patients: Susceptibility to amoxicillin and clarithromycin

AIM: To evaluate susceptibility of Helicobacter pylori to amoxicillin and clarithromycin in end-stage renal disease (ESRD) patients and non-uremic controls. METHODS: The subjects with dyspeptic complaints were 33 ESRD patients and 46 age- and sex-matched non-uremic controls who exhibited H pylori on antral biopsy specimens. The two groups were age and sex matched. The H pylori strains' pattern of susceptibility to amoxicillin and clarithromycin was investigated with the agar dilution technique. RESULTS: None of the H pylori strains from either group showed resistance to amoxicillin with the agar dilution method. Twelve (36.4%) of the ESRD group strains and 7 (15.2%) of the control group strains showed resistance to clarithromycin, and this difference was statistically significant (P<0.05). CONCLUSION: Resistance to amoxicillin does not appear to be an important problem in H py/ori-infected ESRD and non-uremic patients in our region. In contrast, the rates of resistance to clarithromycin are high, particularly in the ESRD population.

Drug utilization of clarithromycin for gastrointestinal disease treatment

AIM： To evaluate the patterns of use of clarithromycin for gastrointestinal disease treatment and promote its rational use.METHODS： Using a structured pro forma, we conducted a two-month survey of the electronic prescriptions containing immediate-release （IR） or sustained-release （SR） product of clarithromycin for outpatients with gastrointestinal diseases in a 2200-bed general hospital. Suitability of the prescription was audited retrospectively. RESULTS： One hundred and sixty-four prescriptions of SR product and 110 prescriptions of IR product were prescribed for gastrointestinal disease treatment. Among prescriptions for anti-Helicobacter pylori （H pylori） therapy, triple therapy take the dominant position （91.8%）, followed by quadruple therapy （4.3%） and dual therapy （3.9%）. Amoxicillin was the most frequently co-prescribed antibiotic.Furazolidone and levofloxacin are used more widely than metronidazole or tinidazole. Clarithromycin SR was administered at inappropriate time points in all prescriptions. Fifty percent of all prescriptions of clarithromycin SR, and 6.4% of prescriptions of clarithromycin IR, were prescribed at inappropriate dosing intervals. Surprisingly, disconcordance between diagnoses and indications was observed in all prescriptions of clarithromycin SR which has not been approved for treating Hpy/ori infection although off-label use for this purpose was reported in literature. On the contrary, only one prescription （0.9%） of clarithromycin IR was prescribed for unapproved indication （i.e. gastro-oesophageal reflux disease）. 1.4% of prescriptions for chronic gastritis or peptic ulcer treatment were irrational in that clarithromycin was not co-prescribed with gastric acid inhibitors. Clinical significant CYP3A based drug interactions with clarithromycin were identified. CONCLUSION： There is a great scope to improve the quality of clarithromycin prescribing in patients with gastrointestinal disease, especially with regard to administration schedule, concordance between indications and diagnoses and management of drug interactions.

Determination of Clarithromycin in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry： Validation and Application in Clinical Pharmacokinetic Study

Aim To develop a liquid chromatographic-tandem mass spectrometric (LC-MS-MS)method to determine clarithromycin in human plasma. Methods The analyte and internal standardroxithromycin were extracted from plasma samples by n-nexane-dichloromethane-isopropanol(300:150:15, V/V/V) and chromalographed on a C_(18) column. The mobile phase consisted ofmethanol-water-formic acid (80 = 20:1, V/V/V) . Detection was performed on a triple quadrupoletandem mass spectrometer via electrospray ionization source (ESI) in the positive mode. Results Themethod had a lower limit of quantification of 10.0 ng·mL^(-1) when 0.2 mL plasma was used. Thelinear calibration curves were obtained in the concentration range of 10.0 - 5000 ng·mL^(-1) . Theintra- and inter-run precisions were lower than 3.3% in terms of relative standard deviation (RSD),and the accuracy ranged +- 0.7% in terms of relative error (RE). T_(max), C_(max), T_(1/2) andAUC_(0-24h), values were found to be (3.1 +- 2.7)h, (8 750+-4734) ng·mL^(-1), (5.3+-2.2) h, and(5932+-2449)ng·mL^(-1), respectively, after a single oral dose of 250 mg clarithromycin tablet to18 volunteers. Conclusion This validated method was successful in the evaluation of phaimacokineticprofiles of clarithromycin tablets administered to 18 healthy male volunteers.

Rabeprazole, clarithromycin, and amoxicillin Helicobacter pylori eradication therapy: Report of an efficacy study

AIM: To investigate the efficacy of a standard triple therapy (comprising rabeprazole, clarithromycin, and amoxicillin) for Helicobacter pylori (H. pylori) eradication, noting factors that influence the outcome and documenting any adverse events.

Characterization of clarithromycin resistance in Malaysian isolates of Helicobacter pylori

AIM： To characterize the types of mutations present in the 23S rRNA genes of Malaysian isolates of clarithromycin-resistant Helicobacter pylori （H pylorl~. METHODS： Clarithromycin susceptibility of H pylori isolates was determined by E test. Analyses for point mutations in the domain V of 23S rRNA genes in clarithromycin-resistant and -sensitive strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using Bsa I and MboI enzymes to detect restriction sites that correspond to the mutations in the clarithromycin- resistant strains. RESULTS： Of 187 isolates from 120 patients, four were resistant to clarithromycin, while 183 were sensitive. The MIC of the resistant strains ranged from 1.5 to 24 pg/mL. Two isolates had an A2142G mutation and another two had A2143G mutations. A T2182C mutation was detected in two out of four clarithromycin-resistant isolates and in 13 of 14 clarithromycin-sensitive isolates. Restriction enzyme analyses with Bsa I and Mbo I were able to detect the mutations. CONCLUSION： Clarithromycin resistance is an uncommon occurrence among Malaysian isolates of Hpylori strains and the mutations A2142G and A2143G detected were associated with low-level resistance.

Omeprazole-based triple therapy with low-versus high-dose of clarithromycin plus amoxicillin for H pylori eradication in Iranian population

AIM： To investigate the efficacy and tolerability of H pylori eradication in an omeprazole-based triple therapy with high and low dose of clarithromycin and amoxicillin. METHODS： One hundred and sixty H pylori positive patients were randomly assigned to two groups based on the following 2 wk investigation; （1） group A or low-dose regimen received omeprazole 20 mg b.i.d, clarithromycin 250 mg b.i.d and amoxicillin 500 mg b.i.d; and （2） group B or high-dose regimen received omeprazole 20 mg b.i.d, clarithromycin 500 mg b.i.d and amoxicillin 1000 mg b.i.d. During the study Hpylori status was assessed by histology and rapid urease test prior and by 13C-urea breath test 6 wk after the therapy. Standard questionnaires were administered to determine the compliance to treatment and possible adverse events of therapy. Data were subject to x^2 to compare the eradication rates in the two groups. The significant level of 95% （P ≤ 0.05） was considered statistically different. RESULTS： We found that the per-protocol eradication rate was 88% （68/77） in group A, and 89% （67/75） in group B. The intension-to-treat eradication rate was 85% （68/80） in group A and 83.75% （67180） in group B. Overall adverse events were 26% in group A and 31% in group B. The adverse events were generally mild in nature and tolerated well in both groups with a compliance of 98% in group A vs 96% in group B. CONCLUSION： The omeprazole-based low dose regimen of darithromycin and amoxicillin for two weeks in Hpylori eradication is as effective as high dose regimen in Iranian population.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23 Sr RNA gene in Helicobacter pylori(H. pylori) by nested-allele specific primer-polymerase chain reaction(nested-ASP-PCR).METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test(RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nestedASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23 SrR NA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates thanASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87(87.88%) and 67(67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.