Molecular basis of clarithromycin-resistance in Mycobacterium avium intracellulare complex.

Nucleotide sequences of domain V and domain II regions of the 23S rRNA gene were determined in both in vitro-made mutants and clinical isolates of Mycobacterium avium and M. intracellulare conferring clarithromycin-resistance. All laboratory-made mutants showed high level resistance to clarithromycin (> 150 micrograms ml-1) and mutation at position 2058 (cognate with Escherichia coli base) in domain V region. In the clinical isolates, while the susceptible ones had no mutation in domain V, the resistant strains showed mutation at 2058 or 2059. Six isolates with low level of resistance exhibited no mutation in domain V. All strains tested had no mutation in domain II region. These results suggested that most of the resistance arose from the mutation in domain V of the 23S rRNA gene, but other unknown mechanisms evidently exist in mycobacteria.     

39株对克拉霉素耐药的结核分枝杆菌临床分离株23SrRNA检测结果分析

目的 分析对克拉霉素耐药的结核分枝杆菌临床分离株23S rRNA的A2058位点的变化。 方法 选择我院菌株库的结核分枝杆菌临床分离株64株,其中10株为对全部抗结核药物敏感的结核分枝杆菌临床分离株;14株为单耐克拉霉素的结核分枝杆菌临床分离株;15株为耐多药,同时耐克拉霉素的结核分枝杆菌临床分离株;15株为耐多药,同时对克拉霉素敏感的结核分枝杆菌临床分离株;10株为广泛耐药菌株,同时对克拉霉素耐药的结核分枝杆菌临床分离株;此外,还有结核分枝杆菌标准株H37Rv 1株。对结核分枝杆菌23S rRNA行PCR检测和测序。 结果 经检测,H37Rv标准株没有A2058突变,只有1株广泛耐药临床分离株检测有A2058A-G的突变,其他临床分离株均没有突变,在耐克拉霉素的结核分枝杆菌临床分离株中占2.56%（1/39）,在广泛耐药结核分枝杆菌临床分离株中占1/10。 结论 结核分枝杆菌临床分离株对克拉霉素耐药的机制中, A2058突变可能不是产生对克拉霉素耐药的主要机制。结核分枝杆菌产生对克拉霉素耐药的机制有待进一步研究。     

幽门螺杆菌对克拉霉素耐药的分子机制研究

目的：研究幽门螺杆菌（Hp)对克拉霉素耐的分子机制。方法：用E－test进行克拉霉素药敏试验,选取治疗前敏感、治疗后耐药的配对菌株及原发耐药Hp菌株进行研究;应用随机扩增多态性DNA（RAPD）分析,确定治疗前后菌株的同一性;用PCR－限制性片段长度多态性（RFLP）分析探讨克拉霉素耐药机制。结果9株克拉霉素耐药菌23SrRNA基因功能区V PCR扩增片段,8株被BsaI酶切,9株均未被BbsI酶切,提示8株在2144位点有A→G突变。结论上海地区大多数克拉霉素耐药Hp菌株存在23SrRNA基因功能区V2144位点A→G突变。     

幽门螺杆菌基因多态性与克拉霉素耐药性关系的研究

目的研究幽门螺杆菌（Helicobacter pylori,Hp）对克拉霉素耐药情况及与23S rRNA基因点突变的关系。方法因上消化道症状进行胃镜检查的189例患者获得胃活检组织,微需氧培养得到坳,提取11例敏感菌和和19例耐药菌的DNA,对23S rRNA基因进行PCR扩增,再对敏感菌和耐药菌的23S rRNA基因进行全基因测序对比和生物信息学分析。结果Hp菌株对克拉霉素的耐药率是29．2％;19个对克拉霉素耐药的却菌株中17株出现23S rRNA基因突变,各种突变的比例分别为A→G36．8％、G→A21．5％、C→T15．8％、A→C10．5％和T→C5．3％。11例敏感株及2例耐药株均未发现23S rRNA基因突变。结论克拉霉素耐药的却菌株比较常见,23SrRNA基因的多个不同位点突变均与跏对克拉霉素耐药有关,而A—G和G—A突变是主要的形式。     

Changing pattern of antimicrobial resistance of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in Korean patients with peptic ulcer diseases

BACKGROUND: Antibiotic resistance of Helicobacter pylori is problematic because it reduces the efficacy of eradication therapy. It has been suggested that the incidence of resistance is rising. In Korea, information on the antimicrobial resistance of H. pylori is rare. The aim of this study was to assess the prevalence of H. pylori antibiotic resistance at a single center in Korea, and the changes in its antimicrobial resistance, and to detect the mutation foci of clarithromycin-resistant strains. METHODS: H. pylori isolates obtained from 224 patients with peptic ulcer disease in Korea between June 1996 and March 2000 were tested for antimicrobial resistance. The minimum inhibitory concentration (MIC) for metronidazole and clarithromycin was determined by the broth microdilution method. Isolates were considered resistant when the MIC was more than 8 microg/ml for metronidazole and more than 1 microg/ml for clarithromycin. To detect H. pylori 23S rRNA mutations, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed. Sequencing was performed on the two strands of the nonrestricted amplicons. RESULTS: Overall, resistance to metronidazole and clarithromycin was detected in 41.9% and 5.4% of patients, respectively. There was no significant difference in metronidazole and clarithromycin resistance according to age group and sex. Six strains were resistant to both metronidazole and clarithromycin. Six of nine clarithromycin-resistant isolates possessed the A2144G mutation in the gene encoding 23S rRNA. Sequencing of the three non-restricted clarithromycin-resistant strains revealed a T-to-C mutation at position 2182. CONCLUSIONS: In Korea, there was no significant increase in the prevalence of metronidazole resistance, but clarithromycin-resistant H. pylori strains had increased relatively over the 5-year period. There was an increasing tendency for the emergence of strains with dual resistance to metronidazole and clarithromycin. Many of the clarithromycin-resistant strains possessed the A2144G mutation.     

突变敏感性分子开关检测肺炎支原体对大环内酯类抗生素的耐药性研究

目的 应用突变敏感性分子开关检测肺炎支原体对大环内酯类抗生素的耐药性。方法 采用微量稀释法检测5种常用大环内酯类抗生素对40株Mp临床分离株的药物敏感性;建立高保真聚合酶和3'硫化修饰引物的分子开关,用分子开关进行Mp临床分离株的PCR扩增,检测其是否存在Mp 23S rRNA 2063、2064和2617 3个热点突变,并通过基因测序进一步确定是否存在点突变,分析点突变与大环内酯类抗生素敏感性之间的关系。结果 5种大环内酯类抗生素中,Mp对14元环的红霉素和克拉霉素耐药程度最高,其MIC≥128 mg/L;对15元环的大环内酯类抗生素阿奇霉素和交沙霉素相对敏感,其中交沙霉素抗Mp活性最高,其MIC≤4 mg/L。用高保真聚合酶和3'硫化修饰引物的分子开关行PCR扩增,检测出35株发生了2063位点基因突变,3株发生2064位点基因突变,未检测出2617位点突变。用基因测序检测到35株Mp发生A2063G的突变,3株发生A2064G的突变,未检测到2617位点突变,与分子开关的检测结果一致,并且2063、2064位点突变Mp株均对大环内酯类药物高度耐药。结论 分子开关可识别23S rRNA基因突变,可用于分析Mp对大环内酯类抗生素的敏感性。     

23S rRNA base pair 2057-2611 determines ketolide susceptibility and fitness cost of the macrolide resistance mutation 2058A-->G

The 23S rRNA A2058G alteration mediates macrolide, lincosamide, and streptogramin B resistance in the bacterial domain and determines the selectivity of macrolide antibiotics for eubacterial ribosomes, as opposed to eukaryotic ribosomes. However, this mutation is associated with a disparate resistance phenotype: It confers high-level resistance to ketolides in mycobacteria but only marginally affects ketolide susceptibility in streptococci. We used site-directed mutagenesis of nucleotides within domain V of 23S rRNA to study the molecular basis for this disparity. We show that mutational alteration of the polymorphic 2057-2611 base pair from A-U to G-C in isogenic mutants of Mycobacterium smegmatis significantly affects susceptibility to ketolides but does not influence susceptibility to other macrolide antibiotics. In addition, we provide evidence that the 2057-2611 polymorphism determines the fitness cost of the 23S rRNA A2058G resistance mutation. Supported by structural analysis, our results indicate that polymorphic nucleotides mediate the disparate phenotype of genotypically identical resistance mutations and provide an explanation for the large species differences in the epidemiology of defined drug resistance mutations.     

粪便基因型检测幽门螺杆菌对克拉霉素耐药性的研究

背景：幽门螺杆菌（Hp）耐药情况日趋严重,选择快速、敏感、价廉的分子生物学技术对Hp耐药进行检测具有重要的临床意义。目的：评价检测粪便Hp基因突变对诊断克拉霉素耐药的有效性,并探讨cagA基因与耐药的相关性。方法：纳入74例13C-尿素呼气试验阳性患者,采集其新鲜粪便标本,提取粪便DNA,采用巢式PCR法扩增Hp23SrRNA,采用PCR—RFLP法检测限制性内切酶BbsI、BceAI、BsaI对23SrRNA扩增产物的酶切情况,采用PCR法扩增cagA基因。结果：74例患者的粪便标本中,60例扩增出Hp23SrRNA367bp片段,其中17例可被BsaI酶切,60例均未被BbsI、BceAI酶切。cagA阳性、阴性表达者的23SrRNA突变率相比差异无统计学意义（P〉0．05）。结论：通过粪便基因型检测Hp对克拉霉素耐药是快速、简便的方法。江苏地区Hp对克拉霉素的耐药机制主要为23SrRNAA2143G突变。cagA基因与Hp对克拉霉素耐药不相关。     

Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure

BACKGROUND: It was recently reported that A to G transition mutations at positions 2143 and 2144 in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori. AIMS: To study the incidence and mechanism of development of clarithromycin resistance by analysing these mutations. SUBJECTS: Eighty two H pylori positive patients who had an endoscopic examination and no history of treatment with macrolide antibiotics. METHODS: Clarithromycin resistance was screened for by polymerase chain reaction-restriction fragment length polymorphism of the 23S rRNA gene coupled with antibiotic susceptibility testing. In clinical isolates with mutations or resistance, mutations in individual colonies were analysed by direct sequencing. RESULTS: Of the 79 amplicons (DNA fragments amplified by polymerase chain reaction), Alw26I and MboII digestion disclosed the mutation in four (5%) and one (1%) respectively. However, the Alw26I cleavage was incomplete in two of the four amplicons, as was the MboII cleavage. Individual colony analysis of the isolates with incomplete cleavage patterns showed the presence of both wild type and mutated strains in the 23S rRNA genes. CONCLUSIONS: Both clarithromycin sensitive and resistant strains colonised in some patients with no history of exposure to macrolides. The results suggest that resistant strains may not be formed but selected by clarithromycin administration.     

Clarithromycin resistance and point mutations in the 23S rRNA gene in Helicobacter pylori isolates from Malaysia

OBJECTIVE: To determine the prevalence of primary clarithromycin resistance amongst Helicobacter pylori (H. pylori) strains in Malaysian patients with gastroduodenal diseases, by using restriction fragment length polymorphism (RFLP) in domain V of 23S rRNA. METHODS: Gastric biopsies were obtained from H. pylori positive patients undergoing gastroscopy. DNA extraction was followed by PCR amplification using the primers Hp23-1 and Hp23-2 flanking a region of 425bp within the bacterial 23S rRNA peptidyltranferase (Hp23S fragment). Analysis of the 23S rRNA gene mutations is based on the generation of restriction sites for two restriction enzymes: BbsI and BsaI, which correspond to the base substitutions characteristic of clarithromycin resistance from A to G at positions 2142 and 2143, respectively. RESULTS: Gastric biopsy samples were obtained from 107 patients. A fragment of size 425bp corresponding to that expected from amplification of domain V of 23S rRNA was PCR-amplified from only 105 samples. The amplicon was subsequently subjected to restriction by BbsI and BsaI. Only 1 sample (0.95%) had the BbsI mutation (base substitution at A2142G) and 2 samples (1.90%) the BsaI mutation (base substitution at A2143G). Thus 3 of 105 (2.9%) samples harbored clarithromycin resistant strains. CONCLUSION: In our experience, PCR-RFLP is a rapid and precise method to detect the resistance of H. pylori to clarithromycin. Using this method, a low prevalence of clarithromycin resistance was detected in our local Malaysian strains. This augurs well for the continued use of clarithromycin as a first line drug in the treatment and eradication of H. pylori infection.     

Skin, soft tissue, and bone infections due to Mycobacterium chelonae chelonae: importance of prior corticosteroid therapy, frequency of disseminated infections, and resistance to oral antimicrobials other than clarithromycin.

Little is known of clinical disease due to Mycobacterium chelonae chelonae. One hundred skin, soft tissue, or bone isolates of this rapidly growing mycobacterium were identified over 10 years. Clinical disease included disseminated cutaneous infection (53%); localized cellulitis, abscess, or osteomyelitis (35%); and catheter infections (12%). Underlying conditions with disseminated infection included organ transplantation, rheumatoid arthritis, and autoimmune disorders; 92% involved corticosteroid use. Trauma and medical procedures were risk factors for localized infections. Corticosteroids and chronic renal failure were risk factors for catheter infections. Overall, 62% of patients were receiving corticosteroids and 72% were immunosuppressed. MICs of six oral antimicrobials were obtained for 180 isolates by broth microdilution. Up to 20% of isolates were susceptible to doxycycline, ciprofloxacin, ofloxacin, and sulfamethoxazole. In contrast, 100% were susceptible to clarithromycin (MICs less than or equal to 1 microgram/mL). Disease due to M. chelonae chelonae usually occurs in the setting of corticosteroid therapy and is often disseminated; the organisms require high MICs of oral antimicrobials other than clarithromycin.     

Azithromycin resistance in Treponema pallidum

PURPOSE OF REVIEW: Although the recommended treatment for syphilis is penicillin, azithromycin has been used as an alternative. We discuss azithromycin-related treatment failures and resistance in Treponema pallidum, and propose ways to meet the resulting clinical and public health challenges. RECENT FINDINGS: Azithromycin treatment failures in syphilis were first noted in San Francisco in 2002 and result from an A-->G mutation at position 2058 of the 23S rRNA gene of T. pallidum. This mutation confers resistance by precluding macrolide binding to the bacterial 50S ribosomal subunit, of which 23S rRNA is a structural component. Azithromycin resistance has also been identified in T. pallidum specimens from elsewhere in the United States, Ireland, and Canada, and the amount of resistant specimens has increased with time. Treatment with azithromycin or other macrolides appears to be a risk factor for presenting with a resistant T. pallidum strain. SUMMARY: Although T. pallidum remains sensitive to penicillin and certain other antibiotics, azithromycin resistance in T. pallidum has emerged and is increasing in the United States, Canada, and Ireland. This poses clinical and public health challenges, and indicates a need for further antibiotic drug development and surveillance for resistance in T. pallidum. If azithromycin is used to treat syphilis, clinicians and public health practitioners should remain vigilant for treatment failures.     

Clinical and molecular analysis of macrolide resistance in Mycobacterium avium complex lung disease

RATIONALE: The clinical features and outcome of macrolide-resistant Mycobacterium avium complex (MAC) lung disease are not known. OBJECTIVES: Characterize patients, treatment, and isolates in macrolide-resistant MAC lung disease. METHODS: Retrospective chart review, susceptibility testing, molecular fingerprinting, and DNA sequence analyses of resistant MAC isolates. MEASUREMENTS AND MAIN RESULTS: We identified 51 patients over a 15-yr period with clarithromycin-resistant MAC (minimum inhibitory concentration (MIC)>or=32 microg/ml) lung disease at a single referral center. Twenty-four (47%) patients had nodular disease with bronchiectasis and 27 (53%) had upper lobe cavitary disease. Most patients (77%) had M. intracellulare. Sequencing of the 23S r-RNA gene showed 49 of 51 isolates (96%) with the expected mutation in adenine 2058 or 2059. Risk factors for resistance included macrolide monotherapy or combination with a quinolone only (39/51 or 76%). Macrolide resistance developed in 12 of 303 (4.0%) patients started on the American Thoracic Society-recommended two companion drugs, with no risk difference in clarithromycin versus azithromycin and daily versus intermittent therapy. Sputum conversion with macrolide-resistant MAC occurred in 11 of 14 (79%) patients who received more than 6 mo of injectable aminoglycoside therapy and lung resection, compared with 2 of 37 (5%) who did not. The 1-yr mortality in patients who remained culture positive was 34% (13/38) compared with 0% (0/13) of patients who became culture negative (converted). CONCLUSIONS: Macrolide resistance rarely occurs in patients also receiving ethambutol and a rifamycin. Macrolide-resistant MAC lung disease requires aggressive drug and surgical therapy for cure.     

Comparison of clarithromycin-sensitive and clarithromycin-resistant Mycobacterium avium strains isolated from AIDS patients during therapy regimens including clarithromycin

OBJECTIVES: Sixteen Mycobacterium avium strains were isolated from the blood of eight AIDS patients over a period of months. All the patients were on combination therapies including clarithromycin, and all had treatment failure and relapses of M.avium bacteremia. Paired clarithromycin-sensitive and resistant M.avium strains isolated at the beginning of treatment and at the first relapse of bacteremia were compared. METHODS: The M.avium isolates were identified after hybridization with DNA probes specific for M.avium rRNA and typed epidemiologically with random amplified polymorphic DNA analyses using three arbitrary primers. The rate of intracellular cell entry or the tumour necrosis factor alpha induction by the M.avium isolates were studied in human monocytes and J774 cells. RESULTS: When the M.avium isolates were hybridized with the rRNA probes, we obtained lower hybridization values with clarithromycin-resistant isolates than with clarithromycin-sensitive isolates. This appeared to be due to smaller amounts of rRNA available for hybridization than to mutation of the 23S rRNA sequences in clarithromycin-resistant strains. The RAPD analyses showed that the clarithromycin-resistant isolates were clonally related to the clarithromycin-sensitive strains in six of the eight patients. The other two patients had a RAPD profile, suggesting a re-infection and/or polyclonal infection. The M.avium isolates obtained on day 0 and after the emergence of resistance to clarithromycin did not differ in terms of their intracellular entry rate, or in terms of tumour necrosis factor alpha induction. CONCLUSIONS: We infer that M.avium strains isolated during bacteraemic relapses on combination therapies including clarithromycin are epidemiologically related to the initial strain and do not show changes in the rate of intracellular cell entry and in terms of tumour necrosis factor alpha induction. Re-infections and/or polyclonal infections however, although less frequent, can also occur.     

Modified allele-specific primer-polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin

BACKGROUND AND AIM: Most clarithromycin-resistant strains of Helicobacter pylori have a mutation from adenine (A) to guanine (G) at position 2142 or 2143 of the 23S rRNA gene. Our aim in this study was to develop a polymerase chain reaction (PCR)-based assay that could determine these mutations in a single reaction tube. METHODS: We designed the forward primer FP2143G and the reverse primer RP2142G, which specifically anneal with the 2143G- and 2142G-mutated sequences, respectively, of the 23S rRNA gene of H. pylori. We also designed the forward primer FP-1 and reverse primer RP-1 upstream and downstream from the positions 2142 and 2143, respectively, to distinguish the wild-type A2142G and A2143G mutations from each other by amplicon sizes. DNA was extracted from 292 gastric tissue samples positive for rapid urease test, and the DNA underwent the PCR reaction. The results were compared with minimum inhibitory concentrations (MIC) for clarithromycin. RESULTS: Helicobacter pylori strains with A2142G, A2143G and wild type could be distinguished by amplicon sizes by a single PCR reaction. The genotyping results were correlated well with the MIC values for clarithromycin. The median MIC for clarithromycin of the wild-type strains was <0.015 microg/mL. Those of strains with 2142G or 2143G were > or =1.0 microg/mL. CONCLUSION: Our new PCR-based assay for 23S rRNA mutations of H. pylori is a useful method for detecting clarithromycin-resistant strains of H. pylori easily.     

New mutation points in 23S rRNA gene associated with Helicobacter pylori resistance to clarithromycin in northeast China

AIM: To investigate the resistance rate of Helicobacter pylori (H pylori ) to clarithromycin, metronidazole, amoxicillin and tetracycline to guide clinical practice, and to study the mechanism of H pylori resistant to clarithromycin. METHODS: Thirty H pylori strains were isolated from the mucosa of peptic ulcer, gastric tumor and chronic gastritis patients, then the minimal inhibitory concentration (MIC) to clarithromycin, metronidazole, amoxicillin and tetracycline was evaluated by E-test method. The sequence analysis of PCR fragments was conducted in 23S rRNA gene of H pylori resistant to clarithromycin to get the resistance mechanism of the bacteria. RESULTS: Among 30 H pylori strains, 7 cases were resistant to clarithromycin, 12 to metronidazole, 2 to tetracycline and no strain was found to be resistant to amoxicillin. The resistance rates were 23.3%, 40%, 6.7% and 0%, respectively. Three new mutation points were found to be related to the clarithromycin resistance in H pylori isolates, which were G2224A, C2245T and T2289C. CONCLUSION: In northeast China, H pylori shows high resistance to metronidazole, while sensitive to amoxicillin. The mechanism of resistance to clarithromycin may be related to the mutation of G2224A, C2245T and T2289C in the 23S rRNA gene.     

Rapid and specific detection of Helicobacter pylori macrolide resistance in gastric tissue by fluorescent in situ hybridisation

BACKGROUND: The development of macrolide resistance in Helicobacter pylori is considered an essential reason for failure of antibiotic eradication therapies. The predominant mechanism of resistance to macrolides, particularly clarithromycin, is based on three defined mutations within 23S rRNA, resulting in decreased binding of the antibiotic to the bacterial ribosome. AIM: To develop an rRNA based whole cell hybridisation method to detect Helicobacter species in situ within gastric tissue, simultaneously with its clarithromycin resistance genotype. METHODS: A set of fluorescent labelled oligonucleotide probes was developed, binding either to H pylori 16S rRNA or 23S rRNA sequences containing specific point mutations responsible for clarithromycin resistance. After hybridisation and stringent washing procedures, labelling of intact single bacteria was monitored by fluorescence microscopy. The new approach was compared with PCR based assays, histology, and microbiological culture. RESULTS: In comparison with the phenotypic resistance measurement by E test, the genotypic clarithromycin resistance correlated perfectly (100%) for 35 H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pylori infection was confirmed by histology (17/27) and correctly detected by whole cell hybridisation. Five clarithromycin resistant strains were identified in gastric tissue specimens directly. Furthermore, non-cultivable coccoid forms of H pylori were easily detectable by whole cell hybridisation. CONCLUSIONS: Whole cell hybridisation of rRNA holds great promise for cultivation independent, reliable, and rapid (three hours) genotypic determination of clarithromycin resistance in H pylori. Compared with PCR techniques it is independent of nucleic acid preparations, not prone to inhibition, and allows semiquantitative visualisation of the bacteria within intact tissue samples.     

CagA＋及VacA＋幽门螺杆菌对克拉霉素的耐药性突变分析

目的分析CagA＋及VacA＋的幽门螺杆菌（Hp）对克拉霉素耐药与23S rRNA基因点突变的关系。方法采集右江民族医学院附属医院2006~2008年确诊为Hp感染患者的胃窦部黏膜样本进行Hp分离培养和鉴定,PCR扩增CagA＋及VacA＋基因,E-test进行药敏实验,PCR方法扩增23S rRNA基因,基因测序检测克拉霉素耐药菌株的点突变。结果对克拉霉素耐药的CagA＋及VacA＋Hp菌株均存在23S rRNA基因v功能区第2144位和第2143位A-G突变,而敏感菌株没有发现该位点突变。结论Hp对克拉霉素耐药的23S rRNA基因A2143G、A2144G点发生突变,与基因分型无关。     

Mutations in genes related to drug resistance in Mycobacterium leprae isolates from leprosy patients in Korea

OBJECTIVE: Identification of the presence and drug resistance of Mycobacterium leprae is key to the diagnosis and treatment of leprosy in non-endemic country like Korea. The aim of this study was to screen the drug target DNA such as folP, rpoB, gyr, and 23S rRNA of drug resistance strain of M. leprae. PATIENTS AND METHODS: Sequences of those genes were analyzed for the 104 bacterial index positive cases out of 171 leprosy patients in Korea using touchdown PCR, single stranded conformational polymorphism. RESULTS: Twenty (19.2%) cases have shown the mutations in folP gene of dapsone-resistant M. leprae in which three (2.89%) cases were mutations in two genes, folP and rpoB, of multidrugs resistant strains to dapsone and rifampin, and two (1.92%) cases in folP and gyr genes of resistance to dapsone and oflaxacin, respectively. Besides double mutation for folP gene was one case (0.96%) and for rpoB gene one case, respectively. There was no mutant isolates in 23S rRNA gene against clarithromycin. CONCLUSIONS: This result should leads to a better understanding of the status of multidrug resistant leprosy in Korea and may assist in the rapid diagnosis of drug resistant M. leprae and the choice of the appropriate treatment regimens.     

Detection of genotypic clarithromycin-resistant Helicobacter pylori by string tests

AIM:To evaluate the utility of the string test to detect genotypic clarithromycin-resistant Helicobacter pylori (H.pylori)by polymerase chain reaction(PCR)-restriction fragment length polymorphism.METHODS:Patients undergoing endoscopic examinations were enrolled in the present study.String tests were done on the next day of endoscopy.Segments of 23S rRNA were amplified from DNA obtained from string tests.PCR-restriction fragment length polymorphism was accomplished by restriction enzymes BbsI and BsaI recognizing the mutation site A to G at 2143or at 2142 of 23S rRNA domain V,respectively.RESULTS:One hundred and thirty-four patients with H.pylori infection underwent string tests.To compare phenotypic resistance,43 isolates were successfully cultured in 79 patients in whom 23S rRNA was successfully amplified.Of five patients with clarithromycinresistant H.pylori,23S rRNA of H.pylori isolates from four patients could be digested by BsaI.In 38 susceptible isolates,23S rRNA of H.pylori isolates from 36 patients could not be digested by either BsaI or BbsI.The sensitivity and specificity of the string test to detect genotypic clarithromycin resistance were 66.7%and97.3%,respectively.Positive and negative predictive values were 80%and 94.7%,respectively.CONCLUSION:String test with molecular analysis is a less invasive method to detect genotypic resistance before treatment.Further large-scale investigations are necessary to confirm our results.