＜Editors' Choice＞ Clinical impact of standardized creatinine on dose adjustment of capecitabine.

Although the Cockcroft-Gault equation is still used for the dose adjustment of many drugs that have been approved prior to creatinine standardization, the clinical impact of standardized creatinine in the dose adjustment of capecitabine is poorly understood. We focused on patients with borderline renal function and evaluated the tolerability and safety of capecitabine in patients who received capecitabine plus oxaliplatin (Cape-Ox). We retrospectively identified patients with resected colorectal cancer who had received adjuvant therapy with Cape-Ox regimen. Creatinine clearance (CrCL) was calculated by the Cockcroft-Gault equation with standardized creatinine measured using enzymatic methods, and adjusted CrCL was estimated by adding 0.2 (mg/dL) to the serum creatinine in the equation. We defined patients with "pseudo-normal" renal function as those who had an adjusted CrCL of ≤50 mL/min in patients with normal renal function (CrCL >50 mL/min). We evaluated the tolerability and grade 2 or severer adverse events of capecitabine treatment. One hundred four patients had normal and 10 had impaired renal function (CrCL <50 mL/min). Among the 104 patients with normal renal function, 23 (22.1%) had pseudo-normal renal function. Seventeen patients completed the eight cycles of Cape-Ox therapy without treatment delay or dose reduction, and all of them had truly normal renal function. The patients with pseudo-normal renal function were more likely to have grade 2 or severer thrombocytopenia than those with truly normal renal function. We should recognize correctly the clinical impact of standardized creatinine in the treatment of borderline renal function with Cape-Ox regimen in patients.

Is area under the curve the best parameter for carboplatin induced emetic risk stratification?

Carboplatin (CBDCA)-induced emetic risk is currently classified on the basis of CBDCA-area under the curve (CBDCA-AUC). We investigated the utility of three CBDCA dosage parameters for predicting emesis by CBDCA. Patients with thoracic cancer treated with CBDCA were included. The endpoints were complete response (CR) and total control (TC). CR was defined as no vomiting and no use of rescue medication during the overall assessment period, whereas TC was defined as no vomiting, nausea, nor use of rescue medication during the overall assessment period. The parameters of CBDCA were defined as follows: (1) CBDCA-AUC; (2) CBDCA/body surface area (BSA): the administered dose of CBDCA per body surface area (mg/m2); and (3) total CBDCA/body: the total administered dose of CBDCA (mg). Eighty-five patients were evaluated. The median CBDCA/BSA but not CBDCA-AUC was higher in patients with non-CR compared to those with CR. Receiver operating characteristic curve analysis revealed that the AUC of CBDCA/BSA for predicting non-CR was higher than that of CBDCA-AUC. CBDCA/BSA shows greater potential for predicting CBDCA-induced emetic risk compared with CBDCA-AUC, which is the parameter in current antiemetic guidelines.

Comparing incidences of infusion site reactions between brand-name and generic vinorelbine in patients with non-small cell lung cancer.

AIM: This study aimed to compare the incidence of infusion site reactions (ISRs) induced by intravenous administration of brand-name and generic vinorelbine (VNR) for treating non-small cell lung cancer. METHOD: This single-centre retrospective cohort study was conducted by medical chart review of VNR infusions. ISRs were defined as symptoms around the infusion site, including pain, redness and swelling. ISRs requiring treatment were defined as ISRs requiring treatments including steroid ointments, vein repuncture and local steroid injections. RESULTS: In all, 1973 VNR infusions were administered to 340 patients (brand-name 141 patients, generic 199 patients). ISRs and ISRs requiring treatment were observed in 161 and 100 patients, respectively. The ISR incidence per patient and per injection was significantly higher in generic VNR-treated patients than in brand-name VNR-treated patients (53.3% vs 39.0%, P = 0.0112 and 15.0% vs 9.9%, P = 0.0008, respectively). The frequency of ISRs requiring treatment was also significantly higher in the generic group (per patient 36.7% vs 19.2%, P = 0.0005; per injection 11.3% vs 5.5%, P < 0.0001). Multivariate analysis revealed that generic VNR was significantly associated with an increased risk of ISRs (per patient adjusted odds ratio [AOR] 1.775, P = 0.0155; per injection AOR 1.672, P = 0.004) and ISRs requiring treatment (per patient AOR 2.422, P = 0.0012; per injection AOR 2.286, P = 0.001). CONCLUSION: Intravenous infusion of generic VNR was associated with an increased risk of ISRs. Further research is needed to elucidate the mechanism underlying the increased incidence of ISRs with generic VNR.

Factors influencing the effectiveness of recombinant human soluble thrombomodulin on disseminated intravascular coagulation: a retrospective study.

BACKGROUND: Although recombinant human soluble thrombomodulin (rTM) has been widely used to treat disseminated intravascular coagulation (DIC) in Japan, there is no consensus regarding rTM efficacy. Therefore, if the factors influencing rTM efficacy is revealed, it may be possible to demonstrate the effectiveness of rTM by limiting the patients who use rTM. This study investigated the factors of rTM treatment which influence DIC status. METHODS: This retrospective case-control study enrolled hospitalized adult patients treated with rTM from October 2010 to May 2020. Among these patients, 227 who were diagnosed with DIC according to the Japanese Association for Acute Medicine DIC scoring system were assessed. The primary endpoint was the 28-day mortality after rTM treatment. For Cox-proportional hazards model, explanatory factors determined using univariate analysis with p <  0.1 were used. In addition, some factors considered to affect DIC-related mortality such as age ≥ 75 years, rTM dose ≥380 U/kg, antithrombin III treatment, and diseases with a poor prognosis (sepsis, solid tumors, and trauma) were added as covariates. RESULTS: Univariate analyses suggested that male sex (p = 0.029), treatment in intensive care unit (p = 0.061), and prothrombin time-international normalized ratio (PT-INR) (p <  0.001) were the factors influencing DIC-related 28-day mortality after rTM treatment. According to Cox-proportional hazard analysis, the adjusted odds ratio for DIC-related 28-day mortality in patients with PT-INR ≥ 1.67 was 2.23 (95% confidence interval: 1.451-3.433, p <  0.001), age ≥ 75 years was 1.57 (95% confidence interval: 1.009-2.439, p = 0.046), and male sex was 1.66 (95% confidence interval: 1.065-2.573, p = 0.025), respectively. As life-threatening bleeding events were not observed, prolonged PT-INR might directly or indirectly affect DIC-related mortality caused by rTM treatment. CONCLUSION: rTM treatment for DIC was less effective in male patients with PT-INR ≥ 1.67 and age ≥ 75 years.

Short hydration with 20 mEq of magnesium supplementation for lung cancer patients receiving cisplatin-based chemotherapy: a prospective study.

BACKGROUND: Intravenous administration of magnesium with a short hydration regimen is recommended for patients receiving high-dose cisplatin to protect against cisplatin-induced nephrotoxicity. However, the optimal dose of magnesium supplementation has not been clarified. The aim of this trial was to investigate the safety and efficacy of a short hydration regimen with 20 mEq of magnesium supplementation for lung cancer patients receiving cisplatin-based chemotherapy. METHODS: The key eligibility criteria included cytologically or histologically diagnosed lung cancer, candidacy for cisplatin-based (≥ 60 mg/m2) chemotherapy or chemoradiotherapy, no prior chemotherapy, aged 20-75 years, and adequate renal function. Cisplatin was administered with pre-hydration with 20 mEq of magnesium sulfate. Mannitol was administered just before cisplatin infusion to enforce diuresis. The primary endpoint was the proportion of patients who underwent cisplatin-based chemotherapy with a short hydration regimen with 20 mEq of magnesium supplementation without a grade 2 or higher elevation in creatinine. RESULTS: Forty patients with a median age of 66 years (range 35-74) were prospectively enrolled. Median baseline creatinine was 0.71 mg/dL. Median dose of cisplatin in the first cycle was 80 mg/m2. In the first cycle, no patients developed grade 2 creatinine toxicity. During the treatment period, one patient developed grade 2 creatinine elevation; thus, the proportion of patients without a grade 2 or higher elevation in creatinine was 97.5% (95% confidence interval 86.8-99.9). CONCLUSION: A short hydration regimen with 20 mEq of magnesium supplementation is safe and feasible for patients with lung cancer receiving cisplatin-based chemotherapy.

Effect of Side Chain Functional Groups on the DPPH Radical Scavenging Activity of Bisabolane-Type Phenols.

Methods for improving the antioxidant activity of phenolic compounds have been widely investigated; however, most studies have focused on the structure⁻activity correlations of substituents on the aromatic rings of catechols or flavonoids. We investigated the influence of side chain functional groups on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of xanthorrhizol and curcuphenol analogues. These compounds were synthesised by the side chain functional group conversion of curcumene, followed by direct oxidation of the aromatic ring. We determined the DPPH radical scavenging activity from the half-maximal effective concentration (EC50) obtained from a DPPH assay in methanol. The positional relationships of the side chain with the aromatic ring and phenolic OH group were determined using density functional theory calculations, and the stability of different conformations was compared. Electron transfer-proton transfer was determined to be the dominant mechanism in the DPPH reaction with xanthorrhizol analogues, based on the correlation between the EC50 and ionisation potential. The radical cation was greatly stabilised in the structure where the side chain functional group was close to the aromatic ring. Stabilisation also depended on the phenolic OH group position. In future antioxidant design, aromatic ring substituent conversion and the use of functional groups far from the OH group or ring should be explored.

Active surveillance in response to the identification of a single carbapenemase-producing Escherichia coli at a Japanese university hospital.

This report described the experience of active surveillance culture implemented in response to the identification of a single carbapenemase-producing Escherichia coli in a Japanese university hospital. It revealed a horizontal transmission event and an additional asymptomatic carrier of carbapenemase-producing Escherichia coli with unique drug susceptibility and resistance gene profiles. Early implementation of active surveillance culture as a part of multifaceted infection control measures appeared to be useful to control further transmission of carbapenemase-producing Escherichia coli even in the low endemic facility. Further investigations on the timing and usefulness of active surveillance culture in the control of carbapenemase-producing Enterobacteriaceae would be warranted.

Molecular typing and genetic characterization of Mycobacterium avium subsp. hominissuis isolates from humans and swine in Japan.

Mycobacterium avium subsp. hominissuis (MAH) causes disease in both humans and swine; however, the genetic variations in MAH isolates are unclear. The aim of this study was to elucidate the genetic variations in MAH isolates from humans and swine in Japan. We analysed the 16S-23S rDNA internal transcribed spacer (ITS) sequence and variable number of tandem repeats (VNTRs) using the Mycobacterium avium tandem repeat loci, prevalence of ISMav6 and clarithromycin resistance for MAH isolates from patients with pulmonary MAC (pMAC) disease (n=69), and HIV-seropositive and blood culture-positive (HIV-MAC) patients (n=28) and swine (n=23). In the minimum spanning tree based on VNTR analysis, swine MAC isolates belonged to a cluster distinguishable from that of human pMAC isolates. Isolates from HIV-MAC were scattered throughout both clusters. The three major distinct sequevars, Mav-A, Mav-B and Mav-F, were determined according to 16S-23S rDNA ITS sequence analysis in addition to three new sequevars, Mav-Q, Mav-R and Mav-S. Mav-A and Mav-F comprised the majority of human pMAC strains; in contrast, Mav-B predominated in swine isolates. Distribution of ITS sequevars in the minimum spanning tree based on VNTR analysis showed similar clusters of isolates from different origins, i.e. human pMAC, HIV-MAC and swine. These results, together with ISMav6 possession and clarithromycin resistance, revealed the genetic diversity of MAH strains recovered from humans and swine. Molecular epidemiology and genetic characterization in the present study showed the distinctive genetic evolutionary lineage of MAH strains isolated from human pMAC diseases and swine.

The first point prevalence survey of health care-associated infection and antimicrobial use in a Japanese university hospital: A pilot study.

BACKGROUND: Point prevalence surveys (PPSs) in Japanese hospitals have not yet been reported. The purpose of this pilot PPS study was to evaluate the epidemiology of health care-associated infections (HAIs) and antimicrobial use in a Japanese tertiary university hospital. METHODS: A 1-day, cross-sectional PPS was performed at a Japanese university hospital. Data on demographics, active HAIs, and antimicrobial use of all inpatients were collected using a data collection form. RESULTS: Of 841 patients, 85 (10.1%) had 90 active HAIs, and 308 patients (36.6%) were administered 494 antimicrobials. Among the 90 HAIs and 58 pathogens, the most frequent infection and isolated pathogen were pneumonia (20.0%) and Enterobacteriaceae (27.6%), respectively. Of the 118 antimicrobials used for treatment of HAIs, carbapenems were the most frequently administered category of antimicrobials (22.9%). In regard to antimicrobials for surgical prophylaxis, 37 of 119 (31.1%) were administered to patients on postoperative day 3 or later, and 48 of 119 (40.3%) were administered orally. CONCLUSIONS: The incidence of HAIs is higher than in other developed countries. The social and medical situation in Japan may affect patient demographics, active HAIs, and antimicrobial use. Multicenter PPSs are necessary to uncover the real epidemiology of HAIs and antimicrobial use in Japan.

Genetic diversity of clinical Mycobacterium avium subsp. hominissuis and Mycobacterium intracellulare isolates causing pulmonary diseases recovered from different geographical regions.

Mycobacterium avium complex (MAC) infections are increasing annually in many countries. MAC strains are the most common nontuberculous mycobacterial pathogens isolated from respiratory samples and predominantly consist of two species, Mycobacterium avium and Mycobacterium intracellulare. The aim of this study was to analyze the molecular epidemiology and genetic backgrounds of clinical MAC isolates collected from The Netherlands, Germany, United States, Korea and Japan. Variable numbers of tandem repeats (VNTR) analysis was used to examine the genetic relatedness of clinical isolates of M. avium subsp. hominissuis (n=261) and M. intracellulare (n=116). Minimum spanning tree and unweighted pair group method using arithmetic averages analyses based on the VNTR data indicated that M. avium subsp. hominissuis isolates from Japan shared a high degree of genetic relatedness with Korean isolates, but not with isolates from Europe or the United States, whereas M. intracellulare isolates did not show any specific clustering by geographic origin. The findings from the present study indicate that strains of M. avium subsp. hominissuis, but not M. intracellulare, exhibit geographical differences in genetic diversity and imply that MAC strains may have different sources, routes of transmission and perhaps clinical manifestations.

Characterization of a novel plasmid, pMAH135, from Mycobacterium avium subsp. hominissuis.

Mycobacterium avium complex (MAC) causes mainly two types of disease. The first is disseminated disease in immunocompromised hosts, such as individuals infected by human immunodeficiency virus (HIV). The second is pulmonary disease in individuals without systemic immunosuppression, and the incidence of this type is increasing worldwide. M. avium subsp. hominissuis, a component of MAC, causes infection in pigs as well as in humans. Many aspects of the different modes of M. avium infection and its host specificity remain unclear. Here, we report the characteristics and complete sequence of a novel plasmid, designated pMAH135, derived from M. avium strain TH135 in an HIV-negative patient with pulmonary MAC disease. The pMAH135 plasmid consists of 194,711 nucleotides with an average G + C content of 66.5% and encodes 164 coding sequences (CDSs). This plasmid was unique in terms of its homology to other mycobacterial plasmids. Interestingly, it contains CDSs with sequence homology to mycobactin biosynthesis proteins and type VII secretion system-related proteins, which are involved in the pathogenicity of mycobacteria. It also contains putative conserved domains of the multidrug efflux transporter. Screening of isolates from humans and pigs for genes located on pMAH135 revealed that the detection rate of these genes was higher in clinical isolates from pulmonary MAC disease patients than in those from HIV-positive patients, whereas the genes were almost entirely absent in isolates from pigs. Moreover, variable number tandem repeats typing analysis showed that isolates carrying pMAH135 genes are grouped in a specific cluster. Collectively, the pMAH135 plasmid contains genes associated with M. avium's pathogenicity and resistance to antimicrobial agents. The results of this study suggest that pMAH135 influence not only the pathological manifestations of MAC disease, but also the host specificity of MAC infection.

[Extensive genetic analysis of clinical tuberculosis isolates and analysis of host factors to evaluate rapid development of multidrug resistance during initial treatment].

INTRODUCTION: In this study, we aimed at determining the cause of resistance to tuberculosis treatment by performing genetic analyses of bacteria obtained from a patient who developed multidrug-resistant tuberculosis (MDR-TB) during the initial course of treatment for tuberculosis. METHODS: Specimens obtained before and after the development of MDR-TB were subjected to spoligotyping, drug-resistance gene analysis, and variable-number tandem repeat (VNTR) typing. The patient's clinical background was also reviewed. RESULTS: After the development of resistance, the bacterial genome had changed with regard to only 1 mutation: S531L in the rpoB gene. Spoligotyping revealed that the genotype was that of the Beijing strain. VNTR typing confirmed all 35 loci. Review of the patient's clinical background showed that diabetes mellitus was present as a complication. DISCUSSION: There was no evidence of reinfection or polyclonal infection. The strain belonged to a sublineage of the Beijing genotype that is a common precipitating cause of MDR-TB due to this genotype. The patient had diabetes mellitus and was thus vulnerable to the development of resistance. Factors associated with both the host and bacteria, therefore, contributed to the development of resistance in this case, which seemed to result in the rapid development of MDR-TB.

Comparative genome analysis of Mycobacterium avium revealed genetic diversity in strains that cause pulmonary and disseminated disease.

Mycobacterium avium complex (MAC) infection causes disseminated disease in immunocompromised hosts, such as human immunodeficiency virus (HIV)-positive patients, and pulmonary disease in persons without systemic immunosuppression, which has been increasing in many countries. In Japan, the incidence of pulmonary MAC disease caused by M. avium is about 7 times higher than that caused by M. intracellulare. To explore the bacterial factors that affect the pathological state of MAC disease caused by M. avium, we determined the complete genome sequence of the previously unreported M. avium subsp. hominissuis strain TH135 isolated from a HIV-negative patient with pulmonary MAC disease and compared it with the known genomic sequence of M. avium strain 104 derived from an acquired immunodeficiency syndrome patient with MAC disease. The genome of strain TH135 consists of a 4,951,217-bp circular chromosome with 4,636 coding sequences. Comparative analysis revealed that 4,012 genes are shared between the two strains, and strains TH135 and 104 have 624 and 1,108 unique genes, respectively. Many strain-specific regions including virulence-associated genes were found in genomes of both strains, and except for some regions, the G+C content in the specific regions was low compared with the mean G+C content of the corresponding chromosome. Screening of clinical isolates for genes located in the strain-specific regions revealed that the detection rates of strain TH135-specific genes were relatively high in specimens isolated from pulmonary MAC disease patients, while, those of strain 104-specific genes were relatively high in those from HIV-positive patients. Collectively, M. avium strains that cause pulmonary and disseminated disease possess genetically distinct features, and it suggests that the acquisition of specific genes during strain evolution has played an important role in the pathological manifestations of MAC disease.

[Identification of novel variable number tandem repeat (VNTR) loci in Mycobacterium avium and development of an effective means of VNTR typing].

INTRODUCTION: To make more effective use of variable number tandem repeat (VNTR) typing, we identified novel VNTR loci in Mycobacterium avium and used them for modified M. avium tandem repeat-VNTR (MATR-VNTR) typing. METHOD: Analysis of a DNA sample extracted from a clinical isolate (strain HN135) with the FLX system genome sequencer (Roche Diagnostic System) led to discovery of several novel VNTR loci. The allelic diversity of the novel VNTR loci was evaluated for 71 clinical isolates and compared with the diversity of the MATR-VNTR loci. To improve efficacy of MATR-VNTR typing, we tested typing using 2 sets of loci selected from the newly identified loci and the MATR loci, i.e., one set containing 7 and another 16 loci. Hunter Gaston's discriminatory index (HGDI) was calculated for these sets. RESULTS: Six VNTR loci were newly identified, of which 5 showed a high diversity. The HGDI was 0.980 for the improved new typing using a set of 7 loci, and 0.995 for another set of 16 loci, while it was 0.992 for the conventional MATR-VNTR typing. DISCUSSION: VNTR typing with the set of the 7 loci enabled a rapid analysis, and another set of 16 loci enabled a precise analysis, as compared with conventional MATR-VNTR typing. A method that uses only VNTR loci with relatively high allelic diversity is considered to be a useful tool for VNTR typing of MAC isolates.

[A study of genetic characteristics of Mycobacterium avium strains from patients with pulmonary M. avium disease in Japan and Korea].

INTRODUCTION: To determine the characteristics of Mycobacterium avium in Japan, we compared the genetic properties of M. avium isolated in different countries. METHODS: A Mycobacterium avium tandem-repeat variable-number tandem-repeat (MATR-VNTR) analysis was performed using South Korean strains (n = 119) and Japanese strains (n = 76). In addition, we compared the frequencies of a new insertion sequence, ISMav6. RESULTS: A phylogenetic analysis identified different clusters between the two countries' strains. The prevalence of ISMav6 was significantly different between them, i.e., 75.0% in Japanese strains and 59.8% in the Korean ones (P < 0.035). The frequency of strains with IS Mav6 in the Shine-Dalgarno (SD) sequence of the cfp29 gene that is involved in the interferon-gamma induction was also different, with stronger significance (Japan: 38.2%, Korea: 12.4%, P < 0.001). DISCUSSION: It is possible that M. avium strains prevalent in Japan and in Korea are genetically distinct. The analyses of the presence of ISMav6, as well as the VNTR patterns of M.avium strains from many different countries would be a promising methodology in elucidating the causes of the recent increase in cases of pulmonary MAC diseases.

[Multicenter study on clinical features and genetic characteristics of Mycobacterium avium strains from patients in Japan with lung disease caused by M. avium].

BACKGROUND: The pulmonary disease caused by Mycobacterium avium shows diverse clinical manifestations. Little is known about the potential association between the genetic characteristics of M. avium strains and disease progression. SUBJECTS AND METHODS: We enrolled 89 patients with disease caused by M. avium, from 12 hospitals in Japan and collected the corresponding M. avium isolates and clinical data. We divided the 89 patients into 2 groups: one group comprising 43 patients with progressive disease despite chemotherapy ("progressive"), and the other group comprising 46 patients with untreated disease ("untreated"). We compared clinical and bacteriological characteristics between these groups. The bacteriological characteristics that we examined were drug susceptibility, variable-number tandem-repeat (VNTR) typing, and presence of the insertion sequence ISMav6. Seventeen patients in the "untreated" group were started on chemotherapy because their condition had clinically deteriorated during follow-up. RESULTS: The result of VNTR typing showed that there was no specific clustering according to geographical region or clinical group in the "untreated" or "progressive" groups. Six out of eight cases those of polyclonal infection, and 11 of 12 isolates that were highly resistant to clarithromycin were isolated from patients with progressive disease. The frequency of isolates with ISMav6 inserted into upstream region of the cfp29 gene, which is involved in the induction of interferon-gamma production, was significantly higher in patients with deteriorating disease than in stable patients in the "untreated" group (p = 0.002). CONCLUSION: Polyclonal infection and clarithromycin resistance may be involved in disease progression. ISMav6 inserted into the cfp29 gene is also suggested to be a factor related to the deterioration of pulmonary Mycobacterium avium complex disease.

Isolation and characterization of Streptomyces, Actinoplanes, and Methylibium strains that are involved in degradation of natural rubber and synthetic poly(cis-1,4-isoprene).

Rubber-degrading bacteria were screened for the production of clearing zones around their colonies on latex overlay agar plates. Novel three bacteria, Streptomyces sp. strain LCIC4, Actinoplanes sp. strain OR16, and Methylibium sp. strain NS21, were isolated. To the best of our knowledge, this is the first report on the isolation of a Gram-negative rubber-degrading bacterium other than γ-proteobacteria. Gel permeation chromatography analysis revealed that these strains degraded poly(cis-1,4-isoprene) to low-molecular-weight products. The occurrence of aldehyde groups in the degradation products by NS21 was suggested by staining with Schiff's reagent and ¹H-nuclear magnetic resonance spectroscopy. The lcp gene of LCIC4, which showed 99% amino acid sequence identity with that of Streptomyces sp. strain K30, was cloned, and contained a putative twin-arginine motif at its N terminus. It is located next to oxiB, which is estimated to be responsible for oxidation of degradation intermediate of rubber in K30. Southern hybridization analysis using LCIC4 lcp probe revealed the presence of a lcp-homolog in OR16. These results suggest that the lcp-homologs are involved in rubber degradation in LCIC4 and OR16.

Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium complex isolates.

OBJECTIVES: Clarithromycin is the key drug in the various treatment regimens of Mycobacterium avium complex (MAC) diseases, and is the only drug for which drug susceptibility has been shown to correlate with clinical response in these diseases. A point mutation at either positions 2058 or 2059 of the 23S rRNA gene has been reported to occur in high-level clarithromycin-resistant isolates. In this study, we examined the correlation between the results from a drug susceptibility test and the mutation of the 23S rRNA gene involved in drug resistance in MAC. Furthermore, we adapted a rapid detection method using amplification refractory mutation system (ARMS)-PCR to identify mutations in the 23S rRNA gene in MAC isolates. METHODS: Using a microdilution method based on the NCCLS/CLSI recommendation, the MIC of clarithromycin was determined for 245 clinical MAC isolates. Of these, 219 clarithromycin-susceptible and 26 clarithromycin-resistant strains were analysed by sequencing of the 23S rRNA gene and ARMS-PCR. RESULTS: The drug susceptibility test revealed a bimodal distribution of MICs for both the susceptible and resistant strains. Sequence analysis of the 23S rRNA gene revealed that all of the clarithromycin-susceptible strains were wild-type whereas 24 of the clarithromycin-resistant strains were mutant type. The sensitivity of the sequence and ARMS-PCR analyses was 92.3% and 84.6%, respectively, and the specificity of both was 100%. CONCLUSIONS: We found a correlation between MICs of clarithromycin and 23S rRNA gene mutations. ARMS-PCR for 23S rRNA mutations of MAC isolates is useful for rapid detection of clarithromycin resistance.

[Clinical application of line probe assay (LiPA) for rifampicin (RFP)-resistant gene examination in sputum from tuberculosis patients].

INTRODUCTION: Preventing the spread of drug-resistant tuberculosis is a clinically important challenge. In this effort, rifampicin (RFP)-resistant gene examination by line probe assay (LiPA) was evaluated for its clinical application for rapid detection of tuberculosis. METHODS: The RFP-resistant gene was examined in a total of 110 samples of sputum obtained from patients that were definitively diagnosed with pulmonary tuberculosis by auto-LiPA. The difference in detection sensitivity between the results of the smear and culture examinations was evaluated. Culture-positive samples were compared with the results of the drug susceptibility test. RESULTS: Smear-positive samples were LiPA positive in 69 of 73 samples (sensitivity: 94.5%), and smear-negative samples were LiPA positive in 25 of 37 samples (67.6%). More than half of the samples were LiPA positive, even those that were culture-negative or contaminated. Comparison of the 76 culture-positive samples with the results of the drug susceptibility test found that all samples were wild type among the RFP-sensitive strains. Among the 8 RFP-resistant strains, 6 were mutation type. All samples shown to be mutation type were obtained from patients with multi-drug resistant tuberculosis. DISCUSSION: Using LiPA, the amount of smear can be used as a factor for detection of RFP-resistant genes. Detection was possible even with culture-negative and contaminated samples, allowing more rapid diagnosis of patients with multi-drug resistant tuberculosis.

Molecular typing of Mycobacterium intracellulare using multilocus variable-number of tandem-repeat analysis: identification of loci and analysis of clinical isolates.

In addition to its known status as a disseminated disease in HIV-positive patients, Mycobacterium avium complex (MAC) is increasingly recognized as a causative pathogen of respiratory disease in HIV-negative patients. MAC is divided into Mycobacterium avium, and the less-epidemiologically studied Mycobacterium intracellulare. Genetic typing for M. intracellulare using variable number of tandem repeats (VNTR) has not yet been developed. The aim of this study was to identify VNTR loci in the genome of M. intracellulare and apply them as an epidemiological tool to clinical isolates. Here, we identified 25 VNTR loci on the M. intracellulare genome, of which 16 showed variations among clinical isolates in the number of tandem repeat motifs. Among the 74 M. intracellulare isolates, 50 genotypes were distinguished using the 16 VNTR loci, resulting in a Hunter Gaston's discriminatory index of 0.988. Moreover, all 16 VNTR loci were stable in different sets of isolates recovered within time intervals ranging from 2 to 1551 days from 14 separate patients. These results indicate that for use as epidemiological markers of M. intracellulare, the loci in this VNTR assay are highly discriminating and stable over time.