Dynamic diversity of SARS-CoV-2 genetic mutations in a lung transplantation patient with persistent COVID-19.

Numerous SARS-CoV-2 variant strains with altered characteristics have emerged since the onset of the COVID-19 pandemic. Remdesivir (RDV), a ribonucleotide analogue inhibitor of viral RNA polymerase, has become a valuable therapeutic agent. However, immunosuppressed hosts may respond inadequately to RDV and develop chronic persistent infections. A patient with respiratory failure caused by interstitial pneumonia, who had undergone transplantation of the left lung, developed COVID-19 caused by Omicron BA.5 strain with persistent chronic viral shedding, showing viral fusogenicity. Genome-wide sequencing analyses revealed the occurrence of several viral mutations after RDV treatment, followed by dynamic changes in the viral populations. The C799F mutation in nsp12 was found to play a pivotal role in conferring RDV resistance, preventing RDV-triphosphate from entering the active site of RNA-dependent RNA polymerase. The occurrence of diverse mutations is a characteristic of SARS-CoV-2, which mutates frequently. Herein, we describe the clinical case of an immunosuppressed host in whom inadequate treatment resulted in highly diverse SARS-CoV-2 mutations that threatened the patient's health due to the development of drug-resistant variants.

Factors Influencing the Indeterminate Results in a T-SPOT.TB test: A Matched Case-control Study.

Objective A T-SPOT.TB can yield indeterminate results under two test observation conditions: a high response to the nil in negative control wells (high nil-control) or a low response to the mitogen in positive control wells (low mitogen-control). The most strongly influential factors for these indeterminate results, however, have yet to be identified. Methods From June 1, 2015, to June 30, 2021, we conducted a 1:1 matched case-control, retrospective study. Patients Patients who underwent a T-SPOT.TB test at Chiba University Hospital. Results The study included 5,956 participants. Indeterminate results were found in 63 participants (1.1%), including high nil-control in 37 and low mitogen-control in 26. Human T-cell leukemia virus type 1 (HTLV-1) positivity was the only influencing factor associated with high nil-control (adjusted odds ratio=98.5, 95% confidence interval: 6.59-1,480). Conclusion Regarding the indeterminate results, all HTLV-1 positive participants had a high nil response and no low mitogen response. It was suspected that abnormally produced interferon γ caused a nonspecific reaction to the negative control well, resulting in a high nil response. Low mitogen-control, conversely, did not appear to have any statistically significant influential factors.

Development of multiplex S-gene-targeted RT-PCR for rapid identification of SARS-CoV-2 variants by extended S-gene target failure.

BACKGROUND: Tracking SARS-CoV-2 variants of concern (VOC) by genomic sequencing is time-consuming. The rapid screening of VOCs is necessary for clinical laboratories. In this study, we developed a rapid screening method based on multiplex RT-PCR by extended S-gene target failure (eSGTF), a false negative result caused by S-gene mutations. METHODS: Three S-gene target (SGT) regions (SGT1, codons 65-72; SGT2, codons 152-159; and SGT3, codons 370-377) and an N-gene region (for internal control) were detected in single-tube. Four types of VOC (Alpha, Delta, Omicron BA.1, and Omicron BA.2) are classified by positive/negative patterns of 3 S-gene regions (eSGTF pattern). RESULTS: The eSGTF patterns of VOCs were as follows (SGT1, SGT2, SGT3; P, positive; N, negative): Alpha, NPP; Delta, PNP; Omicron BA.1, NPN pattern; and Omicron BA.2, PPN. As compared with the S-gene sequencing, eSGTF patterns were identical to the specific VOCs (concordance rate = 96.7%, N = 206/213). Seven samples with discordant results had a minor mutation in the probe binding region. The epidemics of VOCs estimated by eSGTF patterns were similar to those in Japan. CONCLUSIONS: Multiplex RT-PCR and eSGTF patterns enable high-throughput screening of VOCs. It will be useful for the rapid determination of VOCs in clinical laboratories.

Antibody responses and SARS-CoV-2 infection after BNT162b2 mRNA booster vaccination among healthcare workers in Japan.

INTRODUCTION: Vaccine effectiveness against SARS-CoV-2 infections decreases due to waning immunity, and booster vaccination was therefore introduced. We estimated the anti-spike antibody (AS-ab) recovery by booster vaccination and analyzed the risk factors for SARS-CoV-2 infections. METHODS: The subjects were health care workers (HCWs) in a Chiba University Hospital vaccination cohort. They had received two doses of vaccine (BNT162b2) and a booster vaccine (BNT162b2). We retrospectively analyzed AS-ab titers and watched out for SARS-CoV-2 infection for 90 days following booster vaccination. RESULTS: AS-ab titer eight months after two-dose vaccinations had decreased to as low as 587 U/mL (median, IQR (interquartile range) 360-896). AS-ab titer had then increased to 22471 U/mL (15761-32622) three weeks after booster vaccination. There were no significant differences among age groups. A total of 1708 HCWs were analyzed for SARS-CoV-2 infection, and 48 of them proved positive. SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 1.8% and 4.0%, respectively, and were not significant. However, when restricted to those 20-29 years old, SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 2.9% and 13.6%, respectively (p = 0.04). After multivariate logistic regression, COVID-19 wards (adjusted odds ratio (aOR):2.9, 95% confidence interval (CI) 1.5-5.6) and those aged 20-49 years (aOR:9.7, 95%CI 1.3-71.2) were risk factors for SARS-CoV-2 infection. CONCLUSIONS: Booster vaccination induced the recovery of AS-ab titers. Risk factors for SARS-CoV-2 infection were HCWs of COVID-19 wards and those aged 20-49 years. Increased vaccination coverage, together with implementing infection control, remains the primary means of preventing HCWs from SARS-CoV-2 infection.

Tracking SARS-CoV-2 variants by entire S-gene analysis using long-range RT-PCR and Sanger sequencing.

INTRODUCTION: Genomic surveillance of the SARS-CoV-2 virus is important to assess transmissibility, disease severity, and vaccine effectiveness. The SARS-CoV-2 genome consists of approximately 30 kb single-stranded RNA that is too large to analyze the whole genome by Sanger sequencing. Thus, in this study, we performed Sanger sequencing following long-range RT-PCR of the entire SARS-CoV-2 S-gene and analyzed the mutational dynamics. METHODS: The 4 kb region, including the S-gene, was amplified by two-step long-range RT-PCR. Then, the entire S-gene sequence was determined by Sanger sequencing. The amino acid mutations were identified as compared with the reference SARS-CoV-2 genome. RESULTS: The S:D614G mutation was found in all samples. The R.1 variants were detected after January 2021. Alpha variants started to emerge in April 2021. Delta variants replaced Alpha in July 2021. Then, Omicron variants were detected after December 2021. These mutational dynamics in samples collected in the Chiba University Hospital were similar to those in Japan. CONCLUSION: The emergence of variants of concern (VOC) has been reported by the entire S-gene analysis. As the VOCs have unique mutational patterns of the S-gene region, analysis of the entire S-gene will be useful for molecular surveillance of the SARS-CoV-2 in clinical laboratories.

Emergence of Haemophilus influenzae with low susceptibility to quinolones isolated from pediatric patients in Japan.

INTRODUCTION: In 2010, oral fluoroquinolone tosufloxacin (TFX) granules were released as the first oral respiratory quinolone for children in Japan. METHODS: To investigate the recent trend of H. influenzae strains with low susceptibility to quinolones in children, we analyzed the gene sequences of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE of 23 clinical isolates from 15 patients aged <15 years with an MIC of ≥0.5 µg/mL for TFX from 2010 to 2018. RESULTS: Amino acid substitutions were observed in both GyrA and ParC in 13 strains (81%, 13/16), except two strains with a TFX MIC of 0.5 µg/mL with amino acid substitution in only GyrA and one strain with a TFX MIC of 1 µg/mL with no amino acid substitution. Four ST422 strains were observed in 2018, the detection age range was wide (0-7 years), and the residential city was varied. A total of 3/15 patients had a clear history of TFX treatment. CONCLUSIONS: Even for the strain with an MIC of 0.5 µg/mL for TFX, it is highly possible that it harbors a mutation in gyrA, which is the first step toward quinolone resistance, and it may also harbor mutations in both gyrA and parC. Furthermore, several specific sequence type quinolone-resistant H. influenzae strains, particularly ST422, may be widespread among children in Japan. It is necessary to investigate changes in resistance both at the MIC and gene levels. The continuous monitoring of strains and the use of antimicrobial drugs in treatment should be carefully observed.

Pin tract infection caused by Mycobacterium neoaurum in a 14-year-old child: A case report.

Although rapidly growing non-tuberculosis mycobacterium can occasionally cause postoperative infections, Mycobacterium neoaurum is a rare pathogen of surgical site infection. We report a case of pin tract infection caused by M. neoaurum in a 14-year-old girl who was admitted for lengthening of her right fourth metatarsal bone. Pain, redness, and exudate were observed 18 days after external fixator insertion. Repeated exudate cultures revealed M. neoaurum, and she was diagnosed with a mycobacterial pin tract infection. She was initially administered intravenous ciprofloxacin and minocycline, and then was switched to oral trimethoprim-sulfamethoxazole and minocycline for a total of 6 months. Despite the pin tract infection, bone lengthening was completed under antibiotic treatment without removal of the pin; no other complications were noted. There are no prior reports of external fixator pin tract infection by M. neoaurum. While such cases may be rare, this case demonstrates that bone distraction may still be successfully completed using appropriate antibiotic therapy without pin removal.

Bacteriological analysis of Neisseria lactamica isolated from the respiratory tract in Japanese children.

INTRODUCTION: Neisseria lactamica is a commensal bacterium of the upper respiratory tract in humans and is closely related to Neisseria meningitidis. N. lactamica colonization may contribute to preventing N. meningitidis colonization and invasive meningococcal disease. However, the transference of antimicrobial resistance genes from N. lactamica to N. meningitidis has been reported. METHODS: In this study, we aimed to identify N. lactamica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and performed multilocus sequence typing of seven N. lactamica strains isolated from Japanese children. We also analyzed the antimicrobial susceptibility of these strains and the mutations in their antimicrobial resistance genes (penA, gyrA, and parC). RESULTS: All the N. lactamica strains could be identified using MALDI-TOF MS. All strains were of different sequence types (STs), including five new STs. Five strains had intermediate susceptibility, two were resistant to ampicillin, and all had five out of the five known PBP2 mutations. Six strains were resistant to levofloxacin. Among the quinolone-resistant strains, three had GyrA mutations, and three had both ParC and GyrA mutations. CONCLUSIONS: N. lactamica STs may vary in Japanese children, and penicillin- and quinolone-resistant strains may be prevalent. We should pay attention not only to the drug resistance of N. meningitidis but also to the drug susceptibility of N. lactamica whose drug-resistance genes may transfer to N. meningitidis.

Antibiotics prescriptions for pneumonia analyzed by claim information in Japan.

OBJECTIVE: Japan is an aging society, and pneumonia is the leading cause of death, but the suitability of antibiotics for treating community-acquired pneumonia (CAP) in Japan is not clear. The purpose of this study was to investigate antibacterial drugs for treating CAP according to age. MATERIALS AND METHODS: Using the Japanese national database from 2011 to 2014, we analyzed the usage of antibiotics for CAP according to age. RESULTS: The numbers of claim information were 9,386, and 70% of the patients were aged ≥ 75 years. Sulbactam/ampicillin (SBT/ABPC) or ceftriaxone (CTRX) was used in 60%, but broad-spectrum antibiotics, combination therapy, and anti-mycoplasma antibiotics were used in 15 - 28% of all age groups. The 30-day survival rate did not differ between SBT/ABPC or CTRX vs. others. There was no difference in 30-day mortality and risk in any group between the ages of 15 and 64 years. On the other hand, the use of anti-mycoplasma antibiotics reduced the 30-day mortality by 0.50 times (p < 0.01), and the use of two or more antibiotics increased the 30-day mortality by 1.45 times (p = 0.02) at age ≥ 65 years. CONCLUSION: Approximately half of the antibiotics used for CAP requiring hospitalization consisted of CTRX or SBT/ABPC as recommended by the Japanese Respiratory Society (JRS) guidelines. On the other hand, the usage of broad-spectrum antibiotics and combination therapy were relatively frequent at all ages, although their use does not always contribute to survival. Our data provide basic information for analyzing the outcome of pneumonia treatment in terms of an antimicrobial resistance action plan in Japan.

Effect of Sub-MICs of Macrolides on the Sensitivity of Pseudomonas aeruginosa to Nitrosative Stress: Effectiveness against P. aeruginosa with and without Multidrug Resistance.

Sub-MICs of the 14-membered macrolides erythromycin (EM) and clarithromycin (CAM) decreased the growth of Pseudomonas aeruginosa PAO1 and increased its sensitivity to endogenous and exogenous nitrosative stress. However, a 16-membered macrolide, josamycin (JM), was not or less effective. In 9 of 13 non-multidrug-resistant P. aeruginosa (non-MDRP) and 9 of 27 MDRP ST235 strains, the sub-MIC of EM induced significant reductions in bacterial numbers following treatment with a nitric oxide donor.

First report on multidrug-resistant non-encapsulated Streptococcus pneumoniae isolated from a patient with pneumonia.

The non-encapsulated Streptococcus pneumoniae (NESp) has emerged and increased in the clinical setting. The majority of NESp strains have been isolated from the nasopharynxes of healthy carriers and from respiratory specimens of patients with otitis media. NESp strains were shown to be more effective than encapsulated counterparts at forming biofilms. Therefore, NESp should become one of the leading causes of emerging refractory respiratory disease after the introduction of pneumococcal conjugate vaccines. We report the first case of multidrug-resistant - including fluoroquinolone-resistant - NESp isolated from the intrabronchial aspirate of a patient with pneumonia. Drug-resistant NESp infections can possibly emerge as a clinical problem and thus the continuous monitoring of NESp infections is of utmost importance.

Highly sensitive detection of SARS-CoV-2 RNA by multiplex rRT-PCR for molecular diagnosis of COVID-19 by clinical laboratories.

BACKGROUND: The detection of SARS-CoV-2 RNA by real-time reverse transcription-polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA. METHODS: Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control. RESULTS: Good correlation of Cq values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method. CONCLUSION: The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.

Epidemiology and treatment outcome of pneumonia: Analysis based on Japan national database.

Pneumonia is the third leading cause of death in Japan. Mortality increases at an accelerating rate in elderly patients aged ≥65 years. Elderly patients tend to have underlying conditions affecting pneumonia treatment. The national database (NDB) associated with medical services under Japanese universal health insurance is available for research purposes. Our NDB randomly sampled 10% of hospitalized patients every October from 2011 to 2014. In this NDB, we analyzed pneumonia epidemiology in patients aged ≥15 years and 30-day mortality in Japanese hospitals. This study also investigated the factors affecting treatment outcome. A total of 9386 patients were entered. The number of patients from age 65 years and older increased greatly, representing 85% of the total. The thirty-day mortality rate among all patients was 11.7%. Mortality rates at age 15-64, 65-74, 75-84, and ≥85 years were 9.5%, 12.0%, 8.3%, and 14.9%, respectively, showing significant differences (P < 0.001). The underlying conditions varied among age groups. Male gender, age, heart failure, chronic kidney disease (CKD), consciousness disorder, shock and respiratory failure are risk factors for 30-day mortality. Pneumonia develops mainly in people aged 65 years and older in Japan, and treatment outcome is generally poor in elderly patients. The underlying conditions were seen to affect the 30-day mortality rate. CURB-65 and ADROP, a modification of CURB-65 in Japan, have already estimated these risk factors, and heart failure and CKD might be additional factors for estimating pneumonia severity.

A retrospective observational study of antimicrobial treatment for non-tuberculous mycobacteria disease using a nationwide claims database in Japan.

The Macrolides (MLs), clarithromycin and azithromycin, are key drugs for non-tuberculous mycobacteria (NTM) diseases treatment. A three antibiotics regimen including MLs, rifampicin (RFP) and ethambutol (EB) has been recommended for the treatment of NTM diseases in ATS/IDSA guideline. However, anti-biotics are not necessarily prescribed in compliance with the guideline. Inappropriate regimens are risk of introducing MLs resistance. Therefore, we planned this study to evaluate the current Japanese NTM diseases treatment conditions. We used the national database (NDB) from 2011 to 2014. A total of 183 patients were entered into the study. The patients number increased at an accelerating rate in patients aged ≥55 years. Patients aged ≥55 years made up 91.3% of the total NTM diseases. Male and female patients were 61 and 122, respectively, a female/male ratio of 2.00. Clarithromycin, RFP, EB and fluoroquinolones were frequently prescribed, with the numbers of prescriptions being 125, 66, 57 and 45, respectively. The regimen of MLs, RFP and EB recommend by ATS/IDSA guideline 2007 was only followed by 25.1% of the patients. MLs monotherapy was as high as 30.6% of NTM diseases and would be a risk factor leading to an increase of MLs resistance and poor treatment outcome. Without effective NTM disease therapy, the increase of MLs-resistant NTM diseases would be a burden for Japanese health care facilities.

Isolation Rate of Neisseria meningitidis in Japanese Children with Respiratory Tract Infections.

Although invasive meningococcal disease is rare in Japan (0.028 cases per 100,000 population), its incidence is 10 times greater in many other countries. Colonization is a prerequisite for invasive meningococcal disease. However, no study in Japan has involved specifically analyzing the carriage rate of Neisseria meningitidis in children. During 5 months in 2015, the respiratory tract specimens of patients who presented to 3 hospitals with respiratory symptoms were cultured. The bacteria were identified in selective media using a meningococcal detection kit and the serogroup was identified using polymerase chain reaction analysis. In 389 patients aged ≤15 years with respiratory symptoms, the N. meningitidis isolation rate was 0.26% (1/389). The serogroup of the only child who tested positive was Y. In this study, we detected a low meningococcal isolation rate in pediatric patients. Due to increasing globalization, the risk of invasive meningococcal disease is likely increasing in Japan. Accordingly, invasive meningococcal diseases should be continuously monitored in Japan. Future large-scale studies should assess meningococcal isolation rates and corresponding serogroups.

Clinical and bacteriological analyses of bacteremia due to Corynebacterium striatum.

BACKGROUND: Corynebacterium striatum was recently recognized as a potential pathogen of various infectious diseases. However, the clinical entity of this microorganism has not been clearly identified. Therefore, we analyzed C. striatum isolates from blood culture and explored their clinical determinants. METHODS: We reviewed the medical records of all patients from whom C. striatum isolates were recovered from blood culture for analysis of the patients' backgrounds and clinical course including response to antimicrobial therapy and prognosis. RESULTS: During the 5-year study period (January 2010 to December 2014), 24 C. striatum strains were isolated from blood samples, and the frequency of C. striatum bacteremia increased. The majority of the strains were multidrug resistant. All of the tested strains were susceptible to only vancomycin. The age at onset of C. striatum bacteremia encompassed all adult age groups, and at least one underlying condition was documented in all patients. Thirteen of the 24 patients were cured using appropriate antibiotics (true infection group); however, 11 of the 24 patients were cured using inappropriate antibiotic therapy or no antibiotics (contamination group). Malignancy and neutropenia significantly increased the odds of true C. striatum bloodstream infection. CONCLUSIONS: The Corynebacterium species is often considered a contaminant when isolated in culture. Instead, particularly when the strain is isolated from blood, the species should be considered clinically relevant and identified to the species level; in addition, antimicrobial susceptibility testing is recommended.

[Antimicrobial Susceptibility of Streptococcus pneumoniae Isolated from 8 Hospitals in Chiba Prefecture Following the Introduction of 7-valent Pneumococcal Conjugate Vaccine].

We investigated the susceptibility of Streptococcus pneumoniae isolated from 8 hospitals in Chiba prefecture during 2012-2013. We further checked the serotype of S. pneumoniae derived from invasive pneumococcal disease (IPD). We tested for antimicrobial susceptibility in 256 clinical isolates (137 isolates from children, 119 isolates from adults) for 25 drugs. In MIC50 and MIC90, there were very little differences between children and adults, but there were 3 isolates from adults which were resistant to levofloxacin. The most major serotypes were 15A and 3 in IPD. Additionally there was no isolation of the type contained in the 7-valent pneumococcal conjugate vaccine in children, so it seems that the vaccination is very effective for children. Furthermore, in contrast with our preceding report, a decreasing was seen in PCG resistant proportion of S. pneumoniae. The maximum PCG-MIC was 2 µg/mL.

Identification of Nocardia species using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

BACKGROUND: The MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus Nocardia. In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of Nocardia strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC). RESULTS: The newly developed method (Nocardia Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from Nocardia specimens. The Nocardia in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level. CONCLUSIONS: The results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify Nocardia accurately in a short time in combination with a simple processing method and an in-house database.

Direct application of MALDI-TOF mass spectrometry to cerebrospinal fluid for rapid pathogen identification in a patient with bacterial meningitis.

BACKGROUND: Bacterial meningitis is a neurological emergency. Early diagnosis and rapid initiation of antimicrobial therapy are vital. METHODS: Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is increasingly used as a rapid and accurate microbial diagnostic method for species identification of pathogens. Although this technology requires a growth step to obtain bacterial colonies for the acquisition of substantial spectra in most cases, it can also be used to analyze clinical specimens such as urine and cerebrospinal fluid for direct bacterial identification. There are very few reports describing the use of MALDI-TOF MS for the direct detection of microorganisms causing bacterial meningitis. RESULTS: We describe a case of bacterial meningitis caused by Klebsiella pneumoniae in which MALDI-TOF MS provided a rapid bacteriological diagnosis, thus enabling early and appropriate treatment. CONCLUSIONS: Identification of microbes based on MALDI-TOF MS is now an important technology in clinical microbiology laboratories that are required to provide a rapid diagnosis of bacterial meningitis.

Efficacy of Levofloxacin Based Triple and High-Dose PPI-Amoxicillin Dual Eradication Therapy for Helicobacter pylori after Failures of First- and Second-Line Therapies.

Objectives. The aim of this study was to investigate and compare the eradication rate of Helicobacter pylori as the third-line triple therapy with rabeprazole (RPZ) + amoxicillin (AMPC) + levofloxacin (LVFX) and high-dose RPZ + AMPC. Methods. 51 patients who failed Japanese first-line (proton pump inhibitor (PPI) + AMPC + clarithromycin) and second-line (PPI + AMPC + metronidazole) eradication therapy were randomly assigned at a 1 : 1 ratio to one of the following third-line eradication groups: (1) RAL group: RPZ 10 mg (b.i.d.), AMPC 750 mg (b.i.d.), and LVFX 500 mg (o.d.) for 10 days; (2) RA group: RPZ 10 mg (q.i.d.) and AMPC 500 mg (q.i.d.) for 14 days. Patients who failed to respond to third-line eradication therapy received salvage therapy. Results. The rates of eradication success, based on intention to treat (ITT) analysis, were 45.8% in the RAL group and 40.7% in the RA group. The overall eradication rates were 73.9% in the RAL group and 64.0% in the RA group. There was no significant difference between the two groups. Conclusions. The third-line triple therapy with RPZ, AMPC, and LVFX was as effective as that with high-dose RPZ and AMPC.