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Objectives: The emergence of multidrug-resistant Klebsiella pneumoniae has become a serious problem in medical settings worldwide. Methods: A total of 46 isolates of multidrug-resistant K. pneumoniae were obtained from 2 hospitals in Nepal from October 2018 to April 2019. Results: Most of these isolates were highly resistant to carbapenems, aminoglycosides, and fluoroquinolones with the minimum inhibitory concentrations (MICs) of more than 64 µg/mL. These isolates harboured carbapenemase-encoding genes, including blaNDM-1, blaNDM-5, blaOXA-181 and blaOXA-232, and 16S rRNA methyltransferase-encoding genes, including armA, rmtB, rmtC, and rmtF. Multilocus sequence typing revealed that 44 of 46 isolates were high-risk clones such as ST11 (2%), ST14 (4%), ST15 (11%), ST37 (2%), ST101 (2%), ST147 (28%), ST231 (13%), ST340 (4%), and ST395 (28%). In particular, ST395 isolates, which spread across medical settings in Nepal, co-harboured blaNDM-5 and rmtB on IncFII plasmids and co-harboured blaOXA-181/-232 and rmtF on ColKP3 plasmids. Several isolates harboured blaOXA-181 or blaNDM-5 on their chromosomes and multi-copies of blaNDM-1 or genes encoding 16S rRNA methyltransferases on their plasmids. Conclusions: The presented study demonstrates that the high-risk clones of multidrug-resistant K. pneumoniae spread in a clonal manner across hospitals in Nepal.

IntroductionThe SARS-CoV-2 pandemic has had a widespread and severe impact on society, yet there have also been instances of remarkable recovery, even in critically ill patients.Materials and methodsIn this study, we used single-cell RNA sequencing to analyze the immune responses in recovered and deceased COVID-19 patients during moderate and critical stages.ResultsExpanded T cell receptor (TCR) clones were predominantly SARS-CoV-2-specific, but represented only a small fraction of the total repertoire in all patients. In contrast, while deceased patients exhibited monoclonal B cell receptor (BCR) expansions without COVID-19 specificity, survivors demonstrated diverse and specific BCR clones. These findings suggest that neither TCR diversity nor BCR monoclonal expansions are sufficient for viral clearance and subsequent recovery. Differential gene expression analysis revealed that protein biosynthetic processes were enriched in survivors, but that potentially damaging mitochondrial ATP metabolism was activated in the deceased.ConclusionThis study underscores that BCR repertoire diversity, but not TCR diversity, correlates with favorable outcomes in COVID-19.

Abstract Mycobacteroides (Mycobacterium) abscessus, which causes a variety of infectious diseases in humans, is becoming detected more frequently in clinical specimens as cases are spreading worldwide. Taxonomically, M. abscessus is composed of three subspecies of M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense, with different susceptibilities to macrolides. In order to identify rapidly these three subspecies, we determined useful biomarker proteins, including ribosomal protein L29, L30, and hemophore-related protein, for distinguishing the subspecies of M. abscessus using the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiles. Thirty-three clinical strains of M. abscessus were correctly identified at the subspecies-level by the three biomarker protein peaks. This study ultimately demonstrates the potential of routine MALDI-MS-based laboratory methods for early identification and treatment for M. abscessus infections.

Wickerhamiella is a genus of budding yeast that is mainly isolated from environmental samples, and 40 species have been detected. The yeast isolated from human clinical samples usually only contain three species: W. infanticola, W. pararugosa and W. sorbophila. In this study, we isolated W. tropicalis from a blood sample of a six-year-old female with a history of B-cell precursor lymphoblastic leukemia in Japan in 2022. Though the strain was morphologically identified as Candida species by routine microbiological examinations, it was subsequently identified as W. tropicalis by sequencing the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The isolate had amino acid substitutions in ERG11 and FKS1 associated with azole and echinocandin resistance, respectively, in Candida species and showed intermediate-resistant to fluconazole and micafungin. The patient was successfully treated with micafungin. Furthermore, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detected three novel peaks that are specific for W. tropicalis, indicating that MALDI-MS analysis is useful for rapid detection of Wickerhamiella species in routine microbiological examinations.

Abstract Mycobacterium abscessus species (MABS) is the most commonly isolated rapidly growing mycobacteria (RGM) and is one of the most antibiotic-resistant RGM with rapid progression, therefore, treatment of MABS is still challenging. We here presented a new combination treatment with sitafloxacin that targeted rough morphotypes of MABS, causing aggressive infections. Thirty-four clinical strains of MABS were isolated from various clinical samples at the Juntendo university hospital from 2011 to 2020. The susceptibility to a combination of sitafloxacin and antimicrobial agents was compared to that of the antimicrobial agents alone. Out of 34 MABS, 8 strains treated with sitafloxacin–amikacin combination, 9 of sitafloxacin–imipenem combination, 19 of sitafloxacin–arbekacin combination, and 9 of sitafloxacin–clarithromycin combination showed synergistic effects, respectively. Sitafloxacin–arbekacin combination also exhibited the synergistic effects against 10 of 22 Mycobacterium abscessus subspecies massiliense (Mma) strains and 8 of 11 Mycobacterium abscessus subspecies abscessus (Mab) strains, a highly resistant subspecies of MABS. The sitafloxacin–arbekacin combination revealed more synergistic effects in rough morphotypes of MABS (p = 0.008). We demonstrated the synergistic effect of the sitafloxacin–arbekacin combination against MABS. Further, this combination regimen might be more effective against Mab or rough morphotypes of MABS.

There has been a decreasing trend in new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and fatalities worldwide. The virus has been evolving, indicating the potential emergence of new variants and uncertainties. These challenges necessitate continued efforts in disease control and mitigation strategies. We investigated a small cluster of SARS-CoV-2 Omicron variant infections containing a common set of genomic mutations, which provided a valuable model for investigating the transmission mechanism of genetic alterations. We conducted a study at a medical center in Japan during the Omicron surge (sub-lineage BA.5), sequencing the entire SARS-CoV-2 genomes from infected individuals and evaluating the phylogenetic tree and haplotype network among the variants. We compared the mutations present in each strain within the BA.5 strain, TKYnat2317, which was first identified in Tokyo, Japan. From June 29th to July 4th 2022, nine healthcare workers (HCWs) tested positive for SARS-CoV-2 by real-time PCR. During the same period, five patients also tested positive by real-time PCR. Whole genome sequencing revealed that the infected patients belonged to either the isolated BA.2 or BA.5 sub-lineage, while the healthcare worker infections were classified as BF.5. The phylogenetic tree and haplotype network clearly showed the specificity and similarity of the HCW cluster. We identified 12 common mutations in the cluster, including I110V in nonstructural protein 4 (nsp4), A1020S in the Spike protein, and H47Y in ORF7a, compared to the BA.5 reference. Additionally, one case had the extra nucleotide-deletion mutation I27* in ORF10, and low frequencies of genetic alterations were also found in certain instances. The results of genome sequencing showed that the nine HCWs shared a set of genetic mutations, indicating transmission within the cluster. Minor mutations observed in five HCW individuals suggested the emergence of new virus variants. Five amino acid substitutions occurred in nsp3, which could potentially affect virus replication or immune escape. Intra-host evolution also generated additional mutations. The cluster exhibited a mild disease course, with individuals in this case, recovering without requiring any medical treatments. Further investigation is needed to understand the relationship between the genetic evolution of the virus and the symptoms.

Abstract COVID-19 antibody testing has been developed to investigate humoral immune response in SARS-CoV-2 infection. To assess the serological dynamics and neutralizing potency following SARS-CoV-2 infection, we investigated the neutralizing (NT) antibody, anti-spike, and anti-nucleocapsid antibodies responses using a total of 168 samples obtained from 68 SARS-CoV-2 infected patients. Antibodies were measured using an authentic virus neutralization assay, the high-throughput laboratory measurements of the Abbott Alinity quantitative anti-spike receptor-binding domain IgG (S-IgG), semiquantitative anti-spike IgM (S-IgM), and anti-nucleocapsid IgG (N-IgG) assays. The quantitative measurement of S-IgG antibodies was well correlated with the neutralizing activity detected by the neutralization assay (r = 0.8943, p

Background Despite the worldwide campaigns of COVID-19 vaccinations, the pandemic is still a major medical and social problem. The Ortho VITROS SARS-CoV-2 spike-specific quantitative IgG (VITROS S-IgG) assay has been developed to assess neutralizing antibody (NT antibody) against SARS-CoV-2 spike (S) antibodies. However, it has not been evaluated in Japan, where the total cases and death toll are lower than the rest of the world. Methods The clinical performance of VITROS S-IgG was evaluated by comparing with the NT antibody levels measured by the surrogate virus neutralizing antibody test (sVNT). A total of 332 serum samples from 188 individuals were used. Of these, 219 samples were from 75 COVID-19 patients: 96 samples from 20 severe/critical cases (Group S), and 123 samples from 55 mild/moderate cases (Group M). The remaining 113 samples were from 113 healthcare workers who had received 2 doses of the BNT162b2 vaccine. Results VITROS S-IgG showed good correlation with the cPass sVNT assay (Spearman rho = 0.91). Both VITROS S-IgG and cPass sVNT showed significantly higher plateau levels of antibodies in Group S compared to Group M. Regarding the humoral immune responses after BNT162b2 vaccination, individuals who were negative for SARS-CoV-2 nucleocapsid (N)-specific antibodies had statistically lower titers of both S-IgG and sVNT compared to individuals with a history of COVID-19 and individuals who were positive for N-specific antibodies without history of COVID-19. In individuals who were positive for N-specific antibodies, S-IgG and sVNT titers were similar to individuals with a history of COVID-19. Conclusions Although the automated quantitative immunoassay VITROS S-IgG showed a reasonable correlation with sVNT antibodies, there is some discrepancy between Vitros S-IgG and cPass sVNT in milder cases. Thus, VITROS S-IgG can be a useful diagnostic tool in assessing the immune responses to vaccination and herd immunity. However, careful analysis is necessary to interpret the results.

Abstract Background Mycolicibacterium fortuitum is a species of the rapidly growing mycobacteria that can cause pulmonary infection. It is susceptible to multiple antibiotics both in vitro and in clinical practice, so that any combination of susceptible drugs is effective. However, we encountered a case of infection due to fluoroquinolone-resistant M. fortuitum. In this study, we report the case and describe the mechanism of resistance. Case presentation A 65-year-old man with a history of total gastrectomy and immunosuppressant treatment for rheumatoid arthritis developed a recurrence of pulmonary infection caused by M. fortuitum. He was treated with clarithromycin and levofloxacin as a first-line treatment, based on the favorable susceptibility at that time. After recurrence, a high minimum inhibitory concentration to fluoroquinolones was detected. DNA sequencing of the pathogen showed the substitution of serine for tryptophan at residue 83 in the gyrA gene. He was successfully treated with a combination of other antibiotics. Conclusion This is the first report on the treatment of fluoroquinolone-resistant M. fortuitum and investigation of the mechanism of resistance. We suggest that the susceptibility test remains effective for determining the next line of treatment after a pathogen has acquired resistance, and resistance to fluoroquinolones in M. fortuitum can be attributed to a single change of amino acid.

Abstract Background Nontuberculous mycobacteria (NTM) are ubiquitous organisms and the incidence of NTM infections has been increasing in recent years. Mycobacteroides abscessus (M. abscessus) is one of the most antimicrobial-resistant NTM; however, no reliable antibiotic regimen can be officially advocated. We evaluated the efficacy of clarithromycin in combination with various antimicrobial agents against the M. abscessus complex. Results Twenty-nine clinical strains of M. abscessus were isolated from various clinical samples. Of the isolates, 10 (34.5%) were of M. abscessus subsp. abscessus, 18 (62.1%) of M. abscessus subsp. massiliense, and 1 (3.4%) of M. abscessus subsp. bolletii. MICs of three antimicrobial agents (amikacin, imipenem, and moxifloxacin) were measured with or without clarithromycin. The imipenem-clarithromycin combination significantly reduced MICs compared to clarithromycin and imipenem monotherapies, including against resistant strains. The association between susceptibility of the M. abscessus complex and each combination of agents was significant (p = 0.001). Adjusted residuals indicated that the imipenem-clarithromycin combination had the synergistic effect (adjusted residual = 3.1) and suppressed the antagonistic effect (adjusted residual = − 3.1). In subspecies of M. abscessus complex, the association with susceptibility of M. abscessus subsp. massiliense was similarly statistically significant (p = 0.036: adjusted residuals of synergistic and antagonistic effect respectively: 2.6 and − 2.6). The association with susceptibility of M. abscessus subsp. abscessus also showed a similar trend but did not reach statistical significance. Conclusion Our data suggest that the imipenem-clarithromycin combination could be the recommended therapeutic choice for the treatment of M. abscessus complex owing to its ability to restore antimicrobial susceptibility.

Quantitative measurement of SARS-CoV-2 neutralizing antibodies is highly expected to evaluate immune status, vaccine response, and antiviral therapy. The Elecsys® Anti-SARS-CoV-2 S (Elecsys® anti-S) was developed to measure anti-SARS-CoV-2 S proteins. We sought to investigate whether Elecsys® anti-S can be used to predict neutralizing activities in patients' serums using an authentic virus neutralization assay. One hundred forty-six serum samples were obtained from 59 patients with COVID-19 at multiple time points. Of the 59 patients, 44 cases were included in Group M (mild 23, moderate 21) and produced 84 samples (mild 35, moderate 49), while 15 cases were included in Group S (severe 11, critical 4) and produced 62 samples (severe 43, critical 19). The neutralization assay detected 73% positive cases, and Elecsys® anti-S and Elecsys® Anti-SARS-CoV-2 (Elecsys® anti-N) showed 72% and 66% positive cases, respectively. A linear correlation between the Elecsys® anti-S assay and the neutralization assay were highly correlated (r = 0.7253, r2 = 0.5261) than a linear correlation between the Elecsys® anti-N and neutralization assay (r = 0.5824, r2 = 0.3392). The levels of Elecsys® anti-S antibody and neutralizing activities were significantly higher in Group S than in Group M after 6 weeks from onset of symptoms (p < 0.05). Conversely, the levels of Elecsys® anti-N were comparable in both groups. Three immunosuppressed patients, including cancer patients, showed low levels of anti-S and anti-N antibodies and neutralizing activities throughout the measurement period, indicating the need for careful follow-up. Our data indicate that Elecsys® anti-S can predict the neutralization antibodies in COVID-19.