Molecular characterization of rifabutin-resistance in refractory Helicobacter pylori infection in Taiwan.

OBJECTIVES: To explore the molecular characteristics of rpoB, encoding ß-subunit of DNA-directed RNA polymerase, and unravel the link to rifabutin-resistance in patients with refractory Helicobacter pylori infection. METHODS: From January 2018-March 2021, a total of 1590 patients were screened for eligibility to participate in the study. Patients with refractory H. pylori infection were confirmed by using the (13C)-urea breath assay. All enrolled patients underwent esophagogastroduodenoscopy, and biopsies were taken for H. pylori culture and antibacterial susceptibility testing. Sequence analysis of rpoB was conducted for all rifabutin-resistant isolates. RESULTS: In total, 70 patients were diagnosed with refractory H. pylori infection, and 39 isolates were successfully cultured. Amongst, 10 isolates were identified as rifabutin-resistance and nine isolates exhibited at least one amino acid substitution in RpoB. Isolates with a minimal inhibitory concentration >32 mg/l displayed a higher number of mutational changes in RpoB than the others. Additionally, more amino acid substitutions in RpoB correlated with developing a higher minimal inhibitory concentration for H. pylori rifabutin-resistance. CONCLUSION: Our findings highlight the relationship between rifabutin-resistance in refractory H. pylori infection and specific mutations in RpoB, which will aid the clinical selection of appropriate antibacterial agents with better therapeutic effects.

Clostridium scindens metabolites trigger prostate cancer progression through androgen receptor signaling.

Prostate cancer (PCa) is one of the most common malignancies in men; recently, PCa-related mortality has increased worldwide. Although androgen deprivation therapy (ADT) is the standard treatment for PCa, patients often develop aggressive castration-resistant PCa (CRPC), indicating the presence of an alternative source of androgen. Clostridium scindens is a member of the gut microbiota and can convert cortisol to 11ß-hydroxyandrostenedione (11ß-OHA), which is a potent androgen precursor. However, the effect of C. scindens on PCa progression has not been determined. In this study, androgen-dependent PCa cells (LNCaP) were employed to investigate whether C. scindens-derived metabolites activate androgen receptor (AR), which is a pivotal step in the development of PCa. Results showed that cortisol metabolites derived from C. scindens-conditioned medium promoted proliferation and enhanced migration of PCa cells. Furthermore, cells treated with these metabolites presented activated AR and stimulated AR-regulated genes. These findings reveal that C. scindens has the potential to promote PCa progression via the activation of AR signaling. Further studies on the gut-prostate axis may help unravel an alternative source of androgen that triggers CRPC exacerbation.

Multiple amino acid substitutions in penicillin-binding protein-1A confer amoxicillin resistance in refractory Helicobacter pylori infection.

BACKGROUND: Amoxicillin resistance in Helicobacter pylori is mainly associated with mutations in penicillin-binding protein-1A (PBP-1A). However, the specific amino acid substitutions in PBP-1A that confer amoxicillin resistance in H. pylori remain to be investigated. OBJECTIVE: This study aimed to investigate the molecular mechanism underlying amoxicillin resistance in patients with refractory H. pylori infection. METHODS: Esophagogastroduodenoscopy (EGD) was performed in patients with persistent H. pylori infection after at least two courses of H. pylori eradication therapy between January-2018 to March-2021. Refractory H. pylori was cultured from the gastric biopsy specimens. Antibiotic susceptibility testing was conducted to determine the minimum inhibitory concentrations (MICs). Sequence analysis of pbp-1A was performed for amoxicillin-resistant strains. RESULTS: Thirty-nine successfully cultured isolates were classified as refractory H. pylori isolates, and seventeen isolates were resistant to amoxicillin (MIC > 0.125 mg/L). Sequence analysis of resistant strains showed multiple mutations in the C-terminal region of PBP-1A that conferred amoxicillin resistance in H. pylori. However, the number of PBP-1A mutations did not correlate with the high MICs of amoxicillin-resistant isolates. Notably, some amino acid substitutions were identified in all Taiwanese isolates with history of eradication failure but not in published amoxicillin-susceptible strains, suggesting that the mutations may play a role in conferring antibiotic resistance to these strains. CONCLUSIONS: Our results show that amoxicillin resistance in refractory H. pylori is highly correlated with numerous PBP-1A mutations that are strain specific. Continuous improvements in diagnostic tools, particularly molecular analysis approaches, can help to optimize current antimicrobial regimens.

Comparison between the analytical sensitivity and clinical performance of two cobas SARS-CoV-2 tests based on high-throughput and point-of-care systems.

Objectives: This study examined analytical sensitivity, specificity, and the clinical performance in detecting SARS-CoV-2 of the Cobas SARS-CoV-2 Test based on the high-throughput Cobas 6800 system and the Cobas SARS-CoV-2 & Flu A/B Test based on the point-of-care cobas Liat system. Methods: The commercial reagents containing SARS-CoV-2 RNA subgenomes were diluted for assessing the sensitivity of the RT-qPCR assay. 385 nasopharyngeal swab specimens taken from contacts of COVID-19 cases were tested for the SARS-CoV-2 detection with both Cobas SARS-CoV-2 Tests. Results: In analytical sensitivity assays, the Cobas SARS-CoV-2 & Flu A/B Test on the Liat system had a lower limit of detection (12.5-25 copies/mL) than the cobas SARS-CoV-2 Test on the cobas 6800 system (25-50 copies/mL). In clinical performance assays, the cobas SARS-CoV-2 Test demonstrated 89.36% (42 out of 47) PPA (positive percent agreement) and 98.82% (334 out of 338) NPA (negative percent agreement) compared to the results of the Cobas SARS-CoV-2 & Flu A/B test. Among five discordant specimens, four had the positive result of the cobas SARS-CoV-2 test, but the negative result of the cobas SARS-CoV-2 & Flu A/B Test. Moreover, these discordant specimens had the Ct values of greater than 33 for the cobas SARS-CoV-2 Test, implying a very small number of virions in the samples. Remarkably, four specimens with a presumptive positive result of the cobas SARS-CoV-2 test had been confirmed by the Cobas SARS-CoV-2 & Flu A/B Test. Next, the scatter plots of the Ct values showed a highly positive correlation between cobas SARS-CoV-2 & Flu A/B Test and the cobas SARS-CoV-2 Test (R-squared value = 0.954-0.962). Conclusions: Both SARS-CoV2 tests of the cobas 6800 and Liat systems produce reliable high throughput and point-of-care assays respectively for the early virus detection and the personal care decision-making during COVID-19 pandemic.

Haplotype distribution of SARS-CoV-2 variants in low and high vaccination rate countries during ongoing global COVID-19 pandemic in early 2021.

The widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuously impacts our economic and public health. The potential of emerging variants to increase transmissibility and evade vaccine-induced immunity lets us put more effort to research on viral mutations and explore the pathogenic haplotypes. In this study, we characterized the haplotype and sub-haplotype diversity of SARS-CoV-2 global variants in January-March and the areas with low and high COVID19 vaccination rates in May 2021 by analyzing viral proteome of complete genome sequences published. Phylogenetic tree analysis of the proteomes of SARS-CoV-2 variants with Neighbor-Joining and Maximum Parsimony methods indicated that haplotype 2 variant with nsp12 P323L and Spike D614G was dominant (98.81%), including new sub-haplotypes 2A_1 to 2A_3, 2B_1 to 2B_3, and 2C_1 to 2C_2 emerged post-one-year COVID-19 outbreak. In addition, the profiling of sub-haplotypes indicated that sub-haplotype 2A_1 with the mutations at N501Y, A570D, D614G, P681H, T716I, S982A, and D118H in Spike was over 58% in May 2021 in the high partly vaccinated rate group (US, Canada, and Germany). Meanwhile, the new haplotype 2C_3 bearing the mutations at EFR156-158del, T19R, A222V, L452R, T478K, and D614G in Spike occupied over 54.8% in May 2021 in the low partly vaccinated rate group (India, Malaysia, Taiwan, and Vietnam). Sub-haplotypes 2A_1 and 2C_3 had a meaningful alternation of ACE2-specific recognition site, neutralization epitopes, and furin cleavage site in SARS-CoV-2 Spike protein. The results discovered the haplotype diversity and new sub-haplotypes of SARS-CoV-2 variants post one-year pandemic in January-March 2021, showing the profiles of sub-haplotypes in the groups with low and high partly vaccinated rates in May 2021. The study reports the emergence of new SARS-CoV-2 sub-haplotypes during ongoing pandemic and vaccination in early 2021, which might help inform the response to vaccination strategies.

The extent of molecular variation in novel SARS-CoV-2 after the six-month global spread.

The pandemic spread of Coronavirus Disease 2019 (COVID-19) is still ongoing since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is identified as the etiologic pathogen late December 2019. After over six-month spread of COVID-19, SARS-CoV-2 causes critical threats to global public health and economy. The investigations on evolution and genotyping on genetic variations are of great importance, therefore, the present study characterized the molecular variation of SARS-CoV-2 by analyzing 4230 complete genome sequences from the worldwide samples collected during the first 6-month pandemic. Phylogenetic tree analysis with Neighbor-Joining and Maximum-Parsimony methods indicated that the haplotypes of SARS-CoV-2 genome sequences were classified into four clades with the unique nucleotide and amino acid changes: T27879C (ORF8 L84S) in clade 1 (25.34%), A23138G (spike D614G) in clade 2 (63.54%), G10818T (nsp6 L37F), C14540T (nsp12 T442I), and G25879T (ORF3a V251F) in clade 3 (2.58%), and miscellaneous changes in clade 4 (8.54%). Interestingly, subclade 2B with the amino acid changes at nsp2 T85I, Spike D614G, and ORF3a Q57H was firstly reported on March 4, 2020 in United States of America, becoming the most frequent sub-haplogroup in the world (36.21%) and America (45.81%). Subclade 1C with the amino acid changes at nsp13 P504L and ORF8 L84S was becoming the second most frequent sub-haplogroup in the world (19.91%) and America (26.29%). Subclade 2A with the amino acid changes in Spike D614G and Nucleocapsid R203K and G204R was highly prevalent in Asia (18.82%) and Europe (29.72%). The study highlights the notable clades and sub-clades with unique mutations, revealing the genetic and geographical relevant post the six-month outbreak of COVID-19. This study thoroughly observed the genetic feature of SARS-CoV-2 haplotyping, providing an epidemiological trend of COVID-19.