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Objectives: It is crucial to analyze the consequences of repeated messenger RNA (mRNA)-based COVID-19 vaccinations on SARS-CoV-2 spike receptor binding domain (RBD)-specific immunoglobulin (Ig)G subclass and the possible causal relationship with breakthrough infection. Methods: We examined the longitudinal kinetics of RBD-specific IgG subclass antibodies in sera after receiving the second, third, and fourth doses of mRNA-based COVID-19 vaccines in Japanese healthcare workers. Anti-RBD IgG subclass in sera of patients with COVID-19-infected who had not received the COVID-19 vaccine were also examined. We compared anti-RBD IgG subclass antibody titers in the serum of pre-breakthrough-infected vaccinees and non-infected vaccinees. Results: The seropositivity of anti-RBD IgG4 after the vaccination was 6.76% at 1 month after the second dose, gradually increased to 50.5% at 6 months after the second dose, and reached 97.2% at 1 month after the third dose. The seropositivity and titers of anti-RBD IgG1/IgG3 quickly reached the maximum at 1 month after the second dose and declined afterward. The elevated anti-RBD IgG4 Ab levels observed after repeated vaccinations were unlikely to increase the risk of breakthrough infection. Conclusions: Repeated vaccinations induce delayed but drastic increases in anti-RBD IgG4 responses. Further functional investigations are needed to reveal the magnitude of the high contribution of spike-specific IgG4 subclasses after repeated mRNA-based COVID-19 vaccinations.

Objectives: To report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. Methods: We used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. Results: A mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. Conclusion: Rare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate.

Objectives: This study aims to examine whether the parenterally administered mRNA-based COVID-19 vaccines can induce sufficient mucosal-type IgA responses to prevent SARS-CoV-2 transmission. Methods: We examined the longitudinal kinetics of SARS-CoV-2 spike RBD-specific IgA and IgG responses in sera of Japanese healthcare workers (HCWs) after receiving two doses and the third dose of BNT162b2 mRNA vaccines. During the prospective cohort study, Omicron breakthrough infections occurred in 62 participants among 370 HCWs who had received triple doses of the vaccine. Pre-breakthrough sera of infected HCWs and non-infected HCWs were examined for the levels of anti-RBD IgA and IgG titers. Results: The seropositivity of anti-RBD IgA at 1 M after the second vaccine (2D-1M) and after the third dose (3D-1M) was 65.4% and 87.4%, respectively, and wanes quickly. The boosting effect on anti-RBD Ab titers following breakthrough infections was more notable for anti-RBD IgA than for IgG. There were partial cause-relationships between the lower anti-RBD IgA or IgG at pre-breakthrough sera and the breakthrough infection. Conclusions: Parenterally administered COVID-19 vaccines do not generate sufficient mucosal-type IgA responses despite strong systemic IgG responses to SARS-CoV-2. These results demonstrate the necessity and importance of reevaluating vaccine design and scheduling to efficiently increase oral or respiratory mucosal immunity against SARS-CoV-2.

Abstract Background Hypervirulent Klebsiella pneumoniae (HVKp) infections have distinct clinical manifestations from classical K. pneumoniae infections. The hallmark of HVKp infections are liver abscess formation and metastatic infections. Due to the severe sequelae of these complications, method to identify patients at-risk of HVKp infections should be developed. Results A retrospective cohort study of 222 patients with K. pneumoniae bloodstream infections (BSIs) was performed. Patient demographics, clinical manifestations, and bacterial characteristics were investigated. Ten cases of liver abscesses were identified. Characteristics such as community-onset BSIs, hypermucoviscosity phenotype, and capsular serotype K1 were identified as risk factors for HVKp infections. A scoring system was developed based on the risk factors. The area under the receiver operating characteristic curve for the scoring system was 0.90. A score of ≥ 2 points provided sensitivity and specificity of 0.70 and 0.94, respectively. Conclusions Simple scoring system was developed for the diagnosis of HVKp infections. The system allows early identification of patients with K. pneumoniae BSIs in whom hypervirulent infections should be evaluated. Prospective evaluation is expected.

Cervical cancer is the fourth most common cancer in women globally. Based on several epidemiologic studies, human papillomavirus is strongly associated with cervical neoplasia. Aside from HPV, other bacterial infections in the genital tract were associated with cervical neoplasia. This study aimed to determine the prevalence of HPV infection; and co-infection with Ureaplasma spp., Mycoplasma spp., Chlamydia trachomatis, and Neisseria gonorrheae in Filipino cervical cancer patients. Forty-four patients (28 patients with cervical carcinoma and 16 patients with non-malignant cervix) who consulted in the Philippine General Hospital from 2016 to 2017 were included in this study. HPV genotyping and genetic detection of Ureaplasma spp., Mycoplasma spp., C. trachomatis, and N. gonorrheae were done using different PCR assays. The prevalence of HPV 16/18/33/52 was 75% in cervical cancer patients and 25% in control patients. Infection with HPV 16/18/33/52 was significantly associated with having cervical cancer (OR: 9.00; 95% CI: 2.18–37.18; p = 0.0024). HPV-16 was the most prevalent HPV genotype among Filipino cervical cancer patients. HPV-18 and HPV-52 were only detected from cervical cancer patients. Among HPV-positive patients, we noted a 22.73% co-infection with Ureaplasma spp. and 9.09% co-infection with Mycoplasma spp. To our knowledge, this is the first study on the co-infection of HPV and sexually transmitted infections among cervical cancer patients in the Philippines.

Analysis of longitudinal dynamics of humoral immune responses to the BNT162b2 COVID-19 vaccine might provide useful information to predict the effectiveness of BNT162b2 in preventing SARS-CoV-2 infection. Herein, we measure anti-RBD IgG at 1, 3 and 6 months (M) after the second dose of BNT162b2, and at 1 M after a third dose of BNT162b2 vaccination in 431 COVID-19-naïve healthcare workers (HCWs) in Japan. All HCWs mounted high-anti-RBD IgG responses after the two-dose regimen of BNT162b2 vaccinations. Older persons and males presented lower anti-RBD IgG responses than younger adults and females, respectively. The decay in anti-RBD IgG started from 1 M after the second dose of BNT162b2 and anti-RBD IgG titers dropped to nearly one-tenth at 6 M after the second vaccination. Subsequently, the participants received a third dose of BNT162b2 at 8 M after the second dose of BNT162b2 vaccine. Anti-RBD antibody titers 1 M after the third dose of BNT162b2 increased seventeen times that of 6 M after the second dose, and was twice higher than the peak antibody titers at 1 M after the second dose of vaccination. The negative effect of age for the male gender on anti-RBD IgG antibody titers was not observed at 1 M after the third dose of BNT162b2 vaccine. There were no notable adverse events reported, which required hospitalization in these participants. These results suggest that the third dose of BNT162b2 safely improves humoral immunity against SARS-CoV-2 with no major adverse events.

We investigated the drug-resistant mechanisms of intracellular survival of methicillin-resistant S. aureus (MRSA). Our established MRSA clinical strain, OJ-1, with high biofilm-forming ability, and a macrophage cell line, J774A, were used. After ingestion of OJ-1 by J774A, the cells were incubated for ten days with vancomycin at doses 30 times higher than the minimum inhibitory concentration. The number of phagocytosed intracellular OJ-1 gradually decreased during the study but plateaued after day 7. In J774A cells with intracellular OJ-1, the expression of LysoTracker-positive lysosomes increased until day 5 and then declined from day 7. In contrast, LysoTracker-negative and OJ-1-retaining J774A cells became prominent from day 7, and intracellular OJ-1 also escaped from the autophagosome. Electron microscopy also demonstrated that OJ-1 escaped the phagosomes and was localized in the J774A cytoplasm. At the end of incubation, when vancomycin was withdrawn, OJ-1 started to grow vigorously. The present results indicate that intracellular phagocytosed biofilm-forming MRSA could survive for more than ten days by escaping the lysosomes and autophagosomes in macrophages. Intracellular MRSA may survive in macrophages, and accordingly, they could be resistant to antimicrobial drug treatments. However, the mechanisms their escape from the lysosomes are still unknown. Additional studies will be performed to clarify the lysosome-escaping mechanisms of biofilm-forming MRSA.

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks' gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162.

Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA). MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF) and low-biofilm formers (L-BF). These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections.

Background: The epipharynx, with its high expression of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) entry factors angiotensinconverting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), is a primary target for SARS-CoV-2 replication in the early stage of Coronavirus Disease 19 (COVID-19). Epipharyngeal abrasive therapy (EAT) is a treatment for epipharyngitis in Japan which involves applying zinc chloride to the epipharyngeal mucosa. In this study, we evaluated the expression patterns of ACE2 and TMPRSS2 in tissue samples from patients before and after EAT. Patients and Methods: The study subjects were seven patients that had not been treated with EAT and 11 patients that had. For immunohistochemical assessment of the epipharyngeal mucosa, the staining intensity of ACE2 and TMPRSS2 was described as an immunohistochemical score (IHC score). Results: The IHC scores for ACE2 and TEMPRSS2 in the EAT-treated group were 3.40-fold and 1.81-fold lower, respectively, than those in the non-treated group (p=0.0208 and p=0.0244, respectively). Conclusion: EAT down-regulates the expression of SARS-CoV-2 entry factors ACE2 and TMPRSS2. Thus, EAT has potential as a novel COVID-19 preventative method. [ABSTRACT FROM AUTHOR]