Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Host-Pathogen Interactions in K. pneumoniae Urinary Tract Infections: Investigating Genetic Risk Factors in the Taiwanese Population.

BACKGROUND: Klebsiella pneumoniae (K. pneumoniae) urinary tract infections pose a significant challenge in Taiwan. The significance of this issue arises because of the growing concerns about the antibiotic resistance of K. pneumoniae. Therefore, this study aimed to uncover potential genomic risk factors in Taiwanese patients with K. pneumoniae urinary tract infections through genome-wide association studies (GWAS). METHODS: Genotyping data are obtained from participants with a history of urinary tract infections enrolled at the Tri-Service General Hospital as part of the Taiwan Precision Medicine Initiative (TPMI). A case-control study employing GWAS is designed to detect potential susceptibility single-nucleotide polymorphisms (SNPs) in patients with K. pneumoniae-related urinary tract infections. The associated genes are determined using a genome browser, and their expression profiles are validated via the GTEx database. The GO, Reactome, DisGeNET, and MalaCards databases are also consulted to determine further connections between biological functions, molecular pathways, and associated diseases between these genes. RESULTS: The results identified 11 genetic variants with higher odds ratios compared to controls. These variants are implicated in processes such as adhesion, protein depolymerization, Ca2+-activated potassium channels, SUMOylation, and protein ubiquitination, which could potentially influence the host immune response. CONCLUSIONS: This study implies that certain risk variants may be linked to K. pneumoniae infections by affecting diverse molecular functions that can potentially impact host immunity. Additional research and follow-up studies are necessary to elucidate the influence of these risk variants on infectious diseases and develop targeted interventions for mitigating the spread of K. pneumoniae urinary tract infections.

Efficacy of cefoperazone/sulbactam for ESBL-producing Escherichia coli and Klebsiella pneumoniae bacteraemia and the factors associated with poor outcomes.

OBJECTIVE: We aimed to assess the efficacy of cefoperazone/sulbactam (CPZ/SUL) in extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales infections and identify factors influencing outcomes. METHODS: This retrospective multicentre study was conducted in Taiwan (January 2015 to December 2020) and examined the efficacy of CPZ/SUL treatment in ESBL-producing Enterobacterales bacteraemia. The minimum inhibitory concentrations (MICs) were determined using agar dilution; ESBL/AmpC genes were detected using polymerase chain reaction. The primary outcome was clinical success, whereas the secondary outcome was 30-day mortality. Clinical success was defined as the complete resolution of clinical signs and symptoms of K. pneumoniae or E. coli infection, with no evidence of persistent or recurrent bacteraemia. The factors influencing outcomes were identified using a multivariate analysis. RESULTS: CPZ/SUL demonstrated a clinical success rate of 82.7% (91/110) in treating ESBL-producing Enterobacterales bacteraemia, with a 30-day mortality rate of 9.1% (10/110). Among 110 ESBL-producing isolates, a high clinical success rate was observed at an MIC of ≤32/32 mg/L. Multivariate analysis revealed that a Charlson comorbidity index (CCI) of ≥6 was associated with lower clinical success [odds ratio (OR): 5.80, 95% confidence interval (CI): 1.15-29.14, P = 0.033]. High Sequential Organ Failure Assessment scores (≥6) were significantly associated with increased 30-day mortality (OR: 14.34, 95% CI: 1.45-141.82, P = 0.023). DISCUSSION: CPZ/SUL demonstrated a clinical success rate of 82.7% (91/110) in treating ESBL-producing Enterobacterales bacteraemia. Treatment success was evident when the CPZ and SUL MIC was ≤32/32 mg/L. Comorbidities (CCI ≥6) were associated with lower clinical success, while disease severity (Sequential Organ Failure Assessment score ≥6) correlated with higher mortality.

SARS-CoV-2 nucleocapsid protein, rather than spike protein, triggers a cytokine storm originating from lung epithelial cells in patients with COVID-19.

PURPOSE: The aim of this study was to elucidate the factors associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may initiate cytokine cascades and correlate the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) with their serum cytokine profiles. METHODS: Recombinant baculoviruses displaying SARS-CoV-2 spike or nucleocapsid protein were constructed and transfected into A549 cells and THP-1-derived macrophages, to determine which protein initiate cytokine release. SARS-CoV-2-specific antibody titers and cytokine profiles of patients with COVID-19 were determined, and the results were associated with their clinical characteristics, such as development of pneumonia or length of hospital stay. RESULTS: The SARS-CoV-2 nucleocapsid protein, rather than the spike protein, triggers lung epithelial A549 cells to express IP-10, RANTES, IL-16, MIP-1α, basic FGF, eotaxin, IL-15, PDGF-BB, TRAIL, VEGF-A, and IL-5. Additionally, serum CTACK, basic FGF, GRO-α, IL-1α, IL-1RA, IL-2Rα, IL-9, IL-15, IL-16, IL-18, IP-10, M-CSF, MIF, MIG, RANTES, SCGF-ß, SDF-1α, TNF-α, TNF-ß, VEGF, PDGF-BB, TRAIL, ß-NGF, eotaxin, GM-CSF, IFN-α2, INF-γ, and MCP-1 levels were considerably increased in patients with COVID-19. Among them, patients with pneumonia had higher serum IP-10 and M-CSF levels than patients without. Patients requiring less than 3 weeks to show negative COVID-19 tests after contracting COVID-19 had higher serum IP-10 levels than the remaining patients. CONCLUSION: Our study revealed that nucleocapsid protein, lung epithelial cells, and IP-10 may be potential targets for the development of new strategies to prevent, or control, severe COVID-19.

Accelerating pandemic response with the emergency Omicron RT-PCR test: A comprehensive solution for COVID-19 diagnosis and tracking.

The Omicron variant of concern (VOC) has surged in many countries and replaced the previously reported VOC. To identify different Omicron strains/sublineages on a rapid, convenient, and precise platform, we report a novel multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) method in one tube based on the Omicron lineage sequence variants' information. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subvariants were used in a PCR-based assay for rapid identification of Omicron sublineage genotyping in 1000 clinical samples. Several characteristic mutations were analyzed using specific primers and probes for the spike gene, del69-70, and F486V. To distinguish Omicron sublineages (BA.2, BA.4, and BA.5), the NSP1:141-143del in the ORF1a region and D3N mutation in membrane protein occurring outside the spike protein region were analyzed. Results from the real-time PCR assay for one-tube accuracy were compared to those of whole genome sequencing. The developed PCR assay was used to analyze 400 SARS-CoV-2 positive samples. Ten samples determined as BA.4 were positive for NSP1:141-143del, del69-70, and F486V mutations; 160 BA.5 samples were positive for D3N, del69-70, and F486V mutations, and 230 BA.2 samples were without del69-70. Screening these samples allowed the identification of epidemic trends at different time intervals. Our novel one-tube multiplex PCR assay was effective in identifying Omicron sublineages.

A 20-Year Study of Capsular Polysaccharide Seroepidemiology, Susceptibility Profiles, and Virulence Determinants of Klebsiella pneumoniae from Bacteremia Patients in Taiwan.

In this study, we selected bacteremic Klebsiella pneumoniae isolates from the Taiwan Surveillance of Antimicrobial Resistance program. A total of 521 isolates were collected over a period of 2 decades, including 121 from 1998, 197 from 2008, and 203 from 2018. Seroepidemiology showed that the top five capsular polysaccharide types were serotypes K1, K2, K20, K54, and K62, constituting 48.5% of the total isolates, and the respective ratios at each time point have remained similar over the past 2 decades. The antibacterial susceptibility tests showed that K1, K2, K20, and K54 were susceptible to most antibiotics, while K62 was relatively resistant compared to other typeable and nontypeable strains. In addition, six virulence-associated genes, clbA, entB, iroN, rmpA, iutA, and iucA, were predominant in K1 and K2 isolates of K. pneumoniae. In conclusion, serotypes K1, K2, K20, K54, and K62 of K. pneumoniae are the most prevalent serotypes and carry more virulence determinants in bacteremia patients, which may indicate their invasiveness. If further serotype-specific vaccine development is performed, these five serotypes should be included. Since the antibiotic susceptibility profiles were stable over a long duration, empirical treatment may be predicted according to serotype if rapid diagnosis from direct clinical specimens is available, such as PCR or antigen serotyping for serotype K1 and K2. IMPORTANCE This is the first nationwide study to examine the seroepidemiology of Klebsiella pneumoniae using blood culture isolates collected over a period of 20 years. The study found that the prevalence of serotypes remained consistent over the 20-year period, with high-prevalence serotypes associated with invasive types. Nontypeable isolates had fewer virulence determinants than other serotypes. With the exception of serotype K62, the other high-prevalence serotypes were highly susceptible to antibiotics. If rapid diagnosis using direct clinical specimens, such as PCR or antigen serotyping, is available, empirical treatment can be predicted based on serotype, particularly for K1 and K2. The results of this seroepidemiology study could also help the development of future capsule polysaccharide vaccines.

Appropriate antibiotic therapy is a predictor of outcome in patients with Stenotrophomonas maltophilia blood stream infection in the intensive care unit.

BACKGROUND/PURPOSE: The study was to assess the relationship between antibiotic therapy and the outcome in intensive care unit (ICU) patients with Stenotrophomonas maltophilia bloodstream infection (BSI). METHODS: ICU patients with monomicrobial S. maltophilia BSI from January 2004 to December 2019 were included and divided into two groups-those with- and without appropriate antibiotic therapy after BSI-for comparison. The primary outcome was the relationship between appropriate antibiotic therapy and 14-day mortality. The secondary outcome was the influence of different antibiotic therapies: levofloxacin- and trimethoprim-sulfamethoxazole (TMP/SMX)-containing regimens, on 14-day mortality. RESULTS: A total of 214 ICU patients were included. Patients received appropriate antibiotic therapy (n = 133) after BSI had a lower 14-day mortality than those (n = 81) without appropriate antibiotic therapy (10.5% vs. 46.9%, p < 0.001). No difference on 14-day mortality between groups of patients by time of appropriate antibiotic therapy was observed (p > 0.05). After a propensity score matching, the results is consistent that 14-day mortality were lower in patients with appropriate antibiotic therapy than those without appropriate antibiotic therapy (11.5% vs. 39.3%, p < 0.001). Among patients with S. maltophilia BSI receiving appropriate antibiotic therapy, there was a trend levofloxacin-containing regimens is associated with lower mortality than TMP/SMX-containing regimens (HR 0.233, 95% CI 0.050-1.084, p = 0.063). CONCLUSION: Appropriate antibiotic therapy was associated with decreased 14-day mortality in ICU patients with S. maltophilia BSI regardless of time. Levofloxacin-containing regimens may be better choice than TMP/SMX -containing regimens in treating ICU patients with S. maltophilia BSI.

Monitoring algorithm of hospitalized patients in a medical center with SARS-CoV-2 (Omicron variant) infection: clinical epidemiological surveillance and immunological assessment.

Purpose: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major healthcare threat worldwide. Since it was first identified in November 2021, the Omicron (B.1.1.529) variant of SARS-CoV-2 has evolved into several lineages, including BA.1, BA.2-BA.4, and BA.5. SARS-CoV-2 variants might increase transmissibility, pathogenicity, and resistance to vaccine-induced immunity. Thus, the epidemiological surveillance of circulating lineages using variant phenotyping is essential. The aim of the current study was to characterize the clinical outcome of Omicron BA.2 infections among hospitalized COVID-19 patients and to perform an immunological assessment of such cases against SARS-CoV-2. Patients and Methods: We evaluated the analytical and clinical performance of the BioIC SARS-CoV-2 immunoglobulin (Ig)M/IgG detection kit, which was used for detecting antibodies against SARS-CoV-2 in 257 patients infected with the Omicron variant. Results: Poor prognosis was noted in 38 patients, including eight deaths in patients characterized by comorbidities predisposing them to severe COVID-19. The variant-of-concern (VOC) typing and serological analysis identified time-dependent epidemic trends of BA.2 variants emerging in the outbreak of the fourth wave in Taiwan. Of the 257 specimens analyzed, 108 (42%) and 24 (9.3%) were positive for anti-N IgM and IgG respectively. Conclusion: The VOC typing of these samples allowed for the identification of epidemic trends by time intervals, including the B.1.1.529 variant replacing the B.1.617.2 variant. Moreover, antibody testing might serve as a complementary method for COVID-19 diagnosis. The combination of serological testing results with the reverse transcription-polymerase chain reaction cycle threshold value has potential value in disease prognosis, thereby aiding in epidemic investigations conducted by clinicians or the healthcare department.

Preservation of red blood cell antigenicity in a new storage solution in vitro.

INTRODUCTION: Red blood cell (RBC) storage solution is used for suspending and preserving RBCs for later use in in vitro immunohematology testing. Proper RBC preservation is crucial for obtaining accurate results in RBC phenotyping and pretransfusion antibody screening tests. Haemolysis or RBC antigen degradation during storage can result in inaccurate RBC phenotyping, thereby decreasing the sensitivity of pretransfusion antibody screening and identification assays. The conventional RBC storage solutions usually contain adenosine, adenine, and antibiotics. We designed an RBC storage solution and determined whether it could preserve RBC integrity for 70 days. MATERIALS AND METHODS: The new storage solution has a different formula from that of the conventional solution-in particular, it is strengthened with polyethylene glycol (PEG). The extent of haemolysis and hemagglutination reactivity of the RBC antigen systems, Rh, Duffy, Kidd, Lewis, MNS, P1, and the rare antigen Mia (which has a low prevalence antigen in most parts of the world but a higher prevalence in Taiwan), in the new RBC storage solution was compared with that of the conventionally preserved RBC storage solution. RESULTS: The RBCs preserved in the new solution for 70 days retained a similar haemolysis grade as those preserved in the control solution for 28 days. Although both solutions largely preserved RBC antigenicity, the decline in RBC hemagglutination scores in new solution often occurred later than that in the control solution in most antigen phenotyping assays, especially labile antigens such as D, P1, and M. CONCLUSION: The new solution reduces haemolysis more effectively and preserves antigenicity throughout the 70-day storage period. Moreover, Mia antigen is more stable in the experimental group.

The application of a novel 5-in-1 multiplex reverse transcriptase-polymerase chain reaction assay for rapid detection of SARS-CoV-2 and differentiation between variants of concern.

OBJECTIVES: We have established a novel 5-in-1 VOC assay to rapidly detect SARS-CoV-2 and immediately distinguish whether positive samples represent variants of concern (VOCs). METHODS: This assay could distinguish among five VOCs: Alpha, Beta, Gamma, Delta, and Omicron, in a single reaction tube. The five variants exhibit different single nucleotide polymorphisms (SNPs) in their viral genome, which can be used to distinguish them. We selected target SNPs in the spike gene, including N501Y, P681R, K417N, and deletion H69/V70 for the assay. RESULTS: The limit of detection of each gene locus was 80 copies per polymerase chain reaction. We observed a high consistency among the results when comparing the performance of our 5-in-1 VOC assay, whole gene sequencing, and the Roche VirSNiP SARS-CoV-2 test in retrospectively analyzing 150 clinical SARS-CoV-2 variant positive samples. The 5-in-1 VOC assay offers an alternative and rapid high-throughput test for most diagnostic laboratories in a flexible sample-to-result platform. CONCLUSION: The assay can also be applied in a commercial platform with the completion of the SARS-CoV-2 confirmation test and identification of its variant within 2.5 hours.

Human case of Anaplasma phagocytophilum infection in Eastern Taiwan.

Human granulocytic anaplasmosis (HGA) is a tick-borne infection caused by the bacterium Anaplasma phagocytophilum. In this study, we report an indigenous case of clinically diagnosed HGA. The patient was a 41-year-old man who experienced a tick bite and later developed fever, chills, myalgia, malaise, thrombocytopenia, leukocytosis with a left shift, elevated hepatic transaminase levels, and splenomegaly upon admission to the hospital. Immunofluorescence assays detected seroconversion against A. phagocytophilum, whereas tests for spotted fever group rickettsiae, murine typhus, scrub typhus, Q fever, and ehrlichiosis were negative. ELISA and Western blot analysis using recombinant MSP2 protein confirmed the exposure to A. phagocytophilum. Oral doxycycline and intravenous ceftriaxone were prescribed, and the patient made a full recovery. Our findings indicate the presence of HGA on the main island of Taiwan. Precautions against tick bites should be taken when engaging in outdoor activities, and HGA should be considered by physicians in the differential diagnosis.

Gaps in antimicrobial stewardship programmes in Asia: a survey of 10 countries.

Objectives: To determine antimicrobial stewardship (AMS) programme practices in Asian secondary- and tertiary-care hospitals. Methods: AMS programme team members within 349 hospitals from 10 countries (Cambodia, India, Indonesia, Japan, Malaysia, Pakistan, the Philippines, Taiwan, Thailand and Vietnam) completed a questionnaire via a web-based survey link. The survey contained questions as to whether 12 core components deemed essential for AMS programmes were implemented. Results: Overall, 47 (13.5%) hospitals fulfilled all core AMS programme components. There was a mean positive response rate (PRR) of 85.6% for the responding countries in relation to a formal hospital leadership statement of support for AMS activities, but this was not matched by budgeted financial support for AMS activities (mean PRR 57.1%). Mean PRRs were ≥80.0% for the core AMS team comprising a physician or other leader responsible for AMS activities, a pharmacist and infection control and microbiology personnel. Most hospitals had access to a timely and reliable microbiology service (mean PRR 90.4%). Facility-specific antibiotic treatment guidelines for common infections (mean PRR 78.7%) were in place more often than pre-authorization and/or prospective audit and feedback systems (mean PRR 66.5%). In terms of AMS monitoring and reporting, PRRs of monitoring specific antibiotic use, regularly publishing AMS outcome measures, and the existence of a hospital antibiogram were 75.1%, 64.4% and 77.9%, respectively. Conclusions: Most hospitals participating in this survey did not have AMS programmes fulfilling the requirements for gold standard AMS programmes in hospital settings. Urgent action is required to address AMS funding and resourcing deficits.

Critical pediatric neurological illness associated with COVID-19 (Omicron BA.2.3.7 variant) infection in Taiwan: immunological assessment and viral genome analysis in tertiary medical center.

OBJECTIVES: Since April 2022, another wave of the Omicron epidemic has struck Taiwanese society, and children with severe neurological complications have been reported frequently. A few cases even developed acute fulminant encephalitis. To investigate the possible causes of the increased incidence of such complications in Taiwan, we reviewed several cases of pediatric patients with severe neurological symptoms. METHODS: We collected the medical records of pediatric patients with COVID-19 infection who presented with severe neurological symptoms. The COVID-19 infection was diagnosed by nasal swab reverse transcriptase-polymerase chain reaction. The remaining samples were sent for whole genome sequencing and spike (S) protein amino acid variation mapping. RESULTS: The increase of several inflammatory markers was observed in all patients included in this study. However, none of the cerebrospinal fluid samples tested positive for SARS-CoV-2. The result of whole genome sequencing showed that all the sequences belonged to the lineage BA.2.3.7. However, the sequences had a K97E mutation in the S protein that differed from other BA.2.3.7 lineage strains, which was located at the S protein N-terminal domain. CONCLUSION: The new mutation in the S protein, which had not previously been observed but was discovered in this study, potentially explains the sudden increase in incidence of extremely adverse neurological symptoms in pediatric patients.

Boosting with Multiple Doses of mRNA Vaccine after Priming with Two Doses of Protein Subunit Vaccine MVC-COV1901 Elicited Robust Humoral and Cellular Immune Responses against Emerging SARS-CoV-2 Variants.

Confronted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, such as Delta and Omicron, with high infectivity and immune evasion capacity, vaccination remains the most effective tool to prevent infection and severe illness. However, heterologous vaccination of mRNA vaccines primed with protein subunit vaccines had not been evaluated before the current study. Since subunit vaccine MVC-COV1901 (MVC) has been granted emergency use authorization in Taiwan, in this study, we explored the humoral and cellular immune responses to additional third (2× MVC/Mod) and fourth (2× MVC/2× Mod) doses of mRNA-1273 (Mod) after priming with two doses of subunit vaccine MVC against the emerging variants. We found a 12.3- to 16.1-fold increase in antibodies targeting the receptor binding domain (RBD) of the Delta variant with 2× MVC/Mod compared to two doses of MVC (2× MVC) or AZD1222 (2× AZ) regimens and a 26- to 32.2-fold improvement in neutralizing potency against the Omicron variant (BA.1). Besides, the numbers of gamma interferon (IFN-γ)-secreting T cells induced by 2× MVC/Mod were also elevated 3.5-fold and 3.7- to 4.3-fold for the wild type and Delta variant. However, boosting with a fourth dose of Mod (2× MVC/2× Mod) after the 2× MVC/Mod regimen failed to significantly improve the immune responses. Moreover, all vaccination schedules showed reduced neutralizing activity against the Omicron variant. Collectively, our results suggested that the third or fourth dose booster vaccination with mRNA vaccine after priming with two doses of protein subunit vaccine could elicit stronger humoral and cellular immune responses. These findings could provide a future global heterologous boosting strategy against COVID-19. IMPORTANCE Vaccination is the most important strategy to combat the COVID-19 outbreak; however, it remains to be determined whether heterologous prime-boost regimens could induce equal or even stronger immune responses against SARS-CoV-2. Here, we showed that boosting the additional doses of mRNA-1273 (Mod) priming with two doses of MVC-COV1901 (MVC) (2× MVC/Mod) improved humoral and cellular immunity compared to two doses of AZD1222 (2× AZ) or MVC (2× MVC) against SARS-CoV-2 variants. However, the Omicron variant showed strong immune evasion ability for all vaccination schedules. Our findings provided evidence supporting that heterologous vaccination by boosting with mRNA vaccine after priming with two doses of protein subunit vaccine could strongly promote humoral and cellular immune responses against the emerging SARS-CoV-2 variants.

Correlation Between Cefoperazone/Sulbactam MIC Values and Clinical Outcomes of Escherichia coli Bacteremia.

INTRODUCTION: The clinical efficiency of cefoperazone/sulbactam (CPZ/SUL) against Escherichia coli bacteremia was unknown. This study aimed to explore the relationship between CPZ/SUL MIC values and clinical outcomes in Escherichia coli bacteremia. METHODS: A multicenter, retrospective, observational cohort study was conducted in Taiwan between January 2015 and December 2020. Patients treated with CPZ/SUL for E. coli bacteremia were enrolled in the analysis. The CPZ/SUL MICs were determined by using the agar dilution method. The primary outcome was 30-day mortality. RESULTS: Among 247 isolates, 160 (64.8%) isolates were susceptible, 8 (3.2%) were intermediate, and 79 (32.0%) were resistant to cefoperazone. The activity of cefoperazone against cefoperazone-non-susceptible E. coli (n = 87) was restored upon combination with sulbactam, with susceptibility ranging from 0% to 97.7%. The 30-day mortality was 4.5% (11/247) and overall clinical success rate was 91.9% (227/247). Multivariate Cox proportional-hazards model revealed that heart failure [adjusted relative risk (ARR), 5.49; 95% confidence interval (CI) 1.31-23.02; p = 0.020], malignancy (ARR 7.50; 95% CI 2.02-27.80; p = 0.003), SOFA score (ARR 1.29; 95% CI 1.09-1.52; p = 0.003), and CPZ/SUL MIC ≥ 64 mg/L (ARR 11.31; 95% CI 1.34-95.52; p = 0.026) were independently associated with 30-day mortality. No statistically significant differences in 30-day mortality were found between groups with or without cefoperazone susceptibility (3.4% vs. 5.0%, p = 0.751, respectively). CONCLUSIONS: Patients with E. coli bacteremia who were treated with CPZ/SUL had a favorable outcome when the MICs of the isolates were ≤ 16 mg/L and a high risk of mortality with MICs ≥ 64 mg/L.

Humoral, Cellular and Cytokine Immune Responses Against SARS-CoV-2 Variants in COVID-19 Convalescent and Confirmed Patients With Different Disease Severities.

Objectives: To assess humoral and cellular immune responses against SARS-CoV-2 variants in COVID-19 convalescent and confirmed patients, to explore the correlation between disease severity, humoral immunity, and cytokines/chemokines in confirmed patients, and to evaluate the ADE risk of SARS-CoV-2. Methods: Anti-RBD IgG were quantified using an ELISA. Neutralization potency was measured using pseudovirus and real virus. Cellular immunity was measured using ELISpot. Cytokine/chemokine levels were detected using multiplex immunoassays. In vitro ADE assays were performed using Raji cells. Results: One-month alpha convalescents exhibited spike-specific antibodies and T cells for alpha and delta variants. Notably, the RBD-specific IgG towards the delta variant decreased by 2.5-fold compared to the alpha variant. Besides, serum from individuals recently experienced COVID-19 showed suboptimal neutralizing activity against the delta and omicron variants. Humoral immune response, IL-6, IP-10 and MCP-1 levels were greater in patients with severe disease. Moreover, neither SARS-CoV-1 nor SARS-CoV-2 convalescent sera significantly enhanced SARS-CoV-2 pseudovirus infection. Conclusions: Significant resistance of the delta and omicron variants to the humoral immune response generated by individuals who recently experienced COVID-19. Furthermore, there was a significant correlation among disease severity, humoral immune response, and specific cytokines/chemokine levels. No evident ADE was observed for SARS-CoV-2.

Emergency SARS-CoV-2 variants of concern: rapidly direct RT-qPCR detection without RNA extraction, clinical comparison, cost-effective, and high-throughput.

Since the late 2020, the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has been characterized by the emergence of spike protein mutations, and these variants have become dominant worldwide. The gold standard SARS-CoV-2 diagnosis protocol requires two complex processes, namely, RNA extraction and real-time reverse transcriptase polymerase chain reaction (RT-PCR). There is a need for a faster, simpler, and more cost-effective detection strategy that can be utilized worldwide, especially in developing countries. We propose the novel use of direct RT-qPCR, which does not require RNA extraction or a preheating step. For the detection, retrospectively, we used 770 clinical nasopharyngeal swabs, including positive and negative samples. The samples were subjected to RT-qPCR in the N1 and E genes using two different thermocyclers. The limit of detection was 30 copies/reaction for N1 and 60 copies/reaction for E. Analytical sensitivity was assessed for the developed direct RT-qPCR; the sensitivity was 95.69%, negative predictive value was 99.9%, accuracy of 99.35%, and area under the curve was 0.978. This novel direct RT-qPCR diagnosis method without RNA extraction is a reliable and high-throughput alternative method that can significantly save cost, labor, and time during the coronavirus disease 2019 pandemic.

Hospital-acquired infections in patients hospitalized with COVID-19: First report from Taiwan.

BACKGROUND: Coronavirus disease 2019 (COVID-19) inpatients may acquire infections from other pathogens during hospital admission. This is the first research on this subject to be reported from Taiwan. METHODS: Confirmed COVID-19 inpatients were enrolled in this study from January 1, 2020 to July 31, 2021. Various types of pathogens in COVID-19 inpatients, with hospital-acquired infections, were identified and analyzed. The clinical characteristics of COVID-19 patients with and without hospital-acquired infections were reviewed and compared. RESULTS: Of the 204 patients included in the study, 32 (15.7%) patients experienced at least one infectious episode. Of 113 recorded episodes of infection, the predominant type was bacterial (88 of 113 infections, 77.9%); the most frequently isolated bacteria were Acinetobacter spp., followed by Stenotrophomonas maltophilia . With regard to viral infections (19 of 113, 16.8%), the Epstein-Barr virus ranked first place among the identified viruses. Four (3.5%) and 2 (1.8%) of 113 infectious episodes were caused by fungi and atypical pathogens. A multivariate analysis revealed that steroid use was an independent factor in hospital-acquired infections (odds ratio [OR], 6.97; 95% confidence interval [CI], 1.15-42.43; p = 0.035). Patients with hospital-acquired infections were associated with increased 28-day and in-hospital mortality (18.8% vs 5.8% and 31.3% and 5.8%; p = 0.023 and <0.01, respectively), and a longer hospital stay (34 vs 19 days; p < 0.001), compared to those without hospital-acquired infections. CONCLUSION: Our study revealed the unique local epidemiology of hospital-acquired infections among COVID-19 inpatients in Taiwan. These patients were associated with increased mortality and prolonged hospital admissions.

Clinical Characteristics of COVID-19 Patients and Application to an Artificial Intelligence System for Disease Surveillance.

During the coronavirus disease (COVID-19) pandemic, we admitted suspected or confirmed COVID-19 patients to our isolation wards between 2 March 2020 and 4 May 2020, following a well-designed and efficient assessment protocol. We included 217 patients suspected of COVID-19, of which 27 had confirmed COVID-19. The clinical characteristics of these patients were used to train artificial intelligence (AI) models such as support vector machine (SVM), decision tree, random forest, and artificial neural network for diagnosing COVID-19. When analyzing the performance of the models, SVM showed the highest sensitivity (SVM vs. decision tree vs. random forest vs. artificial neural network: 100% vs. 42.86% vs. 28.57% vs. 71.43%), while decision tree and random forest had the highest specificity (SVM vs. decision tree vs. random forest vs. artificial neural network: 88.37% vs. 100% vs. 100% vs. 94.74%) in the diagnosis of COVID-19. With the aid of AI models, physicians may identify COVID-19 patients earlier, even with few baseline data available, and segregate infected patients earlier to avoid hospital cluster infections and to ensure the safety of medical professionals and ordinary patients in the hospital.

Cross-protection induced by highly conserved outer membrane proteins (Omps) in mice immunized with OmpC of Salmonella Typhi or OmpK36 of Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Outer membrane proteins (Omps) are a family of proteins that are highly conserved throughout the evolution of Enterobacteriaceae. Previous studies using sequence comparisons have found a high degree of sequence homology between OmpK36 of Klebsiella pneumoniae and OmpC of Salmonella enterica serovar Typhi. Whether highly conserved OmpC can be directly extrapolated as a common vaccine candidate against K. pneumoniae or other Enterobacteriaceae remains to be verified. METHODS: OmpK36 and OmpC were purified and used to immunize BALB/c mice. After immunization, five mice from each group were injected intraperitoneally with a cell suspension of K. pneumoniae or S. Typhi, and the mice were monitored daily for 14 days to measure the severity of illness and assess their survival. RESULTS: Cross-reacting OmpK36 and OmpC antibodies were identified in the mice immunized with OmpK36 or OmpC. No cross-protection was observed in the mice immunized with OmpC in the presence of K. pneumoniae infection. CONCLUSION: Although a high degree of similarity was observed for the amino acid sequences between OmpK36 and OmpC, our results suggested that no cross-protection occurred in the mice challenged with other species.