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.

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.

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.

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.

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.

Clinical Comparative Evaluation of the LabTurboTM AIO® Reverse Transcription-Polymerase Chain Reaction and World Health Organization-Recommended Assays for the Detection of Emerging SARS-CoV-2 Variants of Concern.

PURPOSE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent behind coronavirus disease-2019 (COVID-19). Single-plex reverse transcription-polymerase chain reaction (RT-PCR)-based assays are widely used for COVID-19 detection but exhibit decreased sensitivity and specificity in detecting the rapidly spreading SARS-CoV-2 variants; in contrast, multiplex RT-PCR reportedly yields better results. Here, we aimed at comparatively analyzing the clinical performance of the LabTurboTM AIO COVID-19 RNA testing kit, a multiplex quantitative RT-PCR kit, including a three-target (E, N1, and RNase P), single-reaction, triplex assay used for SARS-CoV-2 detection, with that of the WHO-recommended RT-PCR assay. MATERIALS AND METHODS: Residual, natural, nasopharyngeal swabs obtained from universal transport medium specimens at SARS-CoV-2 testing centers (n = 414) were collected from May to October 2021. For SARS-CoV-2 qRT-PCR, total viral nucleic acid was extracted. The limit of detection (LOD) and the comparative clinical performances of the LabTurboTM AIO COVID-19 RNA kit and the WHO-recommended RT-PCR assay were assessed. Statistical analysis of the correlation was performed and results with R2 values >0.9 were considered to be highly correlated. RESULTS: The LOD of the LabTurboTM AIO COVID-19 RNA kit was 9.4 copies/reaction for the target genes N1 and E. The results obtained from 102 SARS-CoV-2-positive and 312 SARS-CoV-2-negative samples showed 100% correlation with previous WHO-recommended RT-PCR assay results. CONCLUSION: Multiplex qRT-PCR is a critical tool for detecting unknown pathogens and employs multiple target genes. The LabTurboTM AIO COVID-19 RNA testing kit provides an effective and efficient assay for SARS-CoV-2 detection and is highly compatible with SARS-CoV-2 variants.

Emergency SARS-CoV-2 Variants of Concern: Novel Multiplex Real-Time RT-PCR Assay for Rapid Detection and Surveillance.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread worldwide. Many variants of SARS-CoV-2 have been reported, some of which have increased transmissibility and/or reduced susceptibility to vaccines. There is an urgent need for variant phenotyping for epidemiological surveillance of circulating lineages. Whole-genome sequencing is the gold standard for identifying SARS-CoV-2 variants, which constitutes a major bottleneck in developing countries. Methodological simplification could increase epidemiological surveillance feasibility and efficiency. We designed a novel multiplex real-time reverse transcriptase PCR (RT-PCR) to detect SARS-CoV-2 variants with S gene mutations. This multiplex PCR typing method was established to detect 9 mutations with specific primers and probes (ΔHV 69/70, K417T, K417N, L452R, E484K, E484Q, N501Y, P681H, and P681R) against the receptor-binding domain of the spike protein of SARS-CoV-2 variants. In silico analyses showed high specificity of the assays. Variants of concern (VOC) typing results were found to be highly specific for our intended targets, with no cross-reactivity observed with other upper respiratory viruses. The PCR-based typing methods were further validated using whole-genome sequencing and a commercial kit that was applied to clinical samples of 250 COVID-19 patients from Taiwan. The screening of these samples allowed the identification of epidemic trends by time intervals, including B.1.617.2 in the third Taiwan wave outbreak. This PCR typing strategy allowed the detection of five major variants of concern and also provided an open-source PCR assay which could rapidly be deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, P.1, and B.1.617.2 variants and of four Omicron mutations on the spike protein (ΔHV 69/70, K417N, N501Y, P681H). IMPORTANCE COVID-19 has spread globally. SARS-CoV-2 variants of concern (VOCs) are leading the next waves of the COVID-19 pandemic. Previous studies have pointed out that these VOCs may have increased infectivity, have reduced vaccine susceptibility, change treatment regimens, and increase the difficulty of epidemic prevention policy. Understanding SARS-CoV-2 variants remains an issue of concern for all local government authorities and is critical for establishing and implementing effective public health measures. A novel SARS-CoV-2 variant identification method based on a multiplex real-time RT-PCR was developed in this study. Five SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, and Omicron) were identified simultaneously using this method. PCR typing can provide rapid testing results with lower cost and higher feasibility, which is well within the capacity for any diagnostic laboratory. Characterizing these variants and their mutations is important for tracking SAR-CoV-2 evolution and is conducive to public infection control and policy formulation strategies.

Multicenter study evaluating novel multi-specimen pooling assay for the detection of SARS-CoV-2: High sensitivity and high throughput testing.

BACKGROUND/PURPOSE: Mass screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is important to prevent the spread of coronavirus disease 2019 (COVID-19). Pooling samples can increase the number of tests processed. LabTurbo AIO 48 is an automated platform that allows ribonucleic acid extraction and sample analysis on the same instrument. We created a novel pooling assay on this platform for SARS-CoV-2 detection and demonstrated that the pooling strategy increases testing capacity without affecting accuracy and sensitivity. METHODS: Comparative limit of detection (LoD) assessment was performed on the LabTurbo AIO 48 platform and the current standard detection system based on real-time reverse transcription polymerase chain reaction (rRT-PCR) using 55 clinically positive samples. An additional 330 primary clinical samples were assessed. RESULTS: Six samples pooled into one reaction tube were detected in approximately 2.5 h using the World Health Organization rRT-PCR protocol. LabTurbo AIO 48 also demonstrated a higher throughput than our reference rRT-PCR assay, with an LoD of 1000 copies/mL. The overall percentage agreement between the methods for the 330 samples was 100%. CONCLUSION: We created a novel multi-specimen pooling assay using LabTurbo AIO 48 for the robust detection of SARS-CoV-2, allowing high-throughput results; this assay will aid in better control and prevention of COVID-19. The diagnostic assay was cost-effective and time-efficient; thus, the pooling strategy is a practical and effective method for diagnosing large quantities of specimens without compromising precision.

SARS-CoV-2 variants with T135I nucleocapsid mutations may affect antigen test performance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic. Diagnostic testing for SARS-CoV-2 has continuously been challenged due to several variants with diverse spike (S) and nucleocapsid (N) protein mutations []. SARS-CoV-2 variant proliferation potentially affects N protein-targeted rapid antigen testing. In this study, rapid antigen and reverse transcription PCR (RT-PCR) tests were performed simultaneously in patients with suspected coronavirus disease 2019 (COVID-19). Direct whole genome sequencing was performed to determine the N protein variations, and the viral assemblies were uploaded to GISAID. The genomes were then compared with those of global virus strains from GISAID. These isolates belonged to the B.1.1.7 variant, exhibiting several amino acid substitutions, including D3L, R203K, G204R, and S235F N protein mutations. The T135I mutation was also identified in one variant case in which the rapid antigen test and RT-PCR test were discordantly negative and positive, respectively. These findings suggest that the variants undetected by the Panbio COVID-19 rapid antigen test may be due to the T135I mutation in the N protein, posing a potential diagnostic risk for commercially available antigen tests. Hence, we recommend concomitant paired rapid antigen tests and molecular diagnostic methods to detect SARS-CoV-2. False-negative results could be rapidly corrected using confirmatory RT-PCR results to prevent future COVID-19 outbreaks.

Clinical assessment of SARS-CoV-2 antigen rapid detection compared with RT-PCR assay for emerging variants at a high-throughput community testing site in Taiwan.

OBJECTIVES: With the emergence of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) B.1.1.7 lineage in the ongoing coronavirus disease 2019 (COVID-19) pandemic, Taiwan confronted a COVID-19 flare up in May 2021. Large-scale, accurate, affordable and rapid diagnostic tests such as the lateral flow assay can help to prevent community transmission, but their performance characteristics in real-world conditions and relevant subpopulations remain unclear. METHODS: The COVID-19 Antigen Rapid Test Kit (Eternal Materials, New Taipei City, Taiwan) was used in a high-throughput community testing site; the paired reverse transcription polymerase chain reaction (RT-PCR) results served as a reference for sensitivity and specificity calculations. RESULTS: Of 2096 specimens tested using the rapid antigen test, 70 (3.33%) were positive and 2026 (96.7%) were negative. This clinical performance was compared with the RT-PCR results. The sensitivity and specificity of the rapid antigen test were 76.39% [95% confidence interval (CI) 64.91-85.60%] and 99.26% (95% CI 98.78-99.58%), respectively, with high sensitivity in subjects with cycle threshold values ≤24. Further, the rapid antigen test detected the SARS-CoV-2 B.1.1.7 lineage effectively. CONCLUSIONS: Considering the short turnaround times and lower costs, this simple SARS-CoV-2 antigen detection test for rapid screening combined with RT-PCR as a double confirmatory screening tool can facilitate the prevention of community transmission during COVID-19 emergencies.

Multicenter study evaluating one multiplex RT-PCR assay to detect SARS-CoV-2, influenza A/B, and respiratory syncytia virus using the LabTurbo AIO open platform: epidemiological features, automated sample-to-result, and high-throughput testing.

Since the Coronavirus 19 (COVID-19) pandemic, several SARS-CoV-2 variants of concern (SARS-CoV-2 VOC) have been reported. The B.1.1.7 variant has been associated with increased mortality and transmission risk. Furthermore, cluster and possible co-infection cases could occur in the next influenza season or COVID-19 pandemic wave, warranting efficient diagnosis and treatment decision making. Here, we aimed to detect SARS-CoV-2 and other common respiratory viruses using multiplex RT-PCR developed on the LabTurbo AIO 48 open system. We performed a multicenter study to evaluate the performance and analytical sensitivity of the LabTurbo AIO 48 system for SARS-CoV-2, influenza A/B, and respiratory syncytial virus (RSV) using 652 nasopharyngeal swab clinical samples from patients. The LabTurbo AIO 48 system demonstrated a sensitivity of 9.4 copies/per PCR for N2 of SARS-CoV-2; 24 copies/per PCR for M of influenza A and B; and 24 copies/per PCR for N of RSV. The assay presented consistent performance in the multicenter study. The multiplex RT-PCR applied on the LabTurbo AIO 48 open platform provided highly sensitive, robust, and accurate results and enabled high-throughput detection of B.1.1.7, influenza A/B, and RSV with short turnaround times. Therefore, this automated molecular diagnostic assay could enable streamlined testing if COVID-19 becomes a seasonal disease.

Genomic analysis of early transmissibility assessment of the D614G mutant strain of SARS-CoV-2 in travelers returning to Taiwan from the United States of America.

BACKGROUND: There is a global pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Information on viral genomics is crucial for understanding global dispersion and for providing insight into viral pathogenicity and transmission. Here, we characterized the SARS-CoV-2 genomes isolated from five travelers who returned to Taiwan from the United States of America (USA) between March and April 2020. METHODS: Haplotype network analysis was performed using genome-wide single-nucleotide variations to trace potential infection routes. To determine the genetic variations and evolutionary trajectory of the isolates, the genomes of isolates were compared to those of global virus strains from GISAID. Pharyngeal specimens were confirmed to be SARS-CoV-2-positive by RT-PCR. Direct whole-genome sequencing was performed, and viral assemblies were subsequently uploaded to GISAID. Comparative genome sequence and single-nucleotide variation analyses were performed. RESULTS: The D614G mutation was identified in imported cases, which separated into two clusters related to viruses originally detected in the USA. Our findings highlight the risk of spreading SARS-CoV-2 variants through air travel and the need for continued genomic tracing for the epidemiological investigation and surveillance of SARS-CoV-2 using viral genomic data. CONCLUSIONS: Continuous genomic surveillance is warranted to trace virus circulation and evolution in different global settings during future outbreaks.

Clinical Comparison of Three Sample-to-Answer Systems for Detecting SARS-CoV-2 in B.1.1.7 Lineage Emergence.

PURPOSE: Accurate molecular diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, are needed for epidemiology studies and to support infection-control measures. We evaluated the analytical sensitivity and clinical performance of three sample-to-answer molecular-diagnostics systems for detecting SARS-CoV-2 using 325 nasopharyngeal swab clinical samples from symptomatic patients. METHODS: The BioFire Respiratory Panel 2.1 (RP2.1), cobas Liat SARS-CoV-2 and Influenza A/B, and Cepheid Xpert Xpress SARS-CoV-2/Flu/RSV platforms, which have been granted emergency-use authorization by the US FDA, were tested and compared. RESULTS: The positive percent agreement, negative percent agreement, and overall percent agreement among the three point of care testing systems were 98-100%, including for the wild-type SARS-CoV-2 (non-B.1.1.7) and a variant of concern (B.1.1.7). Notably, the BioFire RP2.1 may fail to detect the SARS-CoV-2 S gene in the B.1.1.7 lineage because of the spike protein mutation. CONCLUSION: All three point of care testing platforms provided highly sensitive, robust, and almost accurate results for rapidly detecting SARS-CoV-2. These automated molecular diagnostic assays can increase the effectiveness of control and prevention measures for infectious diseases.

A Resistance Mechanism in Non-mcr Colistin-Resistant Escherichia coli in Taiwan: R81H Substitution in PmrA Is an Independent Factor Contributing to Colistin Resistance.

Colistin resistance due to the mcr-type genes in Escherichia coli is well characterized. In order to study the resistance mechanism in mcr-negative colistin-resistant E. coli, strains were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. A total of 11 mcr-negative colistin-resistant isolates among 7,942 (0.1%) clinical E. coli isolates were identified between 2008 and 2018. Their prevalence was low and remained stable during the study period. Since 2012, ST131 and ST1193 clones with multiple drug-resistant phenotypes have emerged. All resistant strains displayed higher expression levels of the operons pmrHFIJKLM and pmrCAB than the control MG1655 strain. Although several amino acid substitutions were identified in PmrA or PmrB, only R81H in PmrA was associated with overexpression of pmrHFIJKLM and colistin resistance. The effect of substitution R81H in PmrA in colistin resistance was confirmed by complementation experiments. Although some strains harbored substitutions in PmrB, the identified mutations in pmrB did not contribute to colistin resistance. In conclusion, the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. IMPORTANCE The molecular epidemiology and resistance mechanisms of mcr-negative colistin-resistant E. coli are not well described. In this study, a total of 11 mcr-negative colistin-resistant E. coli isolates were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation. We determined the resistance mechanism of non-mcr colistin-resistant strains using gene knockout and complementation experiments. We observed the occurrence of the global multiple-drug-resistant E. coli clones ST131 and ST1193 starting in 2012. Moreover, for the first time, we proved that the amino acid substitution R81H in PmrA is an independent factor contributing to colistin resistance in non-mcr E. coli. The study results helped to gain an insight into the diversity and complexity of chromosome-encoded colistin resistance in E. coli.

Virulence among different types of hypervirulent Klebsiella pneumoniae with multi-locus sequence type (MLST)-11, Serotype K1 or K2 strains.

BACKGROUND: Two different types of hypervirulent K. pneumoniae (HvKp), the MLST-11 and serotype K1/K2 strains, have been frequently described in recent studies. Although these two types of strains were described to be HvKp, their virulence was not compared. In this study, in vitro and in vivo approaches were used to assess differences in virulence. MATERIALS AND METHODS: A total of twenty-nine isolates, including 6 strains of each of serotype K1 and K2 isolates and 17 strains of ST11 isolates, were selected for this study. Phenotypic tests of virulence were performed by the string test and analysis of the virulent associated genes was detected by PCR. In vitro models of serum resistance and phagocytosis were used as the parameters to assess the virulence. In-frame deletion of virulence-associated genes was performed to study their contributions to virulence. The median lethal dose, i.e., the LD50, in mice was determined following IP injection. RESULTS: Although serotype K1 and K2 strains and ST11 isolates had similar virulence gene profiles, the ST11 isolates showed less serum and phagocytic resistance than the serotype K1/K2 isolates. The mouse lethality test revealed that all ST11 isolates were unable to cause lethality, even at > 107 CFU, while serotypes K1 and K2 showed an LD50 at ≤ 103 CFU. Aerobactin or capsule knockout mutants exhibited a lower LD50 than the parental strain, while capsule mutants showed a more significant decrease in LD50. CONCLUSION: Since there was a significant difference in virulence levels between the two types of HvKp when assessed in in vitro and in vivo models, it may be better to use the designation "HvKp" for some strains based on animal studies to avoid confusion. Virulence and non-virulence could be analysed in a relative manner, especially in comparison studies.