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.

Determinants of quality of life in frontline healthcare providers caring for COVID-19 patients.

AIMS AND OBJECTIVES: To investigate the factors affecting quality of life in healthcare providers who care for patients with COVID-19. BACKGROUND: Healthcare providers caring for COVID-19 patients during the pandemic suffered a deterioration in their quality of life. Several studies have explored their psychological impact of working with COVID patients, but none have examined the causes of this deterioration. DESIGN: A cross-sectional study. METHODS: In the current study, the authors investigated the factors affecting quality of life in 293 healthcare providers recruited from a medical centre in northern Taiwan who had recently cared for patients with suspected or confirmed COVID-19 by analysing their responses to an online self-report questionnaire, using bivariate correlations and structural equation modelling. Reporting of this research adheres to the STROBE guideline. RESULTS: The study identified an important sequence of factors that mediated the effects of perceived success of epidemic prevention policies, family relations problems and education level on quality of life in a sample of healthcare workers caring for COVID-19 patients. The mediators were use of approach-oriented coping strategies and current mental health status. Specifically, use of approach-oriented coping strategies was found to directly cause improved quality of life and indirectly cause improved mental health, whereas use of avoidant coping strategies was found to directly cause worsening of mental health. Poor mental health predicted poor quality of life. CONCLUSIONS: Results suggest that implementation of sound epidemic prevention policies that promote adoption of approach-oriented coping behaviour should lead to a better quality of life in the future for healthcare providers working in challenging circumstances. RELEVANCE TO CLINICAL PRACTICE: Assessment of these policies as well as the providers' family relations are necessary first steps to improving the success of approach-oriented coping behaviour in this population, which in turn can improve their mental health and quality of life. PATIENT OR PUBLIC CONTRIBUTION: Neither patients nor members of the public were involved in the design or execution of the study.

Successful treatment of 28 patients with coronavirus disease 2019 at a medical center in Taiwan.

BACKGROUND: Coronavirus disease-2019 (COVID-19) is a worldwide pandemic. We present the clinical characteristics and outcomes of 28 COVID-19 patients treated in our hospital in Taiwan. METHODS: Patients with COVID-19, confirmed by positive real-time reverse-transcriptase polymerase chain reaction results for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral nucleic acids from oropharyngeal swab specimens between February 4, 2020 and July 6, 2020, were enrolled. Their clinical characteristics and outcomes were reviewed. RESULTS: Seventeen of the 28 patients (60.7%) had pneumonia. The most frequent symptoms were cough (n = 23, 82.1%) and fever (n = 17, 60.7%). The development of pneumonia was associated with age ≥40 years (p < 0.024), body mass index (BMI) ≥25 kg/m2 (p = 0.014), fever (p = 0.007), shortness of breath (p = 0.036), chills ((p = 0.047), and lower platelet counts (<200,000/µL) (p = 0.007). Increased quarantine duration was associated with age ≥40 years (p = 0.026), Charlson index ≥1 (p = 0.037), lower lymphocyte (<1500/uL; p = 0.028) or platelet counts (<200,000/µL) (p = 0.016), lower serum sodium (<140 mEq/L; p = 0.006), and higher C-reactive protein (CRP) level (≥1 mg/dl; p = 0.04). Treatment with hydroxychloroquine or in combination with other medicines did not reduce the quarantine duration. All 28 patients recovered with a median quarantine duration of 27.2 days. CONCLUSION: COVID-19 patients with older age, higher BMI, fever, chills or shortness of breath, lower serum sodium level, lower platelet or lymphocyte count, and higher CRP level may be associated with developing pneumonia or longer quarantine duration.

Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells.

Klebsiella pneumoniae raises significant concerns to the health care industry as these microbes are the source of widespread contamination of medical equipment, cause pneumonia as well as other multiorgan metastatic infections and have gained multidrug resistance. Despite soaring mortality rates, the host cell alterations occurring during these infections remain poorly understood. Here, we show that during in vitro and in vivo K. pneumoniae infections of lung epithelia, microtubules are severed and then eliminated. This destruction does not require direct association of K. pneumoniae with the host cells, as microtubules are disassembled in cells that are distant from the infecting bacteria. This microtubule dismantling is dependent on the K. pneumoniae (Kp) gene ytfL as non-pathogenic Escherichia coli expressing Kp ytfL disassemble microtubules in the absence of K. pneumoniae itself. Our data points to the host katanin catalytic subunit A like 1 protein (KATNAL1) and the katanin regulatory subunit B1 protein (KATNB1) as the gatekeepers to the microtubule severing event as both proteins localise specifically to microtubule cut sites. Infected cells that had either of these proteins knocked out maintained intact microtubules. Taken together, we have identified a novel mechanism that a bacterial pathogen has exploited to cause microtubule destruction within the host epithelia.

Roles of ramR and tet(A) Mutations in Conferring Tigecycline Resistance in Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates.

Tigecycline is regarded as a last-resort treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, but increasing numbers of tigecycline-resistant K. pneumoniae isolates have been reported. The tigecycline resistance mechanisms in CRKP are undetermined. This study aimed to elucidate the mechanisms underlying tigecycline resistance in 16 tigecycline- and carbapenem-resistant K. pneumoniae (TCRKP) isolates. Mutations in tigecycline resistance determinant genes [ramR, acrR, oqxR, tet(A), tet(L), tet(X), tet(M), rpsJ] were assessed by PCR amplicon sequencing, and mutations in ramR and tet(A) exhibited high prevalences individually (81%) and in combination (63%). Eight functional ramR mutation profiles reducing tigecycline sensitivity were verified by plasmid complementation of wild-type and mutant ramR Using a site-specific mutant, the most frequent RamR mutation, A19V (60%), had no significant effect on tigecycline susceptibility or the upregulation of ramA and acrA Two tet(A) variants with double frameshift mutations, type 1 and type 2, were identified; type 2 tet(A) is novel. A parent strain transformed with a plasmid carrying type 1 or type 2 tet(A) increased the tigecycline MIC by 8-fold or 4-fold, respectively. Synergistic effects were observed in strains harboring no ramR gene and a mutated tet(A), with an 8-fold increase in the tigecycline MIC compared with that in strains harboring only mutated tet(A) being seen. Overall, mutations in the ramR and tet(A) efflux genes constituted the major tigecycline resistance mechanisms among the studied TCRKP isolates. The identification of strains exhibiting the combination of a ramR deficiency and widespread mutated tet(A) is concerning due to the possible dissemination of increased tigecycline resistance in K. pneumoniae.

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.

Klebsiella pneumoniae in gastrointestinal tract and pyogenic liver abscess.

To determine the role of gastrointestinal carriage in Klebsiella pneumoniae liver abscess, we studied 43 patients. Bacterial isolates from liver and fecal samples from 10 patients with this condition and 7 healthy carriers showed identical serotypes and genotypes with the same virulence. This finding indicated that gastrointestinal carriage is a predisposing factor for liver abscess.

Seroepidemiology of Klebsiella pneumoniae colonizing the intestinal tract of healthy Chinese and overseas Chinese adults in Asian countries.

BACKGROUND: Capsular serotypes K1 and K2 of Klebsiella pneumoniae are thought to the major virulence determinants responsible for liver abscess. The intestine is one of the major reservoirs of K. pneumoniae, and epidemiological studies have suggested that the majority of K. pneumoniae infections are preceded by colonization of the gastrointestinal tract. The possibility of fecal-oral transmission in liver abscess has been raised on the basis of molecular typing of isolates. Data on the serotype distribution of K. pneumoniae in stool samples from healthy individuals has not been previously reported. This study investigated the seroepidemiology of K. pneumoniae isolates from the intestinal tract of healthy Chinese in Asian countries. Stool specimens from healthy adult Chinese residents of Taiwan, Japan, Hong Kong, China, Thailand, Malaysia, Singapore, and Vietnam were collected from August 2004 to August 2010 for analysis. RESULTS: Serotypes K1/K2 accounted for 9.8% of all K. pneumoniae isolates from stools in all countries. There was no significant difference in the prevalence of K1/K2 isolates among the countries excluding Thailand and Vietnam. The antimicrobial susceptibility pattern was nearly the same in K. pneumoniae isolates. The result of pulsed-field gel electrophoresis revealed no major clonal cluster of serotype K1 isolates. CONCLUSIONS: The result showed that Chinese ethnicity itself might be a major factor predisposing to intestinal colonization by serotype K1/K2 K. pneumoniae isolates. The prevalent serotype K1/K2 isolates may partially correspond to the prevalence of K. pneumoniae liver abscess in Asian countries.

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.

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.

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.

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.

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 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.

Immune response and pathophysiological features of Klebsiella pneumoniae liver abscesses in an animal model.

Capsular serotypes K1 and K2, the rmpA gene (a regulator of the mucoid phenotype) and aerobactin from Klebsiella pneumoniae have been identified as the major virulence factors for pyogenic liver abscesses with high morbidity, mortality and severe complications. The pathological mechanisms remain unclear. In this study, we compared liver immune responses and pathological changes in response to different serotypes of K. pneumoniae infections. A mouse model was used to investigate cytokine and chemokine production, histopathology findings, phagocytic uptake and mortality induced by serotypes K1 (magA(+), rmpA(+), aerobactin(+)), K2 (magA(-), rmpA(+), aerobactin(+)), K62 (magA(-), rmpA(-), aerobactin(-)) and an acapsulated isogenic K1 mutant (ΔK1, magA(+), rmpA(+), aerobactin(+)). K. pneumoniae serotypes K1 and K2 showed lower 50% lethal dose values and more phagocytic resistance to neutrophils than K62 and the ΔK1 mutant. In sequential liver samples, viable bacteria counts increased 3 h to 3 days after low-dose inoculation (<10(1) colony-forming unit (cfu)) with K1 and K2, while K62 and ΔK1 cleared rapidly and became undetectable even with high-dose inoculation (∼2.9 × 10(5) cfu). Time-dependent increases in cytokines and chemokines, including tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, IL-10, keratinocyte-derived chemokines and macrophage inflammatory protein-2, were observed in the serum and liver tissue of K1- and K2-infected mice, and severe disease progression manifesting as microabscesses was also identified. K62 and ΔK1 inoculation did not result in similar immune responses and histological changes. These findings illustrate the critical role of phagocytic resistance against innate immunological defense mechanisms as well as its contribution to the development of liver abscesses.

Molecular typing and virulence analysis of serotype K1 Klebsiella pneumoniae strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan.

Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of <10(2) CFU were from ST23, were resistant to phagocytosis and serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.

Revisiting the importance of virulence determinant magA and its surrounding genes in Klebsiella pneumoniae causing pyogenic liver abscesses: exact role in serotype K1 capsule formation.

BACKGROUND: Mucoviscosity-associated gene A (magA) is proposed to play a decisive role in the pathogenesis of liver abscesses due to Klebsiella pneumoniae. Although some investigators consider MagA to be a putative O-antigen ligase, it is also reportedly associated with the K1 antigen. METHODS: Using magA-positive serotype K1 K. pneumoniae STL43 isolated from a patient with liver abscess, we constructed 3 bacterial mutants by targeting genes within the same transcription unit, including magA, wcaG, and rfbP. The virulence of these mutants was determined by neutrophil phagocytosis and inoculation of mice. Transmission electron microscopy and Western blot analysis were used to define their surface polysaccharides. RESULTS: STL43 was resistant, and all 3 mutants were highly susceptible, to phagocytosis. None of the mutant strains caused death in mice at the lethal dose of STL43. In contrast to previous reports, transmission electron microscopy revealed that all 3 mutants were nonencapsulated. Analysis of surface polysaccharides revealed that all 3 mutants retained their O antigen but lost their K antigen/capsule. Furthermore, amino acid analysis showed that MagA shared a conserved domain of Wzy, the serotype-specific capsular polysaccharide polymerase. CONCLUSIONS: In accordance with the bacterial polysaccharide gene nomenclature (BPGN) scheme, MagA should be renamed Wzy(KpK1), the capsular polymerase specific to K. pneumoniae serotype K1.