The Evolutionary Trend and Genomic Features of an Emerging Lineage of Elizabethkingia anophelis Strains in Taiwan.

The incidence of Elizabethkingia anophelis bacteremia increased significantly in a tertiary hospital, Changhua Christian Hospital (CCH) since 2013. The infection density was 1.3 and 8.1 cases per 100,000 patient-days between 2005 and 2012 and 2013 and 2020, respectively (P < 0.05). During an outbreak investigation, a specific lineage of E. anophelis strains was identified by the pulsed-field gel electrophoresis analysis. To evaluate the evolution of the specific E. anophelis lineage, whole-genome sequencing was performed, and unique genomic features (GRs) were determined by comparative genomic analysis. The specific E. anophelis lineage was novel compared to worldwide strains ever reported by cg-MLST phylogenic and whole-genome comparative analysis. Multiplex PCR using primers designed from unique GRs were performed for prevalence screening among isolates from the CCH and nationwide isolates from the Taiwan surveillance of Antimicrobial Resistance (TSAR) Program. The proportion of the specific E. anophelis lineage increased from 7.9% (3/38) during 2005-2012 to 89.2% (223/250) during 2013-2020 (P < 0.05). Although E. anophelis usually confers resistance to multiple antibiotics with limited therapeutic options, the E. anophelis strains in the specific lineage had higher ciprofloxacin resistance (100% [226/226] versus 27.4% [17/62], P < 0.05) and was associated with a higher 14-day mortality rates (33.2% [37/226] versus 16.1% [10/62], P < 0.05) than other strains at CCH. A similarly increasing trend was also found in the national TSAR program during 2002-2018 (p for trend <0.05). We concluded that a novel lineage of E. anophelis strains has emerged dominantly in Taiwan. The genomic features are important for further investigations of epidemiology, resistance, virulence, and appropriate treatment. IMPORTANCE Elizabethkingia anophelis is an emerging multidrug resistant pathogen caused several global outbreaks recently. E. anophelis was frequently misidentified as E. meningoseptica in the past by conventional culture methods; therefore, the prevalence was often underestimated. Through revised identification, an increasing trend of E. anophelis infection was noted in a tertiary hospital and a dominant lineage of strains was recognized by genotyping. To our best knowledge, the dominant lineage of E. anophelis is novel in comparison to other worldwide strains by whole-genome comparative analysis and several unique genomic regions were found. The whole-genome sequencing data also demonstrated multiple putative virulence factors and genes associated with multidrug resistance. In our study, we identified a specially evolved E. anophelis in Taiwan with increasing nationwide dominance. This study will assist in further epidemiology surveillance and developing corresponsive infection control policies to restrain it potential of global dissemination.

Differentiation of qacA and qacB using high-resolution melt curve analysis, and both qacA and qacB but not qacC or norA types increase chlorhexidine minimal inhibitory concentrations in Staphylococcus aureus isolates.

BACKGROUND: Chlorhexidine is one of the most essential ingredients in infection control applications. Except qacA, the effects of other various efflux-medicated biocide genes (including qacB, qacC, qacEΔ1, qacH or norA) on biguanides resistance are still controversial. In addition, most of the studies have discussed the effect of qacA/B on clinical S. aureus isolates but not that qacA or qacB individually. METHODS: In total, 254 methicillin-resistant Staphylococcus aureus (MRSA), selected 30 methicillin-susceptible S. aureus (MSSA) clinical isolates from different patients during 2014-2015 and 15 S. aureus quality control strains (including Mu3 and Mu50) were included in the study. Various biocide genes, including qacA/B, qacC, qacH, qacEΔ1, and different types of norA, were determined through conventional PCR. S. aureus isolates with qacA/B (+) were analyzed using high-resolution melting curve (HRM) to differentiate qacA from qacB. The chlorhexidine MIC was determined using the agar dilution method. Univariate and multivariate statistics were analyzed to see which biocide resistant genes had effects on chlorhexidine MIC. RESULTS: Results of all HRM analyses (n = 22) were consistent with those of Sanger sequencing for differentiation of qacA from qacB. None of the isolates harbored qacH and only one MRSA harbored qacEΔ1. The harboring rate of qacA, qacB, and qacC among MRSA/MSSA isolates was 7.1% (n = 18)/0%, 38.2% (n = 97)/0%, and 7.5% (n = 19)/3.3% (n = 1), respectively. The most type of norA was norAI (n = 158), followed by norAIII (n = 87) and norAII (n = 9) among MRSA isolates. Based on the results of multivariate logistic regression analyses, only qacA and qacB would increase chlorhexidine MIC from ≤ 1 ug/ml to ≥ 2 ug/ml in MRSA isolates (P < 0.001) but not qacC or norA types (P=0.976 and 0.633 or 0.933, respectively). In addition, only qacA but not qacB was contributed to elevate chlorhexidine from ≤ 1 ug/ml to 4 ug/ml in MRSA isolates (P < 0.001 and 0.017, respectively). CONCLUSION: HRM analysis can be a great method to differentiate qacA from qacB. The biocide gene with the most effect on chlorhexidine MIC in S. aureus isolates was qacA, followed by qacB, but qacC and different types of norA did not have any effect on chlorhexidine susceptibility. Further investigation on the influence of qacB, qacC and types of norA on chlorhexidine susceptibility is necessary.

Copy number alternations of the 17q23-rs6504950 locus are associated with advanced breast cancers in Taiwanese women.

Objective: Breast cancer is one of the most common malignancies and a leading cause of cancer-related death in women worldwide. Both hormone-related factors and genetic aberrations could cause breast cancer. We investigated copy number alternations (CNAs) on four breast cancer-susceptible loci, namely 2q35-rs13387042, 3p24-rs4973768, 17q23-rs6504950, and fibroblast growth factor receptor 2 (FGFR2)-rs2981578, in Taiwanese women. Patients and Methods: Breast cancer tissues and blood samples from 66 patients and their clinical data were collected from a human biobank. The copy numbers of the germline samples (from blood) and cancer tissues from each patient on the susceptible loci - 2q35, 3p24, 17q23, and FGFR2 - were obtained using TaqMan probes in the Applied Biosystems Inc., (ABI) StepOnePlus Real-Time Polymerase Chain Reaction instrument and CopyCaller® Software v1.0 (ABI, CA, USA). Results: The mean copy numbers output by CopyCaller® Software v1.0 of the cancer tissues on these susceptible loci (2q35, 3p24, 17q23, and FGFR2) from the 66 patients were higher than those of the blood samples (2.0 vs. 1.9); however, significantly higher copy numbers for cancer tissues compared with germline samples were discovered only on 2q35-rs13387042 (P = 0.035). In addition, patients with advanced breast cancers had relatively many CNAs between their cancer tissues and germline samples on 17q23-rs6504950 (P = 0.008). Multivariate analysis revealed that the risk factor for patients with advanced breast cancers was CNAs between cancer tissues and germline samples on 17q23-rs6504950 (odds ratio = 13.337, 95% confidence interval: 1.525-122.468). Conclusions: CNAs on 17q23-rs6504950 between cancer tissues and germline samples could affect cancer progression in Taiwanese women with breast cancer. Further investigations regarding the role of CNAs on 17q23-rs6504950 in cancer progression are necessary to elucidate the pathogenesis of breast cancer.

Long-Term Surveillance of Antibiotic Prescriptions and the Prevalence of Antimicrobial Resistance in Non-Fermenting Gram-Negative Bacilli.

The increasing emergence of multidrug-resistant (MDR) bacteria has been recognized as a public health threat worldwide. Hospitalized patients and outpatients are commonly infected by non-fermenting Gram-negative bacilli (NFGNB), particularly the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ACB) and Pseudomonas aeruginosa. Antimicrobial agents are critical for treating the nosocomial infections caused by NFGNB. The aim of this study was to assess antimicrobial resistance and the use of antimicrobial agents. The bacterial isolates of 638,152 specimens from both inpatients and outpatients, retrieved from 2001 to 2008 at a medical center in central Taiwan, were examined for their susceptibility to various antimicrobial agents, including cefepime, imipenem, ciprofloxacin, gentamicin, amikacin, meropenem, and levofloxacin. Administrated prescriptions of the monitored antibiotics were analyzed using the Taiwan National Health Insurance Research Database (NHIRD). Our results show that the defined daily doses (DDDs) for cefepime, imipenem, and ciprofloxacin increased with time, and a trend toward reduced antimicrobial sensitivities of both ACB and P. aeruginosa was noticeable. In conclusion, the antimicrobial sensitivities of ACB and P. aeruginosa were reduced with the increased use of antibiotics. Continuous surveillance of antibiotic prescriptions and the prevalence of emerging resistance in nosocomial infections is warranted.

Identification of a proteomic biomarker associated with invasive ST1, serotype VI Group B Streptococcus by MALDI-TOF MS.

BACKGROUND: Group B Streptococcus (GBS) is an important invasive pathogen in neonates, pregnant women and the elderly. Serotype VI GBS, which has been rarely reported globally, has emerged as a significant pathogen in Asia. However, traditional serologic latex agglutination (LA) methods may fail to type isolates that lack of or low expression of CPS. METHODS: A total of 104 GBS strains were analyzed by MALDI-TOF MS. Multiplex PCR and multilocus sequence typing (MLST) were also performed to confirm their strains. The protein markers were purified with gel electrophoresis and LC-column, followed by identification with nanoLC-MS/MS analysis. RESULTS: Protein peak of 6251-Da was appeared in most (20/24, 92%) serotypes VI (94% ST-1 or single locus variant of ST-1), and protein peak of 6891-Da was appeared in most serotypes III (15/18, 83%) and Ib (19/23, 83%) strains. The protein peak of 6251-Da and 6891-Da were identified as CsbD family protein and UPF0337 protein gbs0600, respectively. CONCLUSIONS: The protein peak of 6251 Da may play a role of emergence of ST-1 clone, serotype VI GBS in central Taiwan and could be useful in rapid identifying invasive serotype VI from III isolates, which is hardly achieved by LA.

Methicillin-resistant Staphylococcus aureus with reduced vancomycin susceptibility in Taiwan.

Staphylococcus aureus is a versatile pathogen which can cause various mild to life-threatening infectious diseases. The evolution of S. aureus resistance is notorious, from penicillin and oxacillin to vancomycin. Vancomycin, introduced in 1956, was once considered a most reliable antibiotic for methicillin-resistant S. aureus (MRSA); unfortunately, the first strain of S. aureus with decreased susceptibility to vancomycin emerged in 1996. Vancomycin has been approved in Taiwan since 1983, and the prevalence rates of heteroresistant vancomycin-intermediate S. aureus (hVISA) and vancomycin-intermediate S. aureus (VISA) in 2003 were 0.7% and 0.2%, respectively. However, a ten-fold increase of hVISA and VISA to 10% and 2.7%, respectively, in 2012-2013 could indicate a challenging clinical situation in Taiwan. The most commonly reported staphylococcal cassette chromosome mec (SCCmec) types of hVISA and VISA are usually SCCmec type III or II, typical nosocomial MRSA strains. Preventing the spread of resistant pathogens through infection control interventions and judicious antibiotic stewardship is a serious medical issue.

Clinical characteristics, treatments, and outcomes of hematogenous pyogenic vertebral osteomyelitis, 12-year experience from a tertiary hospital in central Taiwan.

BACKGROUND: In Taiwan, studies about hematogenous pyogenic vertebral osteomyelitis (HPVO) are limited. We conducted a retrospective study to evaluate the clinical presentations, treatment, and outcomes of patients with the diagnosis of HPVO. METHOD: This 12.5-year retrospective study included patients with a diagnosis of HPVO. Medical records of all HPVO patients were thoroughly reviewed and their clinical data were analyzed by the SPSS software. RESULT: 414 HPVO cases were included and the mean age was 61.6 ± 13.4 years. The mean duration of symptoms was 29 ± 35.3 days and pain over the affected site was reported by most patients (86.0%). Gram-positive bacteria, especially Staphylococcus aureus (162/399 = 40.6%), were the main HPVO pathogens. Escherichia coli (42/399 = 10.5%) was the most common gram-negative isolate. Surgery was performed in 68.8% of cases and the mean duration of total antibiotic treatment was 104.7 ± 77.7 days. All-cause mortality and recurrence rates were 6.3% and 18.8%, respectively. In multivariate analysis, polymicrobial infection (OR: 4.154, 95% CI: 1.039-16.604, p = 0.044), multiple vertebral body involvement (OR: 2.202, 95% CI: 1.088-4.457, p = 0.028), abscess formation treated with antibiotics alone (OR: 2.912, 95% CI: 1.064-7.966, p = 0.037), and the duration of antimicrobial treatment less than 4 weeks (OR: 3.737, 95% CI: 1.195-11.683, p = 0.023) were associated with HPVO recurrence. CONCLUSION: In Taiwan, HPVO mainly affected the elderly and S. aureus remained the most common HPVO pathogen. In patients with risk factors associated with HPVO recurrence, a longer duration (≥6 weeks) of antimicrobial therapy is suggested.

Epidemiology, clinical features, and microbiology of patients with diarrhea in community clinics in Taiwan.

OBJECTIVE: To investigate the clinical features and microbiology of patients with diarrheal diseases in Taiwan. METHODS: From March 2014 to October 2014, patients with diarrheal diseases referred from the community clinics were enrolled into our prospective study. Demographics and clinical features of the participants were acquired. Stool samples were examined by the Luminex Gastrointestinal Pathogen Panel assay. Data were analyzed by SAS version 9.4. RESULTS: A total of 545 patients were enrolled into this study. Male and adults accounted for 52.3% and 82.6% of patients, respectively. The median age was 36 years. Enteropathogen(s) was identified in 43.3% of patients and 8.5% of them had more than one agent in their stool samples. Viruses, especially norovirus GI/GII, were the predominant agents of gastroenteritis. Moreover, Campylobacter species was the most common bacterial agent. Bloody stool was frequently reported in patients with bacterial diarrhea (P = 0.002); contrarily, watery stool was significantly associated with viral diarrhea (P < 0.0001). Regional variation and seasonality of microbiological distribution were also observed. CONCLUSION: In Taiwan, viruses were the predominant pathogens among patients with diarrheal diseases who visited community clinics. The therapeutic strategies for diarrheal patients should be based on the epidemiological and clinical characteristics.

Clinical features, antifungal susceptibility, and outcome of Candida guilliermondii fungemia: An experience in a tertiary hospital in mid-Taiwan.

BACKGROUNDS: Candida guilliermondii is rarely isolated from clinical specimen. C. guilliermondii fungemia is seldom reported in the literature. The aims of this study were to report the clinical features, antifungal susceptibility, and outcomes of patients with C. guilliermondii fungemia. METHODS: From 2003 to 2015, we retrospectively analyzed the clinical and laboratory data of patients with C. guilliermondii fungemia in a tertiary hospital in mid-Taiwan. We performed a multivariable logistic regression analysis to identify the risk factors of mortality. The Sensititre YeastOne microtiter panel assessed the susceptibility of antifungal agents. RESULTS: In this study, we identified 36 patients with C. guilliermondii fungemia. The median age of patients was 50.5 years (range, 17 days to 96 year) and 20 cases (56%) were male. The incidence of C. guilliermondii fungemia was 0.05 per 1000 admissions. Malignancy was the most common co-morbidity, and 25 (69%) patients had central venous catheter in place. Thirty-day overall mortality was 16.7%. In multivariate logistical regression analysis, catheter retention was an independent risk factor of mortality. According to epidemiological cutoff values, most clinical isolates (21/22, 95.5%) belonged to the wild-type MIC distributions for amphotericin B and flucytosine; however, the isolates were less susceptible to fluconazole (68%) and echinocandins (77-91%). CONCLUSION: Despite the lower mortality rate associated with C. guilliermondii fungemia, the removal of a central venous catheter remained an independent factor influencing the outcome of patients. The clinical significance of less susceptibility of C. guilliermondii to triazoles and echinocandins remains to be elucidated.

Characterization of two novel variants of staphylococcal cassette chromosome mec elements in oxacillin-resistant Staphylococcus lugdunensis.

OBJECTIVES: Staphylococcus lugdunensis, a species of CoNS, has become an important hospital pathogen because of increasing resistance to ß-lactam antibiotics such as methicillin and oxacillin. Methicillin resistance is mainly due to the acquisition of the staphylococcal cassette chromosome (SCC) mec (SCCmec). Little is known about the structure of SCCmec in methicillin- or oxacillin-resistant CoNS. METHODS: WGS was performed to determine the structure of SCCmec elements of two clinical S. lugdunensis isolates: CMUH-22 and CMUH-25. RESULTS: These elements were found to be flanked by DRs and IRs with unique mosaic structures and a common integration site in the 3' end of the rlmH gene. The sequences of the regions located between rlmH and the ISSau4-like transposase genes of both elements were similar to those of SCCmec Vt of Staphylococcus aureus PM1. The SCCmec (type V, 5C2&4) of CMUH-25 harboured a novel ccrC complex and a C2-like mec complex in opposite orientations, similar to the type V SCCmec of S. aureus WIS. The sequences of the ccrA4B4 genes and J1 and J2 regions of CMUH-25 were similar to those of the SCC element of Staphylococcus haemolyticus NCTC 11042. In contrast, portions of the sequence of the J1 region of type Vt (5C2) SCCmec in strain CMUH-22 were highly similar to portions of those of Staphylococcus epidermidis RP62A and the composite SCCmec type V of S. aureus WAMRSA40. CONCLUSIONS: These observations suggest that the SCCmec elements of CMUH-25 and CMUH-22 evolved separately and assembled through different recombination events.

Evaluation of double locus (clfB and spa) sequence typing for studying molecular epidemiology of methicillin-resistant Staphylococcus aureus in Taiwan.

BACKGROUND: Pulsed-field gel electrophoresis (PFGE) is the "gold standard" for epidemiological investigation of methicillin-resistant Staphylococcus aureus (MRSA), but several DNA sequence-based methods have been developed in MRSA typing because of the unambiguous results. METHODS: Ninety-one MRSA isolates were collected from the blood cultures of different patients from July 2008 to December 2008 in central Taiwan. The molecular characteristics of each isolate, including double locus sequence typing (DLST; spa and clfB typing), Staphylococcus cassette chromosome mec (SCCmec), and PFGE were determined for comparison. RESULTS: Five major clfB types (types A-E), 18 spa types, 33 DLST genotypes, five SCCmec types, 17 pulsotypes have been observed. Three major DLST genotypes (A1-t002, C0-t037, and B1-t437) and two major pulsotypes (6 and 8) were identified. Most clfB type A isolates (97.1%) were SCCmec type II and all clfB type C isolates (100%) were SCCmec type III. Most clfB type B isolates (88.9%) were SCCmec type IV (59.3%) and VT (29.6%). All (100%) clfB subtypes A1, A2, and C isolates and 70.4% of clfB type B isolates belonged to healthcare-associated-MRSA. The average congruence was 57.7% between DLST and PFGE, and 96.6% between clfB and SCCmec type. The index of discrimination of SCCmec, clfB, spa, PFGE, and DLST was 0.72, 0.79, 0.80, 0.81, and 0.87, respectively. CONCLUSION: ClfB type has high congruence with SCCmec type. The DLST method in this study yielded a higher discriminatory power than PFGE in local investigation of molecular epidemiology of MRSA and a promising alternative to PFGE.

Methicillin-resistant Staphylococcus aureus isolates with SCCmec type V and spa types t437 or t1081 associated to discordant susceptibility results between oxacillin and cefoxitin, Central Taiwan.

Staphylococcus aureus isolates with discordant susceptibility results between oxacillin and cefoxitin obtained using automated microbiology systems are infrequently observed. From April 2013 to December 2014, 1956 methicillin-resistant S. aureus (MRSA) and 1761 methicillin-susceptible S. aureus isolates were obtained from different patients. Forty isolates (1.1% and 2% in case of S. aureus and MRSA, respectively) with discordant susceptibility results (oxacillin susceptible and cefoxitin resistant) and carrying mecA gene were obtained. Except 2 SCCmec type IV isolates, 38 MRSA isolates were all SCCmec type V (VT or non-VT), which were further divided into VT (n=22) and non-VT (n=16). The most common spa type in VT and non-VT isolates were t437 (n=19) and t1081 (n=13), respectively. Only 55% of patients received effective antimicrobial agents; 2 mortalities were not attributable to MRSA infection. Using standard agar dilution, 17 MRSA isolates (0.46% and 0.87% in case of S. aureus and MRSA, respectively) had oxacillin MIC in the susceptible ranges (oxacillin-susceptible MRSA [OS-MRSA]); all carried SCCmec type V (VT, n=8; non-VT, n=9). The most common spa-MLST types of OS-MRSA in VT and non-VT were t437-ST59 (n=4) and t1081-ST45 (n=7), respectively. Concomitant testing by both cefoxitin- and oxacillin-based methods is a practical strategy for OS-MRSA detection in the clinical laboratories. Continuous monitoring of OS-MRSA isolates is necessary to elucidate their impact in clinical infectious diseases.

Clinical manifestations, course, and outcome of patients with neutralizing anti-interferon-γ autoantibodies and disseminated nontuberculous mycobacterial infections.

Neutralizing anti-interferon-γ autoantibody (nAIGA)-associated immunodeficiency is an emerging medical issue worldwide. In the present study, we describe and discuss the clinical features and outcomes of patients with nAIGAs and disseminated infections by nontuberculous mycobacteria (dNTM).We thoroughly reviewed the medical records of all patients. Microorganisms and nAIGAs were identified using previously described methods with modifications. All data were calculated and analyzed using SPSS software.Among 46 adult patients with dNTM infections, we identified 45 cases (97.8%) with nAIGAs. The average patient age was 58.6 years, and there was no sex predominance. Cervical lymphadenitis (81.8%) was the most common clinical manifestation. Endocrine disorder was the leading comorbidity (7 cases). Malignancies were found in 4 patients, and all of the malignancies originated from the T-cell/macrophage lineage. More than half of the identifiable isolates were slow-growing NTMs. Twenty-eight (62.2%) and 18 (40.0%) patients had a history of zoster and salmonellosis, respectively. A high proportion of patients with recurrent episodes of NTM infection or a history of zoster and dNTM infection had initial nAIGA titers ≥10 dilution (P < 0.05). Twenty-seven patients (60.0%) required long-term antimycobacterial therapy and had at least 1 episode of recurrent NTM disease. No mortality was related to dNTM infection.In Taiwan, nAIGAs are a recently recognized mechanism of dNTM infection. Long term of antibiotic treatment and adherence to medical advice are necessary to improve the clinical outcome of patients with nAIGAs.

Validating the Sensitivity of High-Resolution Melting Analysis for JAK2 V617F Mutation in the Clinical Setting.

BACKGROUND: Janus kinase 2 (JAK2) plays an important role in normal hematopoietic growth factor signaling. The detection of the JAK2 V617F mutation (c.1849GNT, GTC → TTC) is crucial for the diagnosis of myeloproliferative neoplasm (MPN) and has become the essential criteria for diagnosis of MPN by the WHO. High-resolution melt (HRM) curve analysis is a nongel-based, closed-tube method, in which PCR amplification and subsequent analysis are sequentially performed in the well, making it more convenient than other scanning methodologies. METHODS: We evaluated JAK2 V617F mutation by HRM. Twenty-nine patients diagnosed with MPN were examined. We studied the analytical sensitivity of the HRM analysis using real-time polymerase chain reaction (PCR) for identifying the JAK2 V617F mutation. Additionally, the sensitivity of HRM analysis and allele-specific PCR (AS-PCR) assay was compared. RESULTS: The JAK2 V617F mutation was successfully discriminated at an abundance of 6% or above in HRM analysis. Both HRM analysis and AS-PCR showed 100% accuracy with detection limits of 6% and 2.5%, respectively. CONCLUSION: HRM analysis is a fast, simple, reliable, and nonexpensive method for the detection of the JAK2 V617F mutation. However, more validation of the detection limits of HRM analysis should be performed before declaration of the analytic sensitivity of the method.

Analysing the mutational status of adenomatous polyposis coli (APC) gene in breast cancer.

BACKGROUND: Breast cancer is a heterogeneous disorder for which the underlying genetic basis remains unclear. We developed a method for identifying adenomatous polyposis coli (APC) mutations and we evaluated the possible association between APC genetic variants and breast cancer susceptibility. METHODS: Genomic DNA was extracted from tumor and matched peripheral blood samples collected from 89 breast cancer patients and from peripheral blood samples collected from 50 controls. All samples were tested for mutations in exons 1-14 and the mutation cluster region of exon 15 by HRM analysis. All mutations were confirmed by direct DNA sequencing. RESULTS: We identified a new single nucleotide polymorphism (SNP), c.465A>G (K155K), in exon 4 and seven known SNPs: c.573T>C (Y191Y) in exon 5, c.1005A>G (L335L) in exon 9, c.1458T>C (Y486Y) and c.1488A>T (T496T) in exon 11, c.1635G>A (A545A) in exon 13, and c.4479G>A (T1493T) and c.5465T>A (V1822D) in exon 15. The following alterations were found in 2, 1, 2, and 1 patients, respectively: c.465A>G, c.573T>C, c.1005A>G, and c.1488A>T. There was no observed association between breast cancer risk and any of these APC SNPs. CONCLUSIONS: APC mutations occur at a low frequency in Taiwanese breast cancer cases. HRM analysis is a powerful method for the detection of APC mutations in breast.

Mutation analysis of 13 driver genes of colorectal cancer-related pathways in Taiwanese patients.

AIM: To investigate the driver gene mutations associated with colorectal cancer (CRC) in the Taiwanese population. METHODS: In this study, 103 patients with CRC were evaluated. The samples consisted of 66 men and 37 women with a median age of 59 years and an age range of 26-86 years. We used high-resolution melting analysis (HRM) and direct DNA sequencing to characterize the mutations in 13 driver genes of CRC-related pathways. The HRM assays were conducted using the LightCycler® 480 Instrument provided with the software LightCycler® 480 Gene Scanning Software Version 1.5. We also compared the clinicopathological data of CRC patients with the driver gene mutation status. RESULTS: Of the 103 patients evaluated, 73.79% had mutations in one of the 13 driver genes. We discovered 18 novel mutations in APC, MLH1, MSH2, PMS2, SMAD4 and TP53 that have not been previously reported. Additionally, we found 16 de novo mutations in APC, BMPR1A, MLH1, MSH2, MSH6, MUTYH and PMS2 in cancerous tissues previously reported in the dbSNP database; however, these mutations could not be detected in peripheral blood cells. The APC mutation correlates with lymph node metastasis (34.69% vs 12.96%, P = 0.009) and cancer stage (34.78% vs 14.04%, P = 0.013). No association was observed between other driver gene mutations and clinicopathological features. Furthermore, having two or more driver gene mutations correlates with the degree of lymph node metastasis (42.86% vs 24.07%, P = 0.043). CONCLUSION: Our findings confirm the importance of 13 CRC-related pathway driver genes in the development of CRC in Taiwanese patients.

Concomitant genotyping revealed diverse spreading between methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus in central Taiwan.

BACKGROUND: Staphylococcus aureus is a versatile bacterium, which can lead to various infectious diseases. Various molecular typing methods are applied to the evolution and epidemiology surveys of S. aureus, mostly for methicillin-resistant S. aureus (MRSA). However, methicillin-susceptible S. aureus (MSSA) is still an important pathogen, but their molecular typing is evaluated infrequently. METHODS: Pulsed-field gel electrophoresis (PFGE), spa typing, and detection of five virulent genes for 95 MRSA and 56 MSSA isolates (July-December 2008 and July 2008-December 2009, respectively) during an overlapping period were performed. RESULTS: More diversity was found in MSSA isolates (23 pulsotypes and 25 spa types, excluding 4 new-type and 1 nontypable isolates for spa typing) than in MRSA isolates (19 pulsotypes and 16 spa types, excluding 1 new-type and 1 nontypable isolates for spa typing). By spa typing, t002 (n = 30), t037 (n = 23), t437 (n = 21), t234 (n = 3), t1081 (n = 3), and t1094 (n = 3) were the six major MRSA clones. For MSSA isolates, t189 (n = 13), t437 (n = 4), t084 (n = 3), t213 (n = 3), t701 (n = 3), and t7200 (n = 3) were the six major types. Combining PFGE and spa typing, there were five combinations (pulsotype + spa type) that contained both MRSA and MSSA isolates (pulsotype 9-t437, pulsotype 15-t037, pulsotype 19-t002, pulsotype 21-t002, and pulsotype 28-t1081). For all 151 S. aureus or 95 MRSA isolates, the PFGE typing had more discrimination power, but spa typing had larger discrimination index for 56 MSSA isolates. CONCLUSION: In conclusion, there were different predominant MRSA and MSSA clones clinically. Continuing longitudinal tracking of molecular typing is necessary for elucidating the evolution of this important clinical pathogen.

Clonal dissemination of invasive and colonizing clonal complex 1 of serotype VI group B Streptococcus in central Taiwan.

BACKGROUND/PURPOSE: The aim of this study was to investigate clinical presentation, serotype distribution and genetic correlation of group B streptococcus (GBS) diseases. Since serotype VI prevalence far exceeded that reported in prior studies, genetic relationship of isolates was further analyzed. METHODS: GBS isolates obtaining from patients with invasive diseases and pregnant women with colonization between June 2007 and December 2010 were analyzed. All isolates were tested for serotypes by multiplex PCR assay and pulsed-field gel electrophoresis (PFGE). Serotype VI isolates were further analyzed by multilocus sequence typing (MLST). RESULTS: A total of 134 GBS isolates were recovered from blood of 126 patients with invasive disease (94.0%) and anogenital swabs of 8 pregnant women (6.0%). Most common serotype was Ib (21.6%), followed by V (20.1%), VI (18.7%), III (15.7%), II (11.9 %), Ia (11.2%), and IX (0.7%). Serotype VI was also the leading type in infants with early onset disease (EOD; 3/8, 37.5%) and colonizing pregnant women (3/8, 37.5%). PFGE distinguished 33 pulsotypes, reflecting genetic diversity among GBS isolates. Among 25 serotype VI isolates tested, 14 were ST-1, seven were ST-679, three were ST-678, one was ST-681, and distributed into four PFGE pulsotypes. ST-678, ST-679, and ST-681 were novel sequence types; ST-678 and ST-679 are single-locus variants of ST-1 that belongs to clonal complex (CC) 1. CONCLUSION: CC1 dissemination of serotype VI GBS thus emerges as an important invasive pathogen in infants and nonpregnant adults in central Taiwan. Serotype prevalence of GBS must be continuously monitored geographically to guide prevention strategy of GBS vaccines.

High-resolution Melting Analysis for Gene Scanning of Adenomatous Polyposis Coli (APC) Gene With Oral Squamous Cell Carcinoma Samples.

BACKGROUND: There have been many different mutations reported for the large adenomatous polyposis coli (APC) tumor suppressor gene. APC mutations result in inactivation of APC tumor suppressor action, allowing the progression of tumorigenesis. The present study utilized a highly efficient method to identify APC mutations and investigated the association between the APC genetic variants Y486Y, A545A, T1493T, and D1822V and susceptibility to oral squamous cell carcinoma (OSCC). METHODS: High-resolution melting (HRM) analysis was used to characterize APC mutations. Genomic DNA was extracted from 83 patient specimens of OSCC and 50 blood samples from healthy control subjects. The 14 exons and mutation cluster region of exon 15 were screened by HRM analysis. All mutations were confirmed by direct DNA sequencing. RESULTS: Three mutations and 4 single nucleotide polymorphisms (SNPs) were found in this study. The mutations were c.573T>C (Y191Y) in exon 5, c.1005A>G (L335L) in exon 9, and c.1488A>T (T496T) in exon 11. Two SNPs, c.4479G>A (T1493T) and c.5465A>T (D1822V), were located in exon 15, whereas c.1458T>C (Y486Y) and c.1635G>A (A545A) were located in exon 11 and 13, respectively. There was no observed association between OSCC risk and genotype for any of the 4 APC SNPs. CONCLUSIONS: The mutation of APC is rare in Taiwanese patients with OSCC. HRM analysis is a reliable, accurate, and fast screening method for APC mutations.

Molecular epidemiological analysis of Saffold cardiovirus genotype 3 from upper respiratory infection patients in Taiwan.

BACKGROUND: Saffold cardiovirus (SAFV) belongs to the Cardiovirus genus of Picornaviridae family, and may be a relevant new human pathogen; Thus far, eleven genotypes have been identified. The SAFV type 3 (SAFV-3) is thought to be the major genotype and is detected relatively frequently in children with acute gastroenteritis and respiratory illness. The epidemiology and pathogenicity of SAFV-3 remain unclear. OBJECTIVES: To investigate the genomic and epidemiologic profiles of SAFV-3 infection in Taiwan. STUDY DESIGN: Virus was detected in respiratory samples from children suffering for URI. SAFV-3 isolates were detected by isolation on cell culture and IF assay. The molecular typing was performed by RT-PCR and was sequenced to compare with reference strains available in the NCBI GeneBank. Serum samples were collected from 2005 to 2013 in Taiwan for seroprevalence investigation. RESULTS: A total of 226 specimens collected from children with URIs, 22 (9.73%) were positive for SAFV-3. The majority of SAFV-3 infections were found in children less than 6 years of age (14 of 22, 63.6%). Genetic analysis of VP1 coding region of Taiwanese isolates shown an 83.2-97.7% difference from other available SAFV-3 sequences in NCBI GenBank. Phylogenetic analysis revealed there is three genetic groups of SAFV-3 co-circulated in Taiwan during the study period. In addition, seroprevalence investigation results indicated that SAFV-3 infection occurs early in life and 43.7-77.8% of children aged between 6 months to 9 years old, had neutralizing antibodies against SAFV-3. CONCLUSION: SAFV-3 may have circulated in Taiwan for some time and it appears to be one of the etiological agents responsible for URIs in children.