Female ; Humans ; Carcinoma, Endometrioid/pathology* ; Endometrial Neoplasms/pathology* ; Molecular Typing/methods*

Objective: To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases. Methods: A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID Results: The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID Conclusion This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.
Capsid Proteins/genetics* ; Enterovirus A, Human/genetics* ; Enterovirus B, Human/genetics* ; Enterovirus C, Human/genetics* ; Enterovirus D, Human/genetics* ; Humans ; Molecular Epidemiology/methods* ; Molecular Typing/methods* ; Reverse Transcriptase Polymerase Chain Reaction/methods*

Bovine tuberculosis is a chronic contagious disease responsible for major agricultural economic losses. Abattoir monitoring and trace-back systems are an appropriate method to control bovine tuberculosis, particularly in beef cattle. In the present study, a trace-back system was applied to bovine tuberculosis cases in Korean native Hanwoo beef cattle. Bovine tuberculosis was detected in three index beef cattle during abattoir monitoring in Jeonbuk Province, Korea, and the original herds were traced back from each index cow. All cattle in each original herd were subjected to tuberculin skin test. The positive rates in the tuberculin skin test were 64.6% (62 of 96), 4.8% (2 of 42), and 8.1% (3 of 37) at farms A, B, and C, respectively. On post-mortem examination of 56 tuberculin-positive cattle, 62% had granulomatous lesions, and Mycobacterium bovis was cultured from 40 (71.4%) of the cattle. Molecular typing by spoligotyping and the mycobacterial interspersed repetitive unit-variable-number tandem repeat assay revealed the genotype of the M. bovis strains from the index cattle were same as the M. bovis genotype in each original herd. The results suggest that tracing back from index cattle to the original herd is an effective method to control bovine tuberculosis in beef cattle.
Abattoirs ; Agriculture ; Animals ; Autopsy ; Cattle ; Disease Outbreaks ; Genotype ; Jeollabuk-do ; Korea ; Methods ; Molecular Typing ; Mycobacterium bovis ; Red Meat ; Skin Tests ; Tandem Repeat Sequences ; Tuberculin ; Tuberculosis, Bovine

BACKGROUND: The increasing morbidity and mortality rates associated with Acinetobacter baumannii are due to the emergence of drug resistance and the limited treatment options. We compared characteristics of colistin-resistant Acinetobacter baumannii (CR-AB) clinical isolates recovered from patients with and without prior colistin treatment. We assessed whether prior colistin treatment affects the resistance mechanism of CR-AB isolates, mortality rates, and clinical characteristics. Additionally, a proper method for identifying CR-AB was determined. METHODS: We collected 36 non-duplicate CR-AB clinical isolates resistant to colistin. Antimicrobial susceptibility testing, Sanger sequencing analysis, molecular typing, lipid A structure analysis, and in vitro synergy testing were performed. Eleven colistin-susceptible AB isolates were used as controls. RESULTS: Despite no differences in clinical characteristics between patients with and without prior colistin treatment, resistance-causing genetic mutations were more frequent in isolates from colistin-treated patients. Distinct mutations were overlooked via the Sanger sequencing method, perhaps because of a masking effect by the colistin-susceptible AB subpopulation of CR-AB isolates lacking genetic mutations. However, modified lipid A analysis revealed colistin resistance peaks, despite the population heterogeneity, and peak levels were significantly different between the groups. CONCLUSIONS: Although prior colistin use did not induce clinical or susceptibility differences, we demonstrated that identification of CR-AB by sequencing is insufficient. We propose that population heterogeneity has a masking effect, especially in colistin non-treated patients; therefore, accurate testing methods reflecting physiological alterations of the bacteria, such as phosphoethanolamine-modified lipid A identification by matrix-assisted laser desorption ionization-time of flight, should be employed.
Acinetobacter baumannii* ; Acinetobacter* ; Bacteria ; Colistin* ; Drug Resistance ; Humans ; In Vitro Techniques ; Lipid A ; Masks ; Methods ; Molecular Typing ; Mortality ; Population Characteristics

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) has been increasingly reported worldwide in the past 10 years, which is an important infection control concern. Since the epidemiology and characteristics of these CPEs vary according to institutes, we aimed to characterize CPEs in a university hospital during the recent 4 years. METHODS: From October 2011 to September 2015, CPE isolates from clinical specimens and hospital surveillance cultures were collected. Carbapenem resistance was confirmed by disk diffusion method and Minimal Inhibitory Concentration (MIC) was determined by agar dilution method. Carbapenemase production was tested by double disk test using aminophenylboronic acid and dipicolic acid. PCR and sequence analysis were performed to detect bla(KPC), bla(IMP-1), bla(VIM-2), bla(NDM-1)-like genes and bla(OXA-48) gene. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were conducted for KPC-producing Klebsiella pneumoniae isolates. RESULTS: Twenty-five isolates (11%) of CPE were identified among 222 carbapenem-resistant Enterobacteriacae isolates during the study period. The most prevalent CPE was KPC-producing K. pneumonia and others were IMP-1, VIM-2, NDM-1 type and OXA-48 producing CPEs. Most of these CPEs showed resistance to carbapenems with variable MICs. The sequence types (STs) of KPC-producing K. pneumoniae were ST307 and ST11. The PFGE of ST11 and ST307 showed clonality in each group suggesting the possibility of in-hospital outbreak. CONCLUSION: The prevalence of CPE has been increasing. In our institute, KPC-producing K. pneumoniae was the most frequently isolated CPE in the recent 4 years. CPE including KPC producers can easily transfer their resistance. Therefore continuous monitoring and more intensified infection control for CPE should be considered.
Academies and Institutes ; Agar ; Carbapenems ; Diffusion ; Drug Resistance, Bacterial ; Electrophoresis, Gel, Pulsed-Field ; Enterobacteriaceae* ; Epidemiology ; Infection Control ; Klebsiella pneumoniae ; Korea* ; Methods ; Molecular Epidemiology* ; Multilocus Sequence Typing ; Pneumonia ; Polymerase Chain Reaction ; Prevalence ; Sequence Analysis

Tuberculosis, caused by the bacterium Mycobacterium tuberculosis, remains one of the most serious global health problems. Molecular typing of M. tuberculosis has been used for various epidemiologic purposes as well as for clinical management. Currently, many techniques are available to type M. tuberculosis. Choosing the most appropriate technique in accordance with the existing laboratory conditions and the specific features of the geographic region is important. Insertion sequence IS6110-based restriction fragment length polymorphism (RFLP) analysis is considered the gold standard for the molecular epidemiologic investigations of tuberculosis. However, other polymerase chain reaction-based methods such as spacer oligonucleotide typing (spoligotyping), which detects 43 spacer sequence-interspersing direct repeats (DRs) in the genomic DR region; mycobacterial interspersed repetitive units–variable number tandem repeats, (MIRU-VNTR), which determines the number and size of tandem repetitive DNA sequences; repetitive-sequence-based PCR (rep-PCR), which provides high-throughput genotypic fingerprinting of multiple Mycobacterium species; and the recently developed genome-based whole genome sequencing methods demonstrate similar discriminatory power and greater convenience. This review focuses on techniques frequently used for the molecular typing of M. tuberculosis and discusses their general aspects and applications.
Base Sequence ; Dermatoglyphics ; Genome ; Global Health ; Methods* ; Molecular Typing ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Repetitive Sequences, Nucleic Acid ; Tandem Repeat Sequences ; Tuberculosis

PURPOSE: The genus Aeromonas is a pathogen that is well known to cause severe clinical illnesses, ranging from gastroenteritis to sepsis. Accurate identification of A. hydrophila, A. caviae, and A. veronii is important for the care of patients. However, species identification remains difficult using conventional methods. The aim of this study was to compare the accuracy of different methods of identifying Aeromonas at the species level: a biochemical method, matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS), 16S rRNA sequencing, and housekeeping gene sequencing (gyrB, rpoB). MATERIALS AND METHODS: We analyzed 65 Aeromonas isolates recovered from patients at a university hospital in Korea between 1996 and 2012. The isolates were recovered from frozen states and tested using the following four methods: a conventional biochemical method, 16S rRNA sequencing, housekeeping gene sequencing with phylogenetic analysis, and MALDI-TOF MS. RESULTS: The conventional biochemical method and 16S rRNA sequencing identified Aeromonas at the genus level very accurately, although species level identification was unsatisfactory. MALDI-TOF MS system correctly identified 60 (92.3%) isolates at the species level and an additional four (6.2%) at the genus level. Overall, housekeeping gene sequencing with phylogenetic analysis was found to be the most accurate in identifying Aeromonas at the species level. CONCLUSION: The most accurate method of identification of Aeromonas to species level is by housekeeping gene sequencing, although high cost and technical difficulty hinder its usage in clinical settings. An easy-to-use identification method is needed for clinical laboratories, for which MALDI-TOF MS could be a strong candidate.
Aeromonas/classification/*genetics/isolation & purification ; DNA, Bacterial/genetics ; Genes, Essential/*genetics ; Humans ; Molecular Typing/*methods ; Phylogeny ; RNA, Ribosomal, 16S/*genetics ; Republic of Korea ; Sensitivity and Specificity ; Sequence Analysis, DNA/*methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods

Animals ; Environmental Microbiology ; Humans ; Malaysia ; Molecular Typing ; methods ; Salmonella Infections ; epidemiology ; microbiology ; Salmonella enterica ; classification ; genetics

This study was aimed to screen human papillomavirus (HPV) types associated with esophageal squamous cell carcinoma of Kazakh in Xinjiang using the gene chip technique and study the clinical significance of this application. The DNAs were collected from esophageal squamous cell carcinoma tissues and healthy esophageal mucosa of Kazakh adults in Xinjiang, and amplified firstly using HPV MY09/11 and then using HPV G5+/6+ to screen positive HPV specimens. These positive specimens were further detected by the gene chip technique to screen highly pathogenic HPV types. After determination with nested PCR amplification with HPV MY09/11 and G5+/6+, the infection rate of HPV was 66.67% in the esophageal squamous cell carcinoma group and 12.12% in the healthy control group. By testing the positive HPV specimens from the esophageal squamous cell carcinoma group, the infection rate of HPV16 was 97.72% and the co-infection rate of HPV16 and HPV18 was 2.27%. HPV16 infection may be involved in the development of esophageal squamous cell carcinoma in Xinjiang Hazakh adults.
Aged ; Asian Continental Ancestry Group ; Carcinoma, Squamous Cell ; ethnology ; genetics ; virology ; Case-Control Studies ; China ; DNA, Neoplasm ; analysis ; genetics ; DNA, Viral ; analysis ; genetics ; Esophageal Neoplasms ; ethnology ; genetics ; virology ; Female ; Host-Pathogen Interactions ; genetics ; Human papillomavirus 16 ; genetics ; Human papillomavirus 18 ; genetics ; Humans ; Male ; Middle Aged ; Molecular Typing ; methods ; statistics & numerical data ; Oligonucleotide Array Sequence Analysis ; methods ; Papillomaviridae ; classification ; genetics ; physiology ; Papillomavirus Infections ; ethnology ; genetics ; virology ; Polymerase Chain Reaction

BACKGROUNDStreptococcus pneumoniae (S. pneumoniae) is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, and has become a major public health concern. We report the pneumococcal serotype and sequence type (ST) distribution, and antimicrobial resistance of 39 S. pneumoniae strains from seven hospitals in China.

METHODSBlood/cerebrospinal fluid (CSF) and sputum isolates from patients were analyzed to determine S. pneumoniae serotypes by polymerase chain reaction (PCR) and the Neufeld Quellung reaction, the multilocus sequence types (MLST) by PCR and sequencing, and susceptibility to antimicrobial agents by the VITEK Gram Positive Susceptibility Card.

RESULTSA total of 39 isolates were collected including 21 blood/CSF and 18 sputum isolates. Conventional serotyping by the Quellung reaction required 749 reactions. In contrast, PCR based typing needed only 106 PCR reactions. The most frequent serotypes from the blood/CSF isolates were 14 (38.1%), 19A (14.3%), 23F (9.5%), and 18C (9.5%). In the sputum isolates the most frequent serotypes were 19F (33.3%), 23F (16.7%), 19A (11.1%), and 3 (11.1%). The incidence of penicillin resistance in the blood/CSF and sputum isolates was 66.7% and 55.6%, respectively. Statistical analysis showed that patients = 5 years old had a higher resistance to penicillin when they compared with the patients = 65 years old (P = 0.011). Serotypes 14, 19A and 19F were significantly associated with penicillin resistance (P < 0.001). ST320, ST271, and ST876 isolates showed high resistant rates to several antibiotics including penicillin (P = 0.006). All of the isolates of serotype 19A were resistant to both penicillin and erythromycin, and they were all multi-drug resistant (MDR) isolates.

CONCLUSIONSThe specificity and sensitivity of multiplex-PCR are good, and this method represents a substantial savings of time and money, and can be widely used in the laboratory and clinical practice. Data from this research showed an extremely high prevalence of penicillin resistance and an increasing prevalence of multi-drug resistant (MDR) rate in S. pneumoniae. A distinctive emergence of serotype 19A was observed which was also associated with the increasing prevalence of antimicrobial resistance. Therefore, nationwide surveillance of pneumococcal resistance and serotypes is strongly warranted.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Drug Resistance, Multiple, Bacterial ; Humans ; Infant ; Microbial Sensitivity Tests ; Middle Aged ; Molecular Typing ; methods ; Multilocus Sequence Typing ; methods ; Pneumococcal Infections ; microbiology ; Serotyping ; Streptococcus pneumoniae ; classification ; drug effects