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*

Mendelian randomization (MR) in epidemiology is the use of genetic variants as instrumental variables (IVs) in non-experimental design to make causality of a modifiable exposure on an outcome or disease. It assesses the causal effect between risk factor and a clinical outcome. The main reason to approach MR is to avoid the problem of residual confounding. There is no association between the genotype of early pregnancy and the disease, and the genotype of an individual cannot be changed. For this reason, it results with randomly assigned case-control studies can be set by regressing the measurements. IVs in MR are used genetic variants for estimating the causality. Usually an outcome is a disease and an exposure is risk factor, intermediate phenotype which may be a biomarker. The choice of the genetic variable as IV (Z) is essential to a successful in MR analysis. MR is named ‘Mendelian deconfounding’ as it gives to estimate of the causality free from biases due to confounding (C). To estimate unbiased estimation of the causality of the exposure (X) on the clinically relevant outcome (Y), Z has the 3 core assumptions (A1-A3). A1) Z is independent of C; A2) Z is associated with X; and A3) Z is independent of Y given X and C; The purpose of this review provides an overview of the MR analysis and is to explain that using an IV is proposed as an alternative statistical method to estimate causal effect of exposure and outcome under controlling for a confounder.
Bias (Epidemiology) ; Case-Control Studies ; Epidemiology ; Genotype ; Mendelian Randomization Analysis ; Methods ; Molecular Epidemiology ; Phenotype ; Pregnancy ; Random Allocation ; Risk Factors

The aim of this study was to describe the genetic diversity of Mycobacterium avium subsp. paratuberculosis (MAP) obtained from individual cows in Korea. Twelve MAP-positive fecal DNA samples and 19 MAP isolates were obtained from 10 cattle herds located in 5 provinces in Korea. In addition, 5 MAP isolates obtained from the Czech Republic and Slovakia and 3 isolates from Australia were genotyped for comparison with the domestic isolates. The most prevalent strains in Korea were of the “bison-type” genotype (23 of 31 fecal DNA/isolates) and were distributed nationwide. The remaining MAP isolates (8) and all of the foreign isolates were identified as “cattle-type”. The bison-type strains which were discriminated only as INMV 68 in variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing. Multilocus short sequence repeat (MLSSR) typing differentiated the bison-type strains into 3 different subtypes. The cattle-type strains were divided into 3 subtypes by MIRU-VNTR and 8 subtypes by MLSSR. The allelic diversities in the MIRU-VNTR and MLSSR results were calculated as 0.567 and 0.866, respectively. These results suggest that MIRU-VNTR typing cannot provide a sufficient description of the epidemiological situation of MAP. Therefore, an alternative method, such as MLSSR, is needed for typing of MAP strains to elucidate the molecular epidemiology of MAP infections. Overall, this study is the first epidemiological survey report in Korea using both MIRU-VNTR and MLSSR typing methods, and it has provided basic data necessary to elucidate the characteristics of MAP infections in Korea.
Animals ; Australia ; Cattle ; Czech Republic ; DNA ; Genetic Variation ; Genotype ; Korea ; Methods ; Molecular Epidemiology ; Mycobacterium avium subsp. paratuberculosis ; Mycobacterium avium ; Mycobacterium ; Paratuberculosis ; Slovakia ; Tandem Repeat Sequences

Staphylococcus aureus is one of the major pathogens causing bovine mastitis and foodborne diseases associated with dairy products. To determine the genetic relationships between human and bovine or bovine isolates of S. aureus, various molecular methods have been used. Previously we developed an rpoB sequence typing (RSTing) method for molecular differentiation of S. aureus isolates and identification of RpoB-related antibiotic resistance. In this study, we performed spa typing and RSTing with 84 isolates from mastitic cows (22 farms, 72 cows, and 84 udders) and developed a molecular prophage typing (mPPTing) method for molecular epidemiological analysis of bovine mastitis. To compare the results, human isolates from patients (n = 14) and GenBank (n = 166) were used for real and in silico RSTing and mPPTing, respectively. Based on the results, RST10-2 and RST4-1 were the most common rpoB sequence types (RSTs) in cows and humans, respectively, and most isolates from cows and humans clearly differed. Antibiotic resistance-related RSTs were not detected in the cow isolates. A single dominant prophage type and gradual evolution through prophage acquisition were apparent in most of the tested farms. Thus, RSTing and mPPTing are informative, simple, and economic methods for molecular epidemiological analysis of S. aureus infections.
Agriculture ; Animals ; Cattle ; Computer Simulation ; Dairy Products ; Databases, Nucleic Acid ; Drug Resistance, Microbial ; Female ; Foodborne Diseases ; Humans ; Mastitis, Bovine ; Methods ; Molecular Epidemiology ; Prophages ; Staphylococcus aureus ; Staphylococcus

Colletotrichum gloeosporioides is an economically important fungal pathogen causing substantial yield losses indifferent host plants. To understand the genetic diversity and molecular epidemiology of this fungus, we have developed a novel, high-resolution multi-locus microsatellite typing (MLMT) method. Bioinformatic analysis of C. gloeosporioides unannotated genome sequence yielded eight potential microsatellite loci, of which five, CG1 (GT)(n), CG2 (GT1)(n), CG3 (TC)(n), CG4 (CT)(n), and CG5 (CT1)(n) were selected for further study based on their universal amplification potential, reproducibility, and repeat number polymorphism. The selected microsatellites were used to analyze 31 strains of C. gloeosporioides isolated from 20 different host plants from India. All microsatellite loci were found to be polymorphic, and the approximate fragment sizes of microsatellite loci CG1, CG2, CG3, CG4, and CG5 were in ranges of 213–241, 197–227, 231–265, 209–275, and 132–188, respectively. Among the 31 isolates, 55 different genotypes were identified. The Simpson's index of diversity (D) values for the individual locus ranged from 0.79 to 0.92, with the D value of all combined five microsatellite loci being 0.99. Microsatellite data analysis revealed that isolates from Ocimum sanctum, Capsicum annuum (chili pepper), and Mangifera indica (mango) formed distinct clusters, therefore exhibited some level of correlation between certain genotypes and host. The developed MLMT method would be a powerful tool for studying the genetic diversity and any possible genotype-host correlation in C. gloeosporioides.
Capsicum ; Colletotrichum* ; Computational Biology ; Fungi ; Genetic Variation ; Genome ; Genotype ; India ; Mangifera ; Methods* ; Microsatellite Repeats* ; Molecular Epidemiology ; Ocimum ; Statistics as Topic

BACKGROUND: It has proven challenging to investigate the molecular epidemiology of Mycobacterium leprae, the causative agent of leprosy, due to difficulties with culturing of the organism and a lack of genetic heterogeneity between strains. Recently, A panel of variable-number tandem-repeat (VNTR) markers and an alternative method, structure-neighbor clustering, which assigns isolates with the most similar genotypes to the same groups and, subsequently, subgroups, without inferring how the strains descended from a common ancestor have been developed. METHODS: A total of 29 samples from Korea found cases were studied by 14 VTRN typing and an alternative method, structure-neighbor clustering with 13 and 14 VNTRs by Structure Program(k=10). RESULTS: Only 286 cases of 522 total cases(including database of Bellingham Research Institute) showed p>0.8(in 13 and 14 VNTRs). Almost Korea found cases(18 cases) were included in group 3(13 VNTRs), in group 9(14 VNTRs)(by Structure Program, k=10). CONCLUSIONS: The structure-neighbor clustering by Structure Program with panels of VNTR is a useful approach for investigating the molecular epidemiology of Mycobacterium leprae.
Cluster Analysis ; Genetic Heterogeneity ; Genotype ; Korea ; Leprosy ; Methods ; Molecular Epidemiology ; Mycobacterium leprae ; Mycobacterium

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

BACKGROUND: Stenotrophomonas maltophilia is one of several opportunistic pathogens of growing significance. Several studies on the molecular epidemiology of S. maltophilia have shown clinical isolates to be genetically diverse. MATERIALS AND METHODS: A total of 121 clinical isolates tentatively identified as S. malophilia from seven tertiary-care hospitals in Korea from 2007 to 2011 were included. Species and groups were identified using partial gyrB gene sequences and antimicrobial susceptibility testing was performed using a broth microdilution method. Multi locus variable number of tandem repeat analysis (MLVA) surveys are used for subtyping. RESULTS: Based on partial gyrB gene sequences, 118 isolates were identified as belonging to the S. maltophilia complex. For all S. maltophilia isolates, the resistance rates to trimethoprime-sulfamethoxazole (TMP/SMX) and levofloxacin were the highest (both, 30.5%). Resistance rate to ceftazidime was 28.0%. 11.0% and 11.9% of 118 S. maltophilia isolates displayed resistance to piperacillin/tazobactam and tigecycline, respectively. Clade 1 and Clade 2 were definitely distinguished from the data of MLVA with amplification of loci. All 118 isolates were classified into several clusters as its identification. CONCLUSION: Because of high resistance rates to TMP/SMX and levofloxacin, the clinical laboratory department should consider providing the data about other antimicrobial agents and treatment of S. maltophilia infections with a combination of antimicrobials can be considered in the current practice. The MLVA evaluated in this study provides a fast, portable, relatively low cost genotyping method that can be employed in genotypic linkage or transmission networks comparing to analysis of the gyrB gene.
Anti-Infective Agents ; Ceftazidime ; Korea ; Levofloxacin ; Methods ; Molecular Epidemiology ; Stenotrophomonas maltophilia* ; Stenotrophomonas* ; Tandem Repeat Sequences*

Malaria is a tropical disease caused by protozoans of the Plasmodium genus. Delayed diagnosis and misdiagnosis are strongly associated with higher mortality. In recent years, a greater importance is attributed to Plasmodium knowlesi, a species found mainly in Southeast Asia. Routine parasitological diagnostics are associated with certain limitations and difficulties in unambiguous determination of the parasite species based only on microscopic image. Recently, molecular techniques have been increasingly used for predictive diagnosis. The aim of the study is to draw attention to the risk of travelling to knowlesi malaria endemic areas and to raise awareness among personnel involved in the therapeutic process.
Asia, Southeastern ; Global Health ; Humans ; Malaria/diagnosis/*epidemiology/*parasitology ; Microscopy/methods ; Molecular Diagnostic Techniques/methods ; Plasmodium knowlesi/*isolation & purification ; Public Health

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