BACKGROUND: The use of the multiplex polymerase chain reaction (PCR) technique for respiratory viruses has become popular in Korea owing to its convenience and sensitivity. However, concerns remain with regard to possible interference due to multiplexing. METHODS: We compared the analytical sensitivity and virus interference of a commercially available, multiplex PCR kit (AdvanSure Respiratory virus real-time PCR kit, LG Life Sciences, Korea) with that of singleplex PCR to detect 11 viruses including coronavirus 229E and OC43; parainfluenza virus 1 (PIV 1), parainfluenza virus 2 (PIV 2), and parainfluenza virus 3 (PIV 3); influenza virus A (INF A) and influenza virus B (INF B); respiratory syncytial virus A (RSV A) and respiratory syncytial virus B (RSV B); adenovirus; and rhinovirus A, B, and C. RESULTS: The lowest detected viral concentrations of coronavirus 229E and OC43, INF A and B, RSV A and B, adenovirus, and rhinovirus A, B, and C were the same for both, multiplex and singleplex systems. However, the lowest detected viral concentrations of PIV1, 2, and 3 differed by 1 dilution factor between the two systems. Threshold cycle (Ct) values for mixed viruses within the same well were not significantly influenced by each other, where the difference between Ct values ranged from 0.24 to 1.99. CONCLUSIONS: Analytical sensitivity of multiplex PCR was comparable to that of singleplex PCR for respiratory viruses. No significant interference was observed with mixed virus samples using multiplexed PCR.
Adenoviridae ; Biological Science Disciplines ; Coronavirus ; Humans* ; Korea ; Multiplex Polymerase Chain Reaction ; Orthomyxoviridae ; Paramyxoviridae Infections ; Polymerase Chain Reaction* ; Real-Time Polymerase Chain Reaction ; Respiratory Syncytial Viruses ; Rhinovirus

PURPOSE: This study aimed to examine the accuracy of rapid influenza diagnostic tests (RIDT) in children with an influenza-like illness and to evaluate factors associated with greater accuracy. METHODS: Pediatric patients, who visited Hallym University Sacred Heart Hospital with an influenza-like illness between June 2011 and May 2016, were enrolled in this study. We tested 798 samples using a real-time polymerase chain reaction (PCR) for respiratory viruses and compared the results with rapid influenza tests. RESULTS: In comparison with the results of the multiplex PCR, the positive agreement rates of RIDT for influenza A and B virus were 75.7% and 60.0%, respectively. The performance of RIDT varied according to days after fever onset. The positive agreement rates of RIDT for influenza A and B tests, performed within 4 days of fever onset, were 77.6% and 73.2%, but the rates for tests performed more than 5 days after fever onset were 66.7% and 21.4%, respectively. CONCLUSIONS: The RIDT is a quick and simple aid to diagnosis, but is less sensitive than the labeled sensitivity. Moreover, test performance varied according to days after fever onset. Test specimens for RIDT should be collected as soon as possible after the onset of symptoms (less than 4 days).
Child ; Diagnosis ; Diagnostic Tests, Routine ; Fever ; Heart ; Herpesvirus 1, Cercopithecine ; Humans ; Immunochromatography ; Influenza, Human ; Multiplex Polymerase Chain Reaction ; Orthomyxoviridae ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Seasons

BACKGROUND: Community-acquired pneumonia (CAP) is an important cause of morbidity and mortality throughout the world in all age groups. Viral causes of CAP are less well characterized than bacterial causes. We analyzed the characteristics of hospitalized patients with CAP who had a viral pathogen detected by multiplex polymerase chain reaction (PCR). METHODS: Multiplex real-time PCR was performed for respiratory viruses in samples collected from 520 adults who developed CAP at Chungnam National University Hospital. Clinical, laboratory, and radiological features at presentation as well as other epidemiological data were analyzed. RESULTS: Of 520 patients with CAP, a viral pathogen was detected in 60 (11.5%), and influenza A was the most common. The virus detection rate in patients with CAP was highest in November. Two or more pathogens were detected in 13 (21.7%) patients. Seven patients had severe disease and were administered in the intensive care unit. Most patients (49/60, 81.7%) had comorbidities. However, nine (15%) patients had no comorbidities, and their age was <60 years. The ground glass opacity pattern was the most common radiological feature. Seven (11.7%) patients died from CAP. CONCLUSION: Viral pathogens are commonly detected in patients with CAP, and a respiratory virus may be associated with the severity and outcome of pneumonia. Careful attention should be paid to the viral etiology in adult patients with CAP.
Adult ; Comorbidity ; Glass ; Humans ; Influenza, Human ; Intensive Care Units ; Multiplex Polymerase Chain Reaction ; Pneumonia ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Viruses

BACKGROUND: Mycobacterium leprae, the etiological agent of leprosy, is noncultivable on axenic media, so, the viability of M. leprae for clinical or experimental applications is often unknown. Quantitative reverse transcriptase PCR (RT-PCR) assays were recently introduced as the new tools for M. leprae viability determination. OBJECTIVE: For evaluating of correlation of results of quantitative real-time PCR(16S rRNA/RLEP) & AFB stain of slit skin smear & histopathology & estimating the viability of M. leprae, the author studied the comparison of results of them METHODS: Of 46 samples from 27 patients(MB 24 cases, PB 3 cases), M. leprae 16S rRNA was used as RNA targets, and M. leprae repetitive element (RLEP) DNA was used to determine relative bacterial numbers and the viability by the quantitative real-time PCR. The ratio of 16S rRNA and RLEP as the indicator of viability was calculated. Student t test and linear Pearson correlation were done by SPSS. RESULTS: There was a correlation between between 16S rRNA/RLEP ratio and BI (r=0.369, p=0.012), and was statistically significant between 16S rRNA/RLEP ratio and histopathological positivity of AFB (p=0.011). However there was no correlation between 16S rRNA/RLEP ratio and MI. CONCLUSIONS: Although the correlation between between 16S rRNA/RLEP ratio and BI and the statistically significant between 16S rRNA/RLEP ratio and histopathological positivity of AFB, there was no correlation between 16S rRNA/RLEP ratio and MI. It needs the further evaluation the correlation about that.
DNA ; Humans ; Leprosy* ; Mycobacterium leprae ; Real-Time Polymerase Chain Reaction* ; Reverse Transcriptase Polymerase Chain Reaction ; RNA ; Skin*

BACKGROUND: One of main mechanisms responsible for acquired azole resistance of Candida glabrata is the increased drug efflux mediated ABS transporters, which are encoded by CgCDR1 and CgCDR2 genes. OBJECTIVES: We compared real-time reverse transcriptase PCR (RT-PCR) with northern hybridization for quantitative analysis of CgCDR1 and CgCDR2 expression in bloodstream isolates of C. glabrata. METHODS: Nineteen blood isolates of C. glabrata were selected, including nine fluconazole susceptible (MIC < or =8 microgram/ml), nine susceptible dose-dependent (S-DD, MIC 16~32 microgram/ml), and one resistant (MIC 128 microgram/ml), isolates. The expression of CgCDR1 and CgCDR2 was quantified using real-time RT-PCR with ROTOR Gene 3000 (Corbettet research, Austria). The results were compared with northern hybridization with sequence-specific probes. RESULTS: Correlation of quantification results between real-time RT-PCR and northern hybridization yielded correlation coefficients of 0.92 for CgCDR1 and 0.82 for CgCDR2 gene. By both methods, no significant differences were observed in the levels of expression of CgCDR1 and CgCDR2 between fluconazole-susceptible isolates and S-DD isolates. In contrast, a strain with high fluconazole resistance (MIC 128 microgram/ml) revealed a greater abundance of CgCDR1 by both methods, compared to the other isolates. Conclusion: This study show that real-time PCR method for C. glabrata RNA quantification correlates well with traditional northern hybridization and can be a valuable alternative to northern hybridization for rapid quantification of CgCDR1 and CgCDR2 genes in clinical isolates of C. glabrata.
Candida ; Candida glabrata ; Chimera ; Danazol ; Fluconazole ; Gene Expression ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; RNA ; Sprains and Strains

BACKGROUND: Novel swine influenza (H1N1) was first identified in Mexico in April 2009. Because of its high infectivity and worldwide distribution, a rapid and efficient screening test is necessary. Here we evaluated the usefulness of a rapid antigen test currently in use, compared to real-time RT-PCR (rRT-PCR) as a screening test for detection of novel swine influenza (H1N1). METHODS: A total of 1,228 patients who visited Hallym University Kangdong Sacred Heart Hospital with influenza-like illness between 14 August 2009 and 30 September 2009, and were tested by both rapid antigen and rRT-PCR tests, were enrolled in this study. RESULTS: Sensitivity, specificity, predictive value of a positive test, and predictive value of a negative test for the rapid antigen test were 30.5%, 99.2%, 86.4% and 90.1%, respectively. Fifty-one (4.2%) patients were positive for both rapid antigen test and rRT-PCR, and 1,053 (85.7%) were negative for both rapid antigen test and rRT-PCR. A total of 124 (10.1%) patients showed a discrepancy between the two tests. Among them, 116 (9.4%) were only positive for rRT-PCR and 8 (0.7%) were only positive for the rapid antigen test. The latter 8 patients all showed negative H1/M2 results in rRT-PCR. There were significant differences in detection rates of the rapid antigen test between different H1 Ct (threshold cycle) interval groups and for different age groups (P<0.05). CONCLUSION: Although the rapid antigen test is easy to perform and provides fast results, its limits as a screening test for detection of novel swine influenza (H1N1) due to its low sensitivity compared to rRT-PCR need to be considered in practical situations.
Heart ; Humans ; Influenza, Human ; Mass Screening ; Mexico ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; RNA-Directed DNA Polymerase ; Sensitivity and Specificity ; Swine

BACKGROUND: Inherited polymorphisms that affect carcinogen metabolism may influence the risk for the development of colorectal cancer. This study was performed to evaluate the potential association between glutathione S-transferase M1 (GSTM1), GSTT1 and N-acetyltransferase 2 (NAT2) polymorphisms and colorectal cancer in Korea. METHODS: The frequencies of GSTM1, GSTT1, and NAT2 polymorphisms were compared between 98 patients with colorectal cancer and age and sex matched 98 healthy controls. GSTM1 and GSTT1 polymorphisms were simultaneously determined by multiplex polymerase chain reaction (PCR) and NAT2 polymorphisms were analyzed by real-time PCR with melting curve analysis. GSTM1 null, GSTT1 null and NAT2 rapid type were considered as risk genotypes. RESULTS: The prevalence of GSTM1 null genotype was 44.9% in the controls, and 58.2% in the cases (odds ratio, OR=1.706, P=0.086). The prevalence of GSTT1 null genotype in the controls and the cases was 48.0% and 54.1%, respectively (OR=1.278, P=0.475). NAT2 rapid type were found in 45.9% of healthy controls and 51.0% of cancer patients (OR=1.227, P=0.568). Relative risk for colorectal cancer increased significantly (P=0.032) as the number of risk genotypes increased. CONCLUSIONS: These results suggest that genetic polymorphisms of GSTM1, GSTT1 and NAT2 have no independent effect on colorectal cancer risk individually, but there exists a dose-response relationship between a combined number of the risk genotypes of GSTM1, GSTT1 and NAT2 and colorectal cancer risk.
Colorectal Neoplasms* ; Freezing ; Genotype ; Glutathione Transferase* ; Humans ; Korea ; Metabolism ; Multiplex Polymerase Chain Reaction ; Polymorphism, Genetic* ; Prevalence ; Real-Time Polymerase Chain Reaction

BACKGROUND: Diarrhea has been the second leading cause of death among children under the age of five, and the rapid and accurate pathogen diagnosis in patients with diarrhea is crucial for reducing morbidity and mortality. A newly developed one-step multiplex real-time PCR assay, the Allplex GI-Virus Assay, was evaluated for its ability to detect six diarrhea-causing viruses (rotavirus, norovirus genogroup I (GI) and genogroup II (GII), enteric adenovirus, astrovirus, and sapovirus) in stool samples. METHODS: The performance of the Allplex assay was compared with those of another multiplex PCR assay (Seeplex Diarrhea-V Ace Detection) and genotyping by sequencing, using 446 stool samples from patients with acute gastroenteritis. RESULTS: The overall agreement rates between the results of the Allplex and Seeplex assays were 98.7% for rotavirus, 99.1% for norovirus GI, 93.3% for norovirus GII, 98.0% for adenovirus, and 99.6% for astrovirus. The overall agreement rates between the Allplex assay and genotyping were 99.1% for rotavirus, 99.1% for norovirus GI, 98.7% for norovirus GII, 89.7% for adenovirus, 98.2% for astrovirus, and 99.8% for sapovirus. In addition, eight rotavirus genotypes, three norovirus GI genotypes, four norovirus GII genotypes, eight adenovirus genotypes, two astrovirus genotypes, and two sapovirus genotypes were detected. CONCLUSIONS: The Allplex assay showed high agreement with Seeplex and genotyping results, and was able to additionally detect sapoviruses. The Allplex assay could be useful in identifying viral gastrointestinal infections in patients with acute gastroenteritis symptoms.
Adenoviridae ; Cause of Death ; Child ; Diagnosis ; Diarrhea ; Gastroenteritis ; Genotype ; Humans ; Mortality ; Multiplex Polymerase Chain Reaction ; Norovirus ; Real-Time Polymerase Chain Reaction* ; Rotavirus ; Sapovirus

A real-time PCR assay using hybridization probe (HybProbe) has been developed to detect Brucella (B.) melitensis strains. The primer and HybProbe sets were designed based on the gap gene of chromosome I with a specific single nucleotide polymorphism of B. melitensis. Specificity of the assay was confirmed by comparison to reference Brucella species and other related strains. In the melting curve analysis, B. melitensis generated a peak at 67degrees C unlike those for other Brucella species observed at 61degrees C. Sensitivity of the assay for B. melitensis ranged from 20 ng to 200 fg of genomic DNA. The ability to identify 94 Mongolian B. melitensis isolates using the real-time PCR assay was identical to that of classical biotyping methods and differential multiplex PCR. These data showed that this new molecular technique is a simple and quick method for detecting B. melitensis, which will be important for the control and prevention of brucellosis.
Brucella ; Brucella melitensis ; Brucellosis ; DNA ; Freezing ; Mongolia ; Multiplex Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity

No abstract available.
Real-Time Polymerase Chain Reaction*