Myung Hyun Nam

Evaluation of a novel real-time RT-PCR using TOCE technology compared with culture and Seeplex RV15 for simultaneous detection of respiratory viruses

Abstract Background Various kinds of commercial molecular systems have been developed for fast and more accurate detection of respiratory viruses. Anyplex™ II RV16 [RV16] was designed for simultaneous detection of 16 respiratory viruses using multiplex PCR coupled with TOCE™ technology. Objectives To compare the performance of RV16 with those of culture and Seeplex® RV15 ACE [RV15] by determining their sensitivity and specificity. Study design Seven hundred and thirty respiratory samples were tested by modified shell vial culture method, RV16, and RV15. For molecular tests, automated nucleic acid extraction and liquid handling system using MICROLAB Nimbus IVD (Hamilton, USA) was adopted to maximize the workflow and accuracy. Performance of each assay was determined against a composite reference standard. Results Two hundred and one samples (28%) out of 730 samples were positive by culture, while additional 281 (39%) were positive by RV16 or RV15. Sensitivities of RV16, RV15, and culture for virus tested were as follows: 100/93/63% for influenza A, 90/80/69% for influenza B, 98/94/63% for RSV, 98/52/23% for adenovirus, and 100/75/46% for PIV. For viruses not covered by culture, sensitivities of RV16 and RV15 were as follows: 99/81% for rhinovirus, 92/100% for coronavirus OC43, 100/56% for coronavirus 229E/NL63, 92/88% for metapneumovirus, 100/62% for bocavirus, and 91/91% for enterovirus. Overall, the specificities of culture, RV16, and RV15 (Seegene) were 100/99.9/99.9%. Conclusions RV16 assay was superior to culture method and RV15 and will be a promising tool for patient management and public health epidemiology.

Evaluation of a rapid diagnostic test specific for Plasmodium vivax

Plasmodium vivax is the only human malaria indigenous to the Republic of Korea (ROK). A rapid and sensitive diagnostic test (RDT) that detects P. vivax is appropriate for evaluating suspected malaria patients with no travel history abroad. The RDTs, SD Malaria Antigen P.v (SD diagnostic, Kyonggi, ROK) specific for P. vivax and the well documented OptiMAL (DiaMed, Cressier, Switzerland) were compared among 282 volunteers for specificity and sensitivity of P. vivax and Plasmodium falciparum malaria infections against Giemsa‐stained blood smears read by an experienced microscopist. A total of 137 volunteers were diagnosed with P. vivax, 45 cases (returned travellers from overseas) were diagnosed with P. falciparum and 100 healthy volunteers were diagnosed as negative for malaria. Correspondingly, the SD Malaria Antigen P.v test identified P. vivax infections in 128/137 malaria patients (93.4%) and 0/100 (0%) healthy volunteers. Three patients identified with P. falciparum also were interpreted as P. vivax by the SD Malaria Antigen P.v test; however, these patients were later confirmed as mixed infections of P. vivax and P. falciparum by polymerase chain reaction. OptiMAL interpreted the three mixed infections only as P. falciparum and detected 130/137 (94.9%) patients with P. vivax. The sensitivity of the SD Malaria Antigen P.v test decreased from 100% (>5000 parasite/μl) to 81.3% (1–100 parasites/μl) as parasitaemia levels declined. For the regions where P. vivax is the primary malaria parasite, the SD P. vivax‐specific rapid diagnostic test may be useful for screening suspected malaria patients when sufficient material and human resources (e.g. trained microscopists) are unavailable for malaria diagnosis.

MicroRNA-194-5p could serve as a diagnostic and prognostic biomarker in myelodysplastic syndromes

Trisomy 8 and trisomy 1q are the most frequent chromosomal abnormalities in Korean patients with myelodysplastic syndrome (MDS). MicroRNA (miRNA) deregulation is involved in the development of hematological malignancies, including MDS, and cancer-associated genomic regions are known to encode miRNAs. The aim of the present study was to investigate the involvement of miRNAs encoded by chromosomes 8 and 1q in MDS. For this, the expression of nine miRNAs encoded by chromosome 8 (miR-30b-5p, miR-30d-5p, miR-101-3p, miR-124-3p, miR-151a-5p, miR-320a, miR-486-5p, miR-596, and miR-875-5p) and three miRNAs encoded by chromosome 1q (miR-29c-3p, miR-194-5p, and miR-214-3p) was compared between 65 MDS patients and 11 controls. We found a significant upregulation of miR-194-5p (5.1-fold, P=0.002) and miR-320a (2.94-fold, P=0.016) in MDS patients compared with controls. The patients with low miR-194-5p expression showed a significantly decreased overall survival (P=0.049). The areas under the miR-194-5p and miR-320a ROC curves were 0.797 (P=0.002) and 0.729 (P=0.016), respectively. Although these findings need to be validated in a larger patient population, our results indicate that miR-194-5p is a candidate diagnostic biomarker for MDS and that low miR-194-5p expression could be associated with poor overall survival for MDS patients.

Evaluation of the AdvanSure™ real-time RT-PCR compared with culture and Seeplex RV15 for simultaneous detection of respiratory viruses

Abstract Recently, AdvanSure™ kit based on multiplex real-time PCR was developed for simultaneous detection of 14 respiratory viruses (RVs). We compared the performance of AdvanSure with those of Seeplex® RV 15 ACE and culture by determining their sensitivities and specificities against a composite reference standard. Four hundred thirty-seven respiratory samples were tested by modified shell vial culture method, RV 15 ACE, and AdvanSure. One hundred fourteen samples (26.2%) out of 437 samples were positive by culture, while additional 91 (20.8%) were positive by AdvanSure or RV15. One hundred twelve of 114 culture-positive samples were positive by AdvanSure except 2 samples (1 adenovirus, 1 respiratory syncytial virus [RSV]). Overall, the sensitivities of culture, RV15, and AdvanSure were 74.5%, 89.8%, and 95.1%, respectively. Sensitivities of culture, RV15, and AdvanSure for each virus tested were as follows: 91/100/96% for influenza A, 60/0/100% for influenza B, 63/95/97% for RSV, 69/81/89% for adenovirus, and 87/93/93% for parainfluenza virus. For viruses not covered by culture, sensitivities of RV15 and AdvanSure were as follows: 77/88% for rhinovirus, 100/100% for coronavirus OC43, 40/100% for coronavirus 229E/NL63, 13/100% for metapneumovirus, and 44/100% for bocavirus. The overall specificities of culture, RV15, and AdvanSure were 100/98.9/99.5%, respectively. Of 45 coinfected specimens, AdvanSure detected 41 specimens (91.1%) as coinfected, while RV15 detected 27 specimens (60.0%) as coinfected. AdvanSure assay demonstrated exquisite performance for the detection of RVs and will be a valuable tool for the management of RV infection.

Clinical performance evaluation of the BD Veritor System Flu A+B assay.

Early identification of influenza is important for optimal patient management and infection control. Rapid influenza antigen tests have been used routinely in clinical settings to confirm clinical suspicion, despite their low sensitivity. To improve sensitivity, various influenza point-of-care test reader systems have been developed. This study evaluated the clinical performance of a digital readout rapid influenza diagnostic test (RIDT), the BD Veritor™ System Flu A+B assay (BD). Nasopharyngeal swabs taken from 250 patients (influenza A positive, n=75; influenza B positive, n=75; and influenza negative, n=100) were analyzed using the BinaxNOW® Influenza A/B antigen kit (BN), SD Influenza Ag A/B kit (SD), BD, real-time reverse transcriptase polymerase chain reaction (RT-PCR), and an influenza virus culture. Compared to RT-PCR, the sensitivities of BN, SD, and BD were 56.0, 53.3, and 72.0%, respectively, for influenza A and 57.3, 65.3, and 69.3%, respectively, for influenza B. No false-positive results were noted with the three rapid antigen tests. For influenza A, the average RT-PCR threshold cycle (Ct) for specimens that tested positive using BD was higher than that for specimens that tested positive using BN and SD. BD is a sensitive and easy method for the early detection of influenza A and B.

Non-ischaemic titrated cardiac injury caused by radiofrequency catheter ablation of atrial fibrillation mobilizes CD34-positive mononuclear cells by non-stromal cell-derived factor-1α mechanism

AIMS It has been known that myocardial ischaemia mobilizes CD34+ bone marrow-derived cells by the stromal cell-derived factor (SDF)-1alpha pathway. We hypothesized that non-ischaemic titrated cardiac injury caused by radiofrequency catheter ablation (RFCA) of atrial fibrillation (AF) recruits CD34+ cells by an alternative mechanism. METHODS AND RESULTS Fifty-six patients (39 males, 53.0 +/- 13.5 years old) who underwent electrophysiology study (EPS; n = 10) or RFCA of AF (n = 46) were included. Peripheral blood CD34+ cell count and multiple serologic markers were evaluated before, immediately after, at 24 h, and 10 days after the procedure. The results are as follows: (i) the per cent increase in CD34+ cells (%DeltaCD34+) was significant after RFCA compared with after EPS (P < 0.01), and correlated with RF duration and troponin I, respectively. (ii) In contrast, SDF-1alpha decreased after RFCA and had no correlation with %DeltaCD34+ cells while matrix metalloproteinase (MMP)-9 (P < 0.0001) and GRObeta (P < 0.001) increased after RFCA and had correlations with 24 h %DeltaCD34+ cells. CONCLUSION Non-ischaemic titrated cardiac injury caused by AF ablation mobilizes CD34+ cells to the peripheral blood through a non-SDF-1alpha pathway associated with MMP-9 and GRObeta.

Comparison between Saliva and Nasopharyngeal Swab Specimens for Detection of Respiratory Viruses by Multiplex Reverse Transcription-PCR

ABSTRACT Nasopharyngeal swabs (NPSs) are being widely used as specimens for multiplex real-time reverse transcription (RT)-PCR for respiratory virus detection. However, it remains unclear whether NPS specimens are optimal for all viruses targeted by multiplex RT-PCR. In addition, the procedure to obtain NPS specimens causes coughing in most patients, which possibly increases the risk of nosocomial spread of viruses. In this study, paired NPS and saliva specimens were collected from 236 adult male patients with suspected acute respiratory illnesses. Specimens were tested for 16 respiratory viruses by multiplex real-time RT-PCR. Among the specimens collected from the 236 patients, at least 1 respiratory virus was detected in 183 NPS specimens (77.5%) and 180 saliva specimens (76.3%). The rates of detection of respiratory viruses were comparable for NPS and saliva specimens (P = 0.766). Nine virus species and 349 viruses were isolated, 256 from NPS specimens and 273 from saliva specimens (P = 0.1574). Adenovirus was detected more frequently in saliva samples (P < 0.0001), whereas influenza virus type A and human rhinovirus were detected more frequently in NPS specimens (P = 0.0001 and P = 0.0289, respectively). The possibility of false-positive adenovirus detection from saliva samples was excluded by direct sequencing. In conclusion, neither of the sampling methods was consistently more sensitive than the other. We suggest that these cost-effective methods for detecting respiratory viruses in mixed NPS-saliva specimens might be valuable for future studies.

Concurrent hypermulticolor monitoring of CD31, CD34, CD45 and CD146 endothelial progenitor cell markers for acute myocardial infarction.

The circulating endothelial progenitor cells (EPCs) in blood of acute myocardial infarction (AMI) patient have been monitored in many previous studies. The number of circulating EPC increases in the blood of patients at onset of the AMI. EPC is originated from bone marrow. It performs vessel regeneration. There are many markers used for detecting EPC. Four of these markers, CD31, CD34, CD45, and CD146, were concurrently detected at the single cell level for the identification of EPC in the present preliminary study. The CD45 negative cell sorting was performed to peripheral blood mononuclear cells (PBMCs) acquired from four AMI patients with a magnetic bead sorter, since, EPCs expressed CD45 negative or dim. The resultant PBMC eluents were treated with quantum-antibody conjugates for the probing four different markers of EPCs and then applied to a high-content single cell imaging cytometer using acousto-optical tunable filter (AOTF). The use of quantum dot, with narrow emission wavelength range and AOTF enabling cellular image at a particular single wavelength, is very advantageous for accurate high-content AMI diagnosis based on simultaneous monitoring of many markers. The number of EPC increased as compared with control in three of four AMI patients. In this approach, two EPC subtypes were found, CD31(+), CD34(+), CD45(-/dim), CD146(-) as early outgrowth EPCs and CD31(+), CD34(+), CD45(-/dim), CD146(+) as late outgrowth EPCs. Patient 1 had CD31(+), CD34(+), CD45(-/dim), CD146(+) cells whose percentage was 4.21% of cells. Patient 2 had 2.38% of CD31(+), CD34(+), CD45(-/dim), CD146(-) cells and patient 3 had 4.28% of CD31(+), CD34(+), CD45(-/dim), CD146(+) cells.

Comparison of the automated fluorescence microscopic viability test with the conventional and flow cytometry methods

The cell viability test is an essential tool in any laboratory, performing cell‐based studies and clinical laboratory tests. The trypan blue exclusion method is the most popular assay for its simple concept among various diagnostic tools. However, several disadvantages include time‐consuming and labor‐intensive steps with low precision. In this study, we evaluated a new technique for the automatic cell viability measurement with microscopic cell counter and microchip. Upon blood draw from 11 healthy volunteers, Mononuclear cells were separated immediately from the heparinized whole blood, and the viable cells were diluted from 100 to 1%. The cell viability tests were performed simultaneously with following three methods: the conventional manual trypan blue exclusion method; the flow cytometry measurement with propidium iodide stain; and the newly developed microscopic cell counter with microchip. Linearities, precisions, and correlations from three methods were analyzed and compared. The correlations data from the microscopic cell counter were in good agreement with both the conventional trypan blue method (r=0.99, P<0.05) and the flow cytometry (r=0.99, P<0.05), respectively. The precision (2.0–6.2%) and linearity from the microscopic cell counter method with microchip were superior in comparison with the conventional method. The microscopic cell counter with microchip performed well with high precision, linearity, and efficient running time than both the manual trypan blue and the flow cytometry methods. J. Clin. Lab. Anal. 25:90–94, 2011.

Clinical Performance Evaluation of the Sofia RSV FIA Rapid Antigen Test for Diagnosis of Respiratory Syncytial Virus Infection

ABSTRACT A recently introduced Sofia respiratory syncytial virus (RSV) fluorescent immunoassay (FIA) was evaluated against the BinaxNOW RSV card and the SD Bioline RSV test using 348 respiratory samples. The Sofia, BinaxNOW, and SD Bioline kits showed sensitivities of 66%, 65%, and 64%, respectively, for detecting RSV-A, and 71%, 63%, and 65% for detecting RSV-B, respectively.