Detección y genotipificación del virus del papiloma humano de alto riesgo mediante PCR multiplex en tiempo real (RT-PCR VPH AR) / High risk human papillomavirus detection and genotyping by multiplex real time PCR (RT-PCR HR HPV)

Utilidad clínica de la prueba La relación causal entre el desarrollo de cáncer de cérvix y la infección con genotipos de alto riesgo (AR) del virus del papiloma humano (VPH), ha llevado al desarrollo de estrategias para su detección y caracterización genotípica, como una medida de prevención de este tipo de cáncer. Dado que la presencia del VPH no puede ser determinada mediante los hallazgos clínicos de la paciente, como tampoco en los hallazgos morfológicos en la citología ni en la detección de anticuerpos específicos contra el VPH (pruebas serológicas), su detección y genotipificación recaen en el uso de pruebas moleculares, las cuales en su mayoría están dirigidas a la detección del ADN de los genotipos de alto riesgo, usando la técnica de reacción en cadena de la polimerasa (PCR) convencional y en tiempo real (RT-PCR) [1]. La técnica de PCR permite la amplificación de regiones específicas del ADN del VPH en los genes L1, E6 y E7, los cuales, por sus variaciones en la secuencia, permiten la genotipificación del virus [2,3]. Las pruebas de detección de ADN y/o genotipificación del VPH son consideradas herramientas de tamización en cáncer de cérvix, que detectan la infección causada por VPH. Su aplicación está enfocada en la clasificación de anormalidades citológicas, monitoreo de infecciones persistentes, seguimiento postratamiento de lesiones intraepiteliales de alto grado y vigilancia epidemiológica en salud pública [4-6]. La utilización de la citología y las pruebas de detección de ADN del VPH, aumenta la sensibilidad de la tamización para la detección de cáncer de cérvix y reduce de manera significativa el riesgo de sufrir lesiones cervicales premalignas por un periodo de 5 años [2,7]

Clinical and laboratory profiles of hospitalized children with acute respiratory virus infection / 소아과

PURPOSE: Despite the availability of molecular methods, identification of the causative virus in children with acute respiratory infections (ARIs) has proven difficult as the same viruses are often detected in asymptomatic children. METHODS: Multiplex reverse transcription polymerase chain reaction assays were performed to detect 15 common respiratory viruses in children under 15 years of age who were hospitalized with ARI between January 2013 and December 2015. Viral epidemiology and clinical profiles of single virus infections were evaluated. RESULTS: Of 3,505 patients, viruses were identified in 2,424 (69.1%), with the assay revealing a single virus in 1,747 cases (49.8%). While major pathogens in single virus-positive cases differed according to age, human rhinovirus (hRV) was common in patients of all ages. Respiratory syncytial virus (RSV), influenza virus (IF), and human metapneumovirus (hMPV) were found to be seasonal pathogens, appearing from fall through winter and spring, whereas hRV and adenovirus (AdV) were detected in every season. Patients with ARIs caused by RSV and hRV were frequently afebrile and more commonly had wheezing compared with patients with other viral ARIs. Neutrophil-dominant inflammation was observed in ARIs caused by IF, AdV, and hRV, whereas lymphocyte-dominant inflammation was observed with RSV A, parainfluenza virus, and hMPV. Monocytosis was common with RSV and AdV, whereas eosinophilia was observed with hRV. CONCLUSION: In combination with viral identification, recognition of virus-specific clinical and laboratory patterns will expand our understanding of the epidemiology of viral ARIs and help us to establish more efficient therapeutic and preventive strategies.

Screening of adult Ph-like acute lymphoblastic leukemia by multiplex real-time quantitative PCR / 中华血液学杂志

Objective@#To investigate the feasibility of multiplex real-time RT-PCR with fluorescent probes in early screening of Ph-like acute lymphoblastic leukemia (ALL) and analyze the clinical feature and prognos.@*Method@#A total of 118 adult B-ALL patients diagnosed between October 2010 and March 2016 were enrolled in this study. Multiplex RT-PCR was used to detect the Ph-like ALL related fusion gene and CRLF2 expression in 58 BCR-ABL and MLL rearrangement negative patients. The clinical features, treatment response and prognosis were analyzed in Ph-like fusion gene positive and/or CRLF2 over-expression patients.@*Result@#Among 58 patients, 9 patients (9/58, 15.5%) showed Ph-like ALL related fusion genes positive and 10 patients (10/58, 17.2%) showed CRLF2 over-expression. There were statistical differences in age, WBC count, immunophenotypes, cytogenetics and risk stratification among Ph-like fusion gene positive or CRLF2 over-expression patients, Ph+ patients, MLL+ patients and B-other patients. The 2-year overall survival rates were 65%, 47%, 64% and 74% respectively among these four groups (P=0.043) . The 2-year relapse free survival rates were 51%, 39%, 62% and 70% respectively among these four groups (P=0.010) .@*Conclusion@#Routine screening of Ph-like ALL by multiplex RTPCR is feasible.

Molecular study of astrovirus, adenovirus and norovirus in community acquired diarrhea in children: One Egyptian center study

Objective To determine the prevalence of astrovirus, norovirus, adenovirus in children below five years old with diarrhea by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) along with rotavirus antigen detection by enzyme linked immunosorbant assay. Methods The study was conducted on children below five years old complaining of acute diarrhea. The study included stool examination by molecular method for detection of norovirus, adenovirus and astrovirus by multiplex RT-PCR. Rotavirus antigen was detected in the stool by enzyme linked immunosorbant assay. Results The study included 100 children below 5 years old with acute diarrhea. Multiplex RT-PCR was positive in 34% of the children. The most frequently detected virus was rotavirus (44%), followed by norovirus (30%), adenovirus (20%) and astrovirus (14%). The clinical symptoms were more significantly associated with viral diarrhea such as fever (P = 0.03), bloody diarrhea (P = 0.025), vomiting (P = 0.000 1) and watery diarrheas (P = 0.05). The frequency of diarrhea with viral pathogen was significantly presented in winter season (39.7%). There were significant frequencies of norovirus and adenovirus in age ranging 1–2 years old (P = 0.04, P = 0.01 respectively). Conclusions The present study spotlights on the prevalence of viral pathogens as an important etiology in diarrhea in children below five years old. Astrovirus, norovirus and adenovirus are common along with rotavirus in this group of patients. Multiplex PCR leads to improve the laboratory diagnosis of these viruses along with antigen detection method. Further longitudinal studies are required to evaluate the epidemiological data associated with these viruses and for proper management of such drastic infection.

Clinical detection of seven porcine diarrhea-associated viruses and evolution analysis of porcine kobuvirus / 生物工程学报

In this study, a multiplex RT-PCR method was developed for detection of seven diarrhea-associated porcine viruses, including porcine teschovirus (PTV), porcine sapovirus (PSV), porcine deltacornavirus (PDCoV), porcine kobuvirus (PKV), porcine sapovirus (PSaV), porcine astrovirus (PAstV) and porcine torovirus (PToV). A total of 419 samples were screened by this method and results showed that PKV had the highest positive rate of 26.98%?45.79% and its mixed infection rate reached 9.52%-18.54%. On account of high positive rate of PKV and its important role in diarrhea disease, complete genomic sequences of three PKV positive samples were further sequenced. Three PKV labeled as PD-PKV, JS-PKV and CM-PKV were classified into porcine kobuvirus genus and had far genetic distance with other kobuviruses. The complete genome homologies among them were 88.1%-89.1%. CM-PKV had the highest identity with the Chinese strain JS-02a-CHN/2013 reported in 2013 while JS-PKV and PD-PKV were most closed to the K-30-HUN/2008/HUN strain reported in Hungary in 2008. This illustrates the significant genetic differences of the different PKV isolates in Shanghai while its relationship with the viral pathogenicity still needs to be explored. This research provides references for further understanding the prevalence of PKV and its role in swine diarrhea.

Establishment of a Multiplex RT-PCR for the Sensitive and Differential Detection of Japanese Encephalitis Virus Genotype 1 and 3

Japanese encephalitis (JE) is a zoonosis that affects the nervous system of humans and other animals. The genotype of JE virus (JEV) has shifted recently from genotype 3 (G3) to genotype 1 (G1) in Asia, including Korea. Thus, a rapid differential assay is required to make an accurate diagnosis of JEV genotype. In this study, we designed common and differential primer sets for JEV G1 and G3 to detect the JEV envelope (E) gene. The specific primer sets for JEV G1 and G3 specifically amplified the target gene. The detection limits of the three primer sets were 10(1.0), 10(2.0), and 10(2.0) TCID₅₀/reaction, respectively. No cross-reactivity was detected with non-JEV reference viruses. The multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay specifically differentiated JEV G1 from G3. Thus, a one-step multiplex RT-PCR assay was established to rapidly and differentially detect JEV. This assay will be useful for confirming JEV infections in animals and checking the JEV genotype in veterinary biological products.

Clinical Characteristics of Acute Respiratory Tract Infections in Full-Term Newborns without Risk Factors

PURPOSE: This study evaluated the characteristics and symptoms of full-term newborns without risk factors who were diagnosed with acute lower respiratory tract infections (ALRI). METHODS: Nasopharyngeal aspirates were obtained from 72 full-term newborns to 30 days of age who were diagnosed with ALRI from September 2011 to November 2013 and analyzed by multiplex real time-polymerase chain reaction (RT-PCR). RESULTS: Viruses were detected in 60 newborns (83.3%). Single viruses were observed in 56 newborns (77.7%). The most commonly detected viral agent was respiratory syncytial virus (RSV) (63.8%), followed by parainfluenza virus (6.9%), rhinovirus A/B (4.1%), and human coronavirus (2.7%). Clinical diagnoses of ALRI in newborns with a single virus included pneumonia (66.07%), bronchiolitis (30.43%), bronchitis (5.35 %), and croup (1.79%). There were no differences in epidemiological characteristics between RSV and other viruses. However, newborns diagnosed with RSV had prolonged hospitalizations and significantly increased respiratory rates. CONCLUSION: Respiratory viruses, especially RSV, are pivotal causes of ALRI in newborns. Further, studies on RSV severity and vaccination are necessary to reduce hospitalization and mortality of full-term infants.

A comparison of three methods for detecting respiratory viruses / 中华微生物学和免疫学杂志

Objective To screen a sensitive method for detecting respiratory viruses from three different methods of singleplex conventional PCR , multiplex conventional PCR and multiplex real-time RT-PCR.Methods Parallel examination of 17 respiratory viruses was performed on 73 throat swab specimens collected from patients with upper respiratory tract infection by the three methods .The detection rates of dif-ferent respiratory viruses were used as evaluating indicator for the three methods .Results The numbers of respiratory viruses detected by singleplex conventional PCR , multiplex conventional PCR and multiplex real-time PCR were 56, 41 and 87, respectively.Conclusion The multiplex real-time RT-PCR might be used for the detection of respiratory viruses in laboratory as its high detection rate in comparison with the other two methods .

Detection of rearrangements of mixed lineage leukemia gene in acute leukemia patients / 白血病·淋巴瘤

Objective To explore the value of fluorescence in situ hybridization (FISH) and multiplex RT-PCR in the detection of mixed lineage leukemia (MLL) gene rearrangement in acute leukemia (AL) patients. Methods Dual-color MLL probe, multiplex RT-PCR and R or G banding techniques were used to detect the MLL gene rearrangement in 189 cases of AL.Results MLL gene rearrangements were detected in 9 cases (5.03 ％) by FISH,and 16 cases (8.47 ％) by multiplex RT-PCR,including MLL/AF9,MLL/AF10,MLL/AF6, MLL/AF7, MLL/ELL, MLL/PTD. R or G banding techniques could find 11q23 in 5 out of 189 patients (2.65 ％). There was no statistic difference in the incidence of 6 common MLL gene rearrangements between ALL (73 cases) and AML patients (116 cases) (P ＞ 0.05).Conclusion Multiplex RT-PCR is a powerful technique in the detection of MLL gene rearrangement for tentatively diagnosed AL.It could not only confirm translocation detected by conventional cytogenetic method, but also detect MLL partial tandem duplication which could not been detected by cytogenetic examination or FISH. It plays an important role in guiding therapy and predicting prognosis for AL.

Simultaneous Detection of Three Arboviruses Using a Triplex RT-PCR Enzyme Hybridization Assay / 中国病毒学·英文版

Arboviruses represent a serious problem to public health and agriculture worldwide.Fast,accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation.We developed a cost-effective,rapid,and highly sensitive one-step triplex RT-PCR enzyme hybridizationassay for simultaneous detections of Japanese Encephallitis virus (JEV,Flaviviridae)Getah virus (GETV,Togaviridae),and Tahyna virus (TAHV,Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction.The analytical sensitivity of this assay was 1 PFU/mL for JEV,10PFU/mL for GETV,and 10 PFU/mL for TAHV.This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods.When “triplex RT-PCR enzyme hybridization” was applied to 29 cerebrospinal fluid(CSF)samples that were JEV-positive by normal RT-PCR assay,all samples were strongly positive for JEV,but negative for GETV and TAHV,demonstrating a good sensitivity,specificity,and performance at CSF specimen detection.

Rapid Detection of Prognostically Significant Fusion Transcripts in Acute Leukemia Using Simplified Multiplex Reverse Transcription Polymerase Chain Reaction

Multiplex reverse transcription polymerase chain reaction (mRT-PCR) has recently emerged as an alternative to cytogenetics. We designed and used simplified mRT-PCR system as a molecular screen for acute leukemia. Fifteen fusion transcripts were included: BCR-ABL1, PML-RARA, ZBTB16-RARA, RUNX1-RUNX1T1, CBFB-MYH11, DEK-NUP214, TCF3-PBX1, ETV6-RUNX1, MLL-AFF1, MLL-MLLT4, MLL-MLLT3, MLL-MLLT10, MLL-ELL, MLL-MLLT1, and MLL-MLLT6. A total of 121 diagnostic acute leukemia specimens were studied, comparing the mRT-PCR system with standard cytogenetics. Fifty-six cases (46.3%) had fusion transcripts revealed by our mRT-PCR assay. The concordance rate between mRT-PCR and cytogenetics was 91.7%. However, false negative results were found in three cases who have inv(16), t(4;11) or t(11;19)(q23;p13.1), respectively. Seven cryptic translocations including ETV6-RUNX1, MLL-MLLT3, MLL-MLLT4, and PML-RARA were detected. This mRT-PCR assay is a useful screening tool in acute leukemia because it provides rapid and reliable detection of clinically important chimeric transcripts. In addition, cryptic translocations provide additional genetic information that could be clinically useful.

Genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in Ernakulam, Kerala, South India.

Dengue fever, a mosquito-borne viral infection, causes significant morbidity and has become endemic in the Indian subcontinent. Virus strains currently circulating in many parts of the country are not well studied at the molecular level. In the present study, genetic characterization of virus strains from a dengue outbreak that occurred in and around a tertiary care hospital in Ernakulam, Kerala in the year 2008 has been reported. By reverse transcription polymerase chain reaction (RT-PCR), 37 out of 75 (49.3%) clinically suspected cases were positive for dengue viral RNA. Among these, 21 (56.8%) samples showed concurrent infection with multiple serotypes of the virus. Majority of the combined infections were caused by dengue serotype 2 and 3. Co-infections with type 1 and 2 in two patients, and type 1, 2 and 3 in one patient were also observed. The core-pre-Membrane (CprM) junction nucleotide sequencing and phylogenetic analysis revealed that the type 1 strains were related to the viral strains reported from Delhi-2001 and Gwalior-2002 dengue outbreaks, while the type 2 strains were related to the strains from Gwalior-2001 epidemic. Sequences of type 3 strains did not show clear relation to any of the previous Indian isolates, and in the phylogenetic analysis, they formed a distinct lineage within the Indian type 3 strains. This study indicates hyperendemicity of dengue in the region with the presence of multiple serotypes and high rates of co-infection, and local genomic evolution of the viral strains involved in this outbreak.

Comparison of SD BIOLINE Rapid Influenza Antigen Test Using Two Different Specimens, Nasopharyngeal Swabs and Nasopharyngeal Aspirates / 대한임상미생물학회지

BACKGROUND: The pandemic swine origin influenza A/H1N1 2009 virus (H1N1 2009) was rapidly spread out all over the world after it was first found in April, 2009. This study was made to compare the performance of nasopharyngeal swabs and nasopharyngeal aspirates for the SD Bioline rapid influenza antigen test. METHODS: From Aug to Nov, 2009 the SD Bioline rapid influenza antigen tests were conducted with the nasopharyngeal swabs and the nasopharyngeal aspirates from the 244 specimens of patients who had come to the hospital with influenza-like illness. The data from the examination were compared with the multiplex RT-PCR as a reference standard to obtain sensitivity, specificity, positive predictive value and negative predictive value. RESULTS: The sensitivity and the specificity of the SD Bioline rapid influenza antigen tests with the nasopharyngeal swabs were 75.8%, and 93.3% respectively, and the sensitivity and specificity with the nasopharyngeal aspirates were 61.3%, and 98.3% respectively. CONCLUSION: Even if the nasopharyngeal aspirates showed the lower sensitivity than the nasopharyngeal swabs, since the specificity is higher, the nasopharyngeal aspirates are more useful because we can reduce false positive rate.

Application of multiplex reverse transcription polymeruse chain reaction in acute myeloid leukemia / 白血病·淋巴瘤

Objective To analyse the fusion genes derived from chromosome structural aberrations in acute myeloid leukemia(AML) and the relationship between fusion genes and the MICM classification, clinical diagnosis, chemotherapy and prognosis. Methods The expression of fusion gene in bone marrow samples was detected with multiplex RT-PCR technique and chromosome karyotypes, immunological phenotypes and clinical data were analyzed in 60 acute myeloid leukemia newly diagnosed. Results 37 cases(61.67 %) of 60 patients carried 5 kinds of fusion genes consisting of MLL-AF9, TLS-ERG, CBFβ-MYH1, AML1-ETO and PML-RARα. The activation of oncogene HOX11 was detected in 13 AML cases, three of them with other chromosome aberration simultaneously.23 cases of 31 patients carrying AML1-ETO or PML-RARα, reached complete remission(CR) after chemotherapy and without relapse. Conclusion Gene typing is the most precise classification method that can direct clinical treatment and evaluate prognosis. Multiplex RT-PCR technique, which can quickly screen 29 kinds of fusion gene derived from chromosome structural aberrations at one time, maybe helpful to improve M1CM classification and guide the choice of treatment.

Comparison of Nasopharyngeal Aspirates and Nasal Swabs for the Detection of Respiratory Viruses by Multiplex RT-PCR / 소아알레르기및호흡기학회지

PURPOSE: Although the nasopharyngeal aspirate (NPA) is more commonly used because of relatively higher accuracy, the nasal swab (NS) is a less painful and easier method than NPA. A few recent reports have shown that NS is more effective than NPA for the detection of respiratory virus using immunofluorescence or viral culture. The objective of the present study was to compare the results of NPA and NS sampling specimens in children for respiratory viruses detection using multiplex RT-PCR. METHODS: From December 2008 to June 2009 Paired NPA and NS specimens were collected from 250 children admitted with symptoms of acute respiratory infections at the Department of Pediatrics, Soonchunhyang University Cheonan Hospital, Cheonan, Korea. The sensitivity and agreement of virus detection between NPA and NS using multiplex RT-PCR were compared and analyzed. RESULTS: The median age of the subjects was 1.3 years (range, 20 days to 16.5 years), and 228 patients (91.2%) were under the age of 5 years. The agreement of virus detection between NPA and NS was excellent (Cohen's kappa >0.8) for parainfluenza virus type 3 or substantial (0.6 to 0.8) for rhinovirus A, RSV A and RSV B, moderate (0.4 to 0.6) for adenovirus and metapneumovirus and poor (<0.4) for influenza A. The overall sensitivity of detection of respiratory viruses was significantly higher in NPA (0.96) than in NS (0.59, P<0.05). CONCLUSION: We recommend NPA may be more accurate specimen for detection of respiratory viruses in hospitalized children. NS might be used in limited cases at a office setting or for larger epidemiological studies. However, results obtained from NS for influenza virus type A, metapneumovirus and adenovirus, should be interpreted carefully.

Comparison of Nasopharyngeal Aspirates and Nasal Swabs for the Detection of Respiratory Viruses by Multiplex RT-PCR / 소아알레르기및호흡기학회지

PURPOSE: Although the nasopharyngeal aspirate (NPA) is more commonly used because of relatively higher accuracy, the nasal swab (NS) is a less painful and easier method than NPA. A few recent reports have shown that NS is more effective than NPA for the detection of respiratory virus using immunofluorescence or viral culture. The objective of the present study was to compare the results of NPA and NS sampling specimens in children for respiratory viruses detection using multiplex RT-PCR. METHODS: From December 2008 to June 2009 Paired NPA and NS specimens were collected from 250 children admitted with symptoms of acute respiratory infections at the Department of Pediatrics, Soonchunhyang University Cheonan Hospital, Cheonan, Korea. The sensitivity and agreement of virus detection between NPA and NS using multiplex RT-PCR were compared and analyzed. RESULTS: The median age of the subjects was 1.3 years (range, 20 days to 16.5 years), and 228 patients (91.2%) were under the age of 5 years. The agreement of virus detection between NPA and NS was excellent (Cohen's kappa >0.8) for parainfluenza virus type 3 or substantial (0.6 to 0.8) for rhinovirus A, RSV A and RSV B, moderate (0.4 to 0.6) for adenovirus and metapneumovirus and poor (<0.4) for influenza A. The overall sensitivity of detection of respiratory viruses was significantly higher in NPA (0.96) than in NS (0.59, P<0.05). CONCLUSION: We recommend NPA may be more accurate specimen for detection of respiratory viruses in hospitalized children. NS might be used in limited cases at a office setting or for larger epidemiological studies. However, results obtained from NS for influenza virus type A, metapneumovirus and adenovirus, should be interpreted carefully.

Characterization of Respiratory Viral Infection in Children in Gwangju

BACKGROUND: This investigation was to perform the epidemiological surveillance and genetic analysis on respiratory viral agents from children with acute respiratory infections in Gwangju. MATERIALS AND METHODS: For this study, 3,695 specimens obtained from patients with acute respiratory infections were collected by collaboration with pediatric hospitals in Gwangju between 2005 and 2007. Specimens were screened for 8 respiratory viruses including influenza viruses (IFV), human rhinoviruses (HRV), human coronaviruses (HCoV), adenoviruses (ADV), parainfluenza viruses (PIV), human enteroviruses (HEV), respiratory synthitial viruses (RSV) and human bocaviruses (HBoV). Respiratory viruses were detected using multiplex (RT) PCR with viral specific primers. RESULTS: Out of 3,695 specimens, the ratio of virus detection was 24.9% (919). Overall, HRV (35.5%) and IFV (34.9%) were the most common viruses found, followed by HBoV (14.8%), HCoV (10.6%), RSV (3.7%), ADV (3.4%), PIV (3.2%) and HEV (3.0%). In addition, multiple infections were detected in 80 patients (8.7%). When the prevalence was analyzed according to season, HBoV, IFV and HCoV showed two epidemic points in late spring and early winter. ADV, HRV, RSV PIV and HEV, however, were all found to have only one epidemic point, with RSV being most common during winter and the others being most prominent during spring. CONCLUSIONS: Through this epidemiological surveillance, the respiratory viruses prevalent in children in Gwangju area were investigated. We strongly recommend the development of nationwide policy for the management of prevalent respiratory virus that includes long term collection of data and samples, vaccine development and prevention education of the misuse of antibiotics.

Characterization of Respiratory Viral Infection in Children in Gwangju

BACKGROUND: This investigation was to perform the epidemiological surveillance and genetic analysis on respiratory viral agents from children with acute respiratory infections in Gwangju. MATERIALS AND METHODS: For this study, 3,695 specimens obtained from patients with acute respiratory infections were collected by collaboration with pediatric hospitals in Gwangju between 2005 and 2007. Specimens were screened for 8 respiratory viruses including influenza viruses (IFV), human rhinoviruses (HRV), human coronaviruses (HCoV), adenoviruses (ADV), parainfluenza viruses (PIV), human enteroviruses (HEV), respiratory synthitial viruses (RSV) and human bocaviruses (HBoV). Respiratory viruses were detected using multiplex (RT) PCR with viral specific primers. RESULTS: Out of 3,695 specimens, the ratio of virus detection was 24.9% (919). Overall, HRV (35.5%) and IFV (34.9%) were the most common viruses found, followed by HBoV (14.8%), HCoV (10.6%), RSV (3.7%), ADV (3.4%), PIV (3.2%) and HEV (3.0%). In addition, multiple infections were detected in 80 patients (8.7%). When the prevalence was analyzed according to season, HBoV, IFV and HCoV showed two epidemic points in late spring and early winter. ADV, HRV, RSV PIV and HEV, however, were all found to have only one epidemic point, with RSV being most common during winter and the others being most prominent during spring. CONCLUSIONS: Through this epidemiological surveillance, the respiratory viruses prevalent in children in Gwangju area were investigated. We strongly recommend the development of nationwide policy for the management of prevalent respiratory virus that includes long term collection of data and samples, vaccine development and prevention education of the misuse of antibiotics.

Studies on the Development of Viral Detection Markers for the Quality Control of Blood

According to the serological screening methods of antigen-antibody reaction such as ELISA, it has been known that the complete detection of viral infections of HBV, HCV, and HIV-1 viruses in the blood and blood related-products is not much reliable. Therefore, nucleic acid amplification testing methods (NAT) adopted to detect the small quantitative viral nucleic acids could support the basis of using and supplying the blood and its related products safely. This research work is basically designed to describe the simultaneous blood screening system by multiplex or duplex tests for detection of HBV, HCV, and HIV-1 viruses in the blood at one time with low price and labor. It is aimed at easy detection by using the conventional agarose gel electrophoresis. Thus, we tried to detect and identify the viral components in the blood sample according to their different size of PCR products. We decided a set of consensus sequences to recognize each viral DNA fragments after running the multiplex PCR in one tube. This was done by nested RT-PCR using two different RNA viral genomic templates followed by multiplex PCR with addition of viral DNA and their primers after purifying the viral genomic nucleic acids. Those specific primers could be used without any interference to amplify each viral genome in the blood samples. The sensitivities with different viral loads were evaluated on the agarose gel electrophoresis. Three different viral agents in the blood samples could be tested by this multiplex (RT)-PCR with three different primers.

Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Chromosomal Translocation in Hematologic Malignancies / 华中科技大学学报（医学）（英德文版）

Multiplex reverse transcription-polymerase chain reaction (M-RT-PCR) has been proved to possess great clinical potential for simultaneous screening of 29 chromosomal translocations in acute leukemia. To evaluate the clinical value of M-RT-PCR in hematologic malignancies, bone marrow samples from 90 patients with various hematologic malignancies, including 25 acute myeloge nous leukemia (AML), 22 acute lymphoblastic leukemia (ALL), 27 chronic myelogenous leukemia (CML), 4 myeloproliferative diseases (MPD), 3 chronic lymphoblastic leukemia (CLL), 3 and 1 malignant histocytosis (MH) were subjected to both M-RT-PCR and chromosome karyotypic analysis. Some of cases were subjected to follow-up examination of M-RT-PCR during the period of ukemia. In our hand, 12 of 29chromosomal translocation transcripts including TEL/PDGFR, DEK/CAN, MLL/AF6, AML1/ETO,F9, BCR/ABL, MLL/MLL, PML/RARα, TLS/ERG, E2A/HLF, EVIl and HOXI1 were detected in 57 cases (63.3 %) of the 90 samples, which were in consistence with the results of karyore, M-RT-PCR had also shown good clinical relevance when used as an approach to detect minimal residual leukemia. We concluded that M-RT-PCR could be used as an effiy in the initial diagnosis of hematologic malignancies but also in subsequent monitor of minimal residual leukemia.