We attempted to detect and identify virus types quickly by improving an RT-PCR-based dot-blot hybridization test for echoviruses, important human pathogens mainly causing aseptic meningitis. This test was applied to reference viruses of seven echovirus serotypes prevalent in Korea (E6, 7, 9, 11, 13, 25, and 30) and seventy isolates of echovirus isolated in Korea between 2002 and 2004. The primers for target DNA and hybridization probes (25mer, 50mer, and 70mer) were designed within the VP1 region of the echovirus. In RT-PCR, a nonradioactive digoxigenin-DNA labeling mix was added instead of dNTP to initiate PCR. The PCR product was then hybridized against 25mer, 50mer, and 70mer probe DNA spotted on nylon membranes and the reaction was observed. To investigate the optimal conditions for hybridization, various concentrations of target DNA (0.1, 1, 10, and 100 ng/micron l), size of probe DNA (25mer, 50mer, and 70mer), concentrations of probe DNA (10~50 pM), and reaction time were included. In the test zone, the optimal condition in terms of time and cost was a reaction time of 1 h with 10 ng/micron l target DNA concentration and 10 pM of a 50mer probe. We found 100% diagnosis of the serotypes for seven reference echoviruses and 90% (63/70) sensitivity for clinical isolates. Also, tests with this probe for reactivity with seven reference echoviruses by using DNA chips showed that diagnostic identification was possible without other serotype cross-reactivity. Therefore, efficiency analysis of probe and target DNA on clinical specimens by using dot-blot analysis indicated that this system can be applied to the prestages of the DNA chip and that the dot blot analysis itself can be used in applications to develop a tool for diagnosing specific viral serotypes.
Diagnosis ; DNA ; Enterovirus B, Human* ; Humans ; Korea ; Membranes ; Meningitis, Aseptic ; Nylons ; Oligonucleotide Array Sequence Analysis ; Polymerase Chain Reaction ; Reaction Time

Human cytomegalovirus (HCMV) is a common human pathogen that causes morbidity and mortality in hematopoietic stem cell transplantation (HSCT) recipients. Early diagnosis of HCMV infection or reactivation, and setting threshold values for effective pre-emptive therapies, are required for appropriate HCMV disease prevention in HSCT recipients. We compared the HCMV infections detected by the two methods, LightCycler-based PCR (LC PCR) and in-house immediate early protein PCR (in-house IE PCR) with the results of a pp65 antigenemia assay as the reference. The sensitivity and specificity for the in-house IE PCR were 79.3% and 72.7%, respectively, and 82.9% and 40.7%, respectively, for the LC PCR. The correlation between the HCMV viral load and pp65 antigenemia in HSCT recipients was r=0.603 with in-house IE PCR and r=0.525 with LC PCR. The discordant results between methods and relatively low (r) values suggest that we need more study to set threshold values according to the using methods with clinical outcome.
Cytomegalovirus ; Early Diagnosis ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Viral Load

Human cytomegalovirus (HCMV) is a common human pathogen that causes morbidity and mortality in hematopoietic stem cell transplantation (HSCT) recipients. Early diagnosis of HCMV infection or reactivation, and setting threshold values for effective pre-emptive therapies, are required for appropriate HCMV disease prevention in HSCT recipients. We compared the HCMV infections detected by the two methods, LightCycler-based PCR (LC PCR) and in-house immediate early protein PCR (in-house IE PCR) with the results of a pp65 antigenemia assay as the reference. The sensitivity and specificity for the in-house IE PCR were 79.3% and 72.7%, respectively, and 82.9% and 40.7%, respectively, for the LC PCR. The correlation between the HCMV viral load and pp65 antigenemia in HSCT recipients was r=0.603 with in-house IE PCR and r=0.525 with LC PCR. The discordant results between methods and relatively low (r) values suggest that we need more study to set threshold values according to the using methods with clinical outcome.
Cytomegalovirus ; Early Diagnosis ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; Humans ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Sensitivity and Specificity ; Viral Load

BACKGROUND: Host genetic polymorphisms in the HIV-1 co-receptor CCR5 and CCR2b and SDF-1, ligand for co-receptor CXCR4, have been known to be associated with the resistance of HIV infection and/or the delayed disease progression in HIV-infected patients. METHODS: We examined the frequencies of SDF1-3'A and CCR2b-64I alleles of 354 Koreans including 100 HIV-uninfected persons, 13 discordant spouses of HIV-infected persons, and 241 HIV-infected persons. The genotyping assays of SDF1 and CCR2b genes were carried out by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: The frequencies of CCR2b-64I and SDF1-3'A alleles in Koreans were very high compared with Caucasians and blacks. Observed frequencies of CCR2b-64I and SDF1-3'A allelic variants were 25.1% and 28.7%, respectively. The frequency of the CCR2b-64I allele in Koreans was 2~4 times higher than those of other ethnic groups with the exception of Asian. The frequencies of CCR2b-64I and SDF1- 3'A genotypes did not show the significant difference between HIV-infected and uninfected Koreans. However, the prevalence of CCR2b-64I genotype of the LTNP group was about two times higher than that of the remainder group (P < 0.05). Four (45%) out of 9 LTNPs (long-term nonprogressors) showed having the SDF1-3'A allele and 7 (78%) out of 9 LTNPs carried the CCR2b-64I allele. 3 (33%) out of 9 LTNPs had both SDF1-3'A and CCR2b-64I alleles. But none of 5 RPs (rapid progressors) appeared to have both SDF1-3'A and CCR2b-64I alleles. CONCLUSION: The different genetic backgrounds in study populations may affect the disease progression and the AIDS epidemic in each country. Further studies need to define whether high frequencies of CCR2b-64I and SDF1-3'A allelic variants may affect the HIV disease progression.
African Continental Ancestry Group ; Alleles ; Asian Continental Ancestry Group ; Chemokine CXCL12* ; Disease Progression ; Ethnic Groups ; Genotype ; HIV Infections* ; HIV* ; HIV-1 ; Humans ; Polymorphism, Genetic ; Prevalence ; Spouses

BACKGROUND: The global effect of HIV infection on the host cell gene expression profiles in healthy HIV-infected patients, as long-term non-progressors, remains largely unknown. To identify the cellular genes related with HIV infection and delayed disease progression in vivo, the host gene expression profiles between healthy HIV-infected Koreans and AIDS patients were investigated. METHODS:Differential expression gene analysis was performed via oligonucleotide microarray with using Magic-oligo 10K chip. Ten HIV-uninfected persons and 10 HIV-infected patients (healthy HIV-infected patients vs. AIDS patients. respectively) were studied. RESULTS: Only 10.8% (1,097 genes) of the total genes, that is, 331 up-regulated genes and 766 down- regulated genes were differentially expressed with more than a two-fold change in the HIV-infected persons as compared to those of the HIV-uninfected persons. Especially, 97 genes (8.8%) among 1,097 genes were commonly up- or down-regulated in both the healthy HIV-infected patients and the AIDS patients. 187 genes were differently expressed on the gene expression analysis between the healthy HIV-infected patients and the AIDS patients. Twenty-eight genes out of them showed very significant differences with a P value <0.01. Especially, tripartite motif (TRIM) 14 protein and interferon gamma receptor 2 were dramatically up-regulated in healthy HIV-infected patients, while death-associated protein, DNA directed RNA polymerase II polypeptide A and STAT were over-expressed in AIDS patients. CONCLUSION: Although this microarray study has some limitations, the above results will be helpful for performing more detailed, future functional studies on the differentially expressed genes related to HIV infection and delayed disease progression in vivo.
Disease Progression ; DNA-Directed RNA Polymerases ; Gene Expression ; HIV Infections ; Humans ; Interferons ; Oligonucleotide Array Sequence Analysis ; Transcriptome

Human immunodeficiency virus type 1 (HIV-1) virus causes severe defect in the immune system and affects the host cell gene expression profoundly. The gene expression pattern will be characterized by changes in cellular mRNA levels that are dependent on both the stage of infection and the biological state of the infected cells. The expression levels of 7,404 cellular RNA transcripts were assessed in H9 cells at different time points after HIV-1 IIIB infection. In total 7 time-points, 959/7,404 (13%) genes were a 2-fold or greater expressed. 387 of 959 genes (40.4%) were up-regulated, and other 572 genes (59.6%) were down-regulated. Three hundred seventeen genes were up-regulated a 2-fold or greater at 72 hr postinfection and 2 to 139 genes were up-regulated at the other time-points. In contrast, 126 to 349 genes were down-regulated a 2-fold or greater in all time-points, excepting 6 hr postinfection. Twenty-three genes were up-regulated a 2-fold or greater over at least four of seven time-points, which were mostly ribosomal proteins and MHCs. Especially, MHCs including HLA-DRA were steadily up-regulated from 24 hr postinfection. Thirty genes were down-regulated a 2-fold or greater in all the time-points, which were mainly related with synthesis and metabolism. These results show that host cell gene expression was altered by HIV-1 infection according to time-points and will provide a framework for studies on interactions between host and HIV-1 infection.
Gene Expression ; HIV-1 ; HLA-DR alpha-Chains ; Immune System ; Metabolism ; Oligonucleotide Array Sequence Analysis ; Ribosomal Proteins ; RNA ; RNA, Messenger

The human immunodeficiency viruses (HIV) exhibit tremendous genetic variability in their hosts. It is mainly due to two factors: the error-prone nature of the viral reverse transcriptase enzyme and the effects of environmental constraints such as antiviral therapy, cellular tropism, or HIV-specific immune responses. These quasispecies show fluctuation both in the overall divergence and diversity between individual sequences with different duration after primary infection. For better understand the viral quasispecies, we have performed the longitudinal genetic analysis of HIV-1 env gene V1-C5 region (1.2 kb) by two molecular cloning methods. Diversity indicated that 'single clone per PCR' value was higher than that of 'multiple clones per PCR' in subjects: 0.58-3.15 in subject 1 (P<0.05) and 0.28-2.25 in subject 2 (P<0.05). But divergence was similar in both molecular cloning methods. Phylogenetic analysis of longitudinal sequences at different sampling stages revealed the existence of different topologies individually. These data suggested that 'single clone per PCR' is more efficient than 'multiple clones per PCR' in genetic diversity analysis.
Clone Cells* ; Cloning, Molecular ; Genes, env* ; Genetic Variation ; HIV ; HIV-1* ; Polymerase Chain Reaction* ; RNA-Directed DNA Polymerase ; Tropism

BACKGROUND: In spite of active HIV/AIDS control and managements, UNAIDS estimate that 40 million people were living worldwide with HIV at the end of 2001. In Korea, The member of HIV- infected adults are continuously growing. For improvement of HIV screening and prevention, we analyzed over times the relationship between the changes in initial CD4+ T cell counts of newly HIV- diagnosed adults, sex, and exposure route. METHODS: We selected 1011 newly HIV-diagnosed adults whose initial CD4+ T cell count was determined within 6 months of HIV diagnosis between 1990 and June, 2002. Based on CD4+ T cell counts, the selected people were grouped into 4 as follows: <200 cells/mm3, 200-349 cells/mm3, 350-699 cells/mm3, and >700 cells/mm3. The relationship between initial CD4+ T cell counts, age, sex, and HIV risk category were studied by regression statistic methods. RESULTS: The median initial CD4+ T cell counts decreased over times (P<0.001). In each major group, over 50% of initial CD4+ T cell counts were below 350 cells/mm3. For homosexually infected adults, the median age did not statistically increase (P=0.062). However, in heterosexually infected adults, the median age increased throughout the time period examined (P<0.001) with an exception of female group (P=0.427). The multi-regression analyses revealed that older age (P<0.001) and male sex (P<0.001) were independently associated with lower initial CD4+ T cell counts, but not exposure group (P=0.483). For each year cohort of newly diagnosed adults, the median initial CD4+ T cell counts in subsequent years decreased until 1998 and then increased thereafter. CONCLUSION: These results show that a large proportion of HIV-infected adults are being diagnosed late in the course of HIV infection, particularly heterosexually infected male group. Therefore, we should continuously enforce screening, prevention and prompt diagnosis of high risk groups.
Adult ; Cell Count* ; Cohort Studies ; Diagnosis ; Female ; HIV ; HIV Infections ; Homosexuality ; Humans ; Korea ; Male ; Mass Screening

BACKGROUND: In spite of active HIV/AIDS control and managements, UNAIDS estimate that 40 million people were living worldwide with HIV at the end of 2001. In Korea, The member of HIV- infected adults are continuously growing. For improvement of HIV screening and prevention, we analyzed over times the relationship between the changes in initial CD4+ T cell counts of newly HIV- diagnosed adults, sex, and exposure route. METHODS: We selected 1011 newly HIV-diagnosed adults whose initial CD4+ T cell count was determined within 6 months of HIV diagnosis between 1990 and June, 2002. Based on CD4+ T cell counts, the selected people were grouped into 4 as follows: <200 cells/mm3, 200-349 cells/mm3, 350-699 cells/mm3, and >700 cells/mm3. The relationship between initial CD4+ T cell counts, age, sex, and HIV risk category were studied by regression statistic methods. RESULTS: The median initial CD4+ T cell counts decreased over times (P<0.001). In each major group, over 50% of initial CD4+ T cell counts were below 350 cells/mm3. For homosexually infected adults, the median age did not statistically increase (P=0.062). However, in heterosexually infected adults, the median age increased throughout the time period examined (P<0.001) with an exception of female group (P=0.427). The multi-regression analyses revealed that older age (P<0.001) and male sex (P<0.001) were independently associated with lower initial CD4+ T cell counts, but not exposure group (P=0.483). For each year cohort of newly diagnosed adults, the median initial CD4+ T cell counts in subsequent years decreased until 1998 and then increased thereafter. CONCLUSION: These results show that a large proportion of HIV-infected adults are being diagnosed late in the course of HIV infection, particularly heterosexually infected male group. Therefore, we should continuously enforce screening, prevention and prompt diagnosis of high risk groups.
Adult ; Cell Count* ; Cohort Studies ; Diagnosis ; Female ; HIV ; HIV Infections ; Homosexuality ; Humans ; Korea ; Male ; Mass Screening

PURPOSE: Allergic diseases are triggered by Th2-mediated immune reactions to allergens and orchestrated by various immunological factors, including immune cells and cytokines. Although many reports have suggested that childhood is the critical period in the onset of allergic diseases and aging leads to alter the susceptibility of an individual to allergic diseases, age-related changes in various immunological factors in healthy individuals as well as their difference between healthy and allergic children have not yet been established. METHODS: We investigated the ratio of Th1/Th2 cells and the levels of 22 allergy-related cytokines across all age groups in individuals who were classified as clinically non-atopic and healthy. We also examined their differences between healthy and allergic children to evaluate immunological changes induced by the development of allergic diseases during childhood. RESULTS: The Th1/Th2 ratio rose gradually during the growth period including childhood, reaching peak values in the twenties-thirties age group. Th1/Th2 ratios were significantly lower in allergic children than in healthy controls, whereas 14 of 22 cytokines were significantly higher in allergic children than in healthy controls. On the other hand, there were no differences in Th1/Th2 ratios and cytokines between healthy and allergic adolescents. CONCLUSIONS: In this study, age-related changes in Th1/Th2 ratios were found in normal controls across all age groups, and decreases in Th1/Th2 ratio were observed with increasing of 14 cytokines in allergic children. The results of this study may be helpful as reference values for both monitoring immunological changes according to aging in healthy individuals and distinguishing between normal and allergic subjects in terms of immune cells and soluble factors.
Adolescent ; Aging ; Allergens ; Child ; Critical Period (Psychology) ; Cytokines ; Hand ; Humans ; Hypersensitivity ; Immunologic Factors* ; Reference Values