Adolescent ; Adult ; Antigens, Viral ; blood ; Cytomegalovirus ; isolation & purification ; DNA, Viral ; analysis ; Female ; Fluorescence ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Middle Aged ; Phosphoproteins ; blood ; Polymerase Chain Reaction ; methods ; Viral Load ; Viral Matrix Proteins ; blood

OBJECTIVETo study the molecular epidemiological characteristics of hantavirus seen during 2000-2003 in Qingdao region of Shandong province.

METHODSSera were collected from 64 patients with hemorrhagic fever with renal syndrome (HFRS) and viral RNA was extracted from the sera. HTN and SEO universal primers were designed as outer primers and HTN and SEO specific primers as inner primers. G1 gene region of M segment from hantavirus was amplified by using RT-nest-PCR for sequencing. The data of nucleotide sequences were analyzed by DNA star software.

RESULTSSix cases were positive by HTN specific primer of total cases (9%); 25 of 64 cases by SEO specific primer (39%); total positive rate was 48%. In general, SEO type was a prevalent type of hantavirus in Qingdao region. The variation of the nucleotide sequences among SEO viruses (nucleotide sequence divergence ranged from 0.3% approximately 8.9%) was lower than that among HTN type (nucleotide sequence divergence ranged from 2.6% approximately 11.2% ).

CONCLUSIONMajority of hantavirus found in Qingdao region belonged to SEO type and still a few strains belonged to HTN type. Most of the HTN viruses were detected in Jiaonan county.

China ; Genotype ; Hantavirus ; genetics ; Hemorrhagic Fever with Renal Syndrome ; blood ; virology ; Humans ; RNA, Viral ; blood ; genetics ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA ; Viral Matrix Proteins ; genetics

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Cytomegalovirus/genetics/*isolation & purification ; Cytomegalovirus Infections/*diagnosis/virology ; DNA, Viral/blood ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Phosphoproteins/analysis ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; Reproducibility of Results ; Sensitivity and Specificity ; Viral Load/*methods ; Viral Matrix Proteins/analysis/blood

BACKGROUND: Rapid and accurate laboratory tests are essential to detect cytomegalovirus (CMV) infections in solid organs and haematopoietic stem cell transplant recipients. We assessed the realtime quantitative PCR (RQ-PCR) technology for its usefulness in detecting CMV DNA. METHODS: We evaluated the analytical performance of CMV RQ-PCR using Real-Q Cytomegalovirus Quantification kit (BioSewoom Inc., Korea). To evaluate its clinical utility, we also compared it to pp65 antigenemia test, an immunostaining method, on 343 samples of total 84 patients, including 63 transplant recipients. RESULTS: The detection limit of RQ-PCR was 63 copies/mL and none of hepatitis B virus, hepatitis C virus, or human immunodeficiency virus showed a cross-reactivity with CMV. Total coefficient of variation (CV) was 10.4-19.5%. It detected CMV DNA in a linear range from 1 x 10(2) to 5 x 10(11) copies/mL (P<10(-13), R2=0.9994). The qualitative positive rates of pp65 antigenemia test and RQ-PCR were 4.7%, 16.3%, respectively and concordance rate between the two tests was 84.8% (K=0.221, P<10(-6)). In comparison of quantitative results, the correlation between two tests was significant (r=0.45, P<10(-17)). In comparison among three groups by pp65 antigen level, CMV DNA level obtained with RQ-PCR increased significantly (P<10(-3) and P<10(-7), respectively). CONCLUSIONS: The RQ-PCR is easier to perform than the immunostaining method, has good analytical performance and reflects the blood level of viral DNA well. It may be a new method substituting the pp65 antigenemia test. Further studies determining RQ-PCR value starting pre-emptive therapy will be required.
Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Cytomegalovirus/genetics/*isolation & purification ; Cytomegalovirus Infections/*diagnosis/virology ; DNA, Viral/blood ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Phosphoproteins/analysis ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; Reproducibility of Results ; Sensitivity and Specificity ; Viral Load/*methods ; Viral Matrix Proteins/analysis/blood

Successful preemptive therapy for cytomegalovirus (CMV) infection in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infection. Although the pp65 antigenemia assay has been widely used for this purpose, real-time quantification of CMV DNA has recently been recognized as an alternative diagnostic approach. However, the guidelines for antiviral therapy based on real-time quantitative polymerase chain reaction (RQ-PCR) have yet to be established. From November 2004 to March 2005, a total of 555 whole blood samples from 131 hematopoietic stem cell transplant (HSCT) recipients were prospectively collected. RQ-PCR was conducted using an Artus(R) CMV LC PCR kit (QIAGEN). Both qualitative and quantitative correlations were drawn between the two methods. Exposure to the antiviral agent influenced the results of the two assays. Additionally, the discrepancy was observed at low levels of antigenemia and CMV DNA load. Via ROC curve analysis, the tentative cutoff value for preemptive therapy was determined to be approximately 2x10(4) copies/mL (sensitivity, 80.0%; specificity, 50.0%) in the high risk patients, and approximately 3x10(4) copies/mL (sensitivity, 90.0%; specificity, 70.0%) in the patients at low risk for CMV disease. Further study to validate the optimal cutoff value for the initiation of preemptive therapy is currently underway.
Adolescent ; Adult ; Child ; Child, Preschool ; Cytomegalovirus/genetics/*isolation & purification ; Cytomegalovirus Infections/*diagnosis/therapy ; DNA, Viral/*blood ; Female ; *Hematopoietic Stem Cell Transplantation ; Humans ; Infant ; Male ; Middle Aged ; Phosphoproteins/analysis/immunology ; Polymerase Chain Reaction/*methods ; ROC Curve ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Viral Matrix Proteins/analysis/immunology