Estudio comparativo entre dos técnicas comerciales para la cuantificación de carga viral plasmática en pacientes infectados por el virus de inmunodeficiencia humana tipo 1 / Comparative study of two comercial technical for quantification of viral load in patients infected with Human Immunodeficiency Virus Type 1

Los ensayos de cuantificación de ARN plasmáticos de VIH-1 son importantes para el control de pacientes infectados, así como el monitoreo de la respuesta a la terapia antirretroviral. Por lo tanto, los ensayos comerciales empleados para este propósito deben presentar buena correlación entre si, para dar lugar al manejo terapéutico apropiado. El objetivo del estudio consistió en correlacionar los resultados obtenidos mediante el ensayo de amplificación de señal (bDNA) y PCR en tiempo real (RT-PCR), ambas casas comerciales aprobadas por la FDA y con diferente diana de detección del VIH-1. La validación se realizó con 180 muestras clínicas de pacientes referidos al INHRR. Los resultados fueron comparados con la subpoblación de linfocitos TCD4+ determinados mediante citometría de flujo. El análisis estadístico se realizó empleando el coeficiente de regresión lineal de Pearson (R2) y el valor de contraste de hipótesis con una significancia del 95 %, usando el programa SPSS Statistics v10.0. Se observó una buena correlación entre los ensayos (R2=0.961, p<0.05), siendo la RTPCR más sensible. Las diferencias cuantitativas de carga viral entre las técnicas ensayadas fue menor de 0.5 log10 copias/ml para el 89% de las muestras, y >1 log10 copias/ml solo en dos pacientes, no indicando necesariamente cambio terapéutico. Adicionalmente, se encontró una correlación inversa entre los linfocitos TCD4+ y carga viral del VIH-1 medida por bDNA (R2= 0.20, p<0.05) y RT-PCR (R2= 0.15, p<0.05). Los ensayos evaluados mostraron que ambas técnicas puedes ser empleadas indistintamente para el control de los pacientes VIH positivo.

The assay for quantification of plasma HIV-1 RNA are important for the control of patients infected, as well as the monitoring of the response to antiretroviral therapy. Therefore, the commercial assays used for this purpose must submit good correlation between to give place to the appropriate therapeutic management. In this study, we correlate the results obtained through the testing of signal amplification (bDNA) and real-time PCR (RT-PCR), two comercial technical approved by the FDA and with different targets of detection HIV-1. The validation was carried out with 180 clinical samples of patients referred to the INHRR. The results were compared with the subpopulation of lymphocytes TCD4+ determined by flow cytometry. The statistical analysis was performed using the program SPSS Statistics v10. It was observed good correlation between the tests studied (R2=0.961, p<0.05), with RT-PCR more sensitive. The quantitative differences in viral load between the techniques tested was less than 0.5 log10 copies/ml for the 89% of the samples, and >1 log10 copies/ml in only two patients. Additionally, it was found an inverse correlation between lymphocytes TCD4+ and viral load of HIV-1, measured by bDNA (R2= 0.20, p<0.05) and RT-PCR (R2= 0.15, p<0.05). Therefore, these assays can be employed for the patient control HIV.

Comparison of the reverse transcription-PCR with the branched DNA assay for measurement of human immunodeficiency virus type 1 RNA levels in plasma of Korean patients

Viral load testing of human immunodeficiency virus (HIV) is an essential tool for initiating and monitoring the antiretroviral therapy for HIV patients. To this end, several methods including polymerase chain reaction (PCR), branched DNA (bDNA), nucleic acid sequence based amplification assay (NASBA) and internally controlled virion PCR (ICV PCR) have become available. Of these methods, the standard reverse transcription-PCR (RT-PCR) assay has been widely used in Korea. However, no comparison study has been performed among the various detection methods currently used in Korean patients. We evaluated the correlation and agreement between the PCR and the branched DNA (bDNA) assay for measurement of HIV RNA in Korean patients. Eighty randomly selected samples from HIV-1-seropositive patients visiting Yonsei Medical Center Severance Hospital were studied. We found that these assays show good agreement, have a reliable correlation (r = 0.92, mean difference in log10 copies/ mL +/- 2 standard deviation = 0.098 +/- 0.805) and produce values whose relationship is given by the following equation: log10v3 bDNA = -0.3405 + 1.0601 x log10RT-PCR. Thus, we conclude that these two methods may allow direct comparison of the results obtained from different assay systems.

The observation of therapeutic effect of interferon-? on adult patients with HBV associated glomerulonephritis / 中国实用内科杂志

Objective To observe the therapeutic effect of interferon-? on adult patients with HBV associated glomerulonephritis.Methods Nine patients with HBV associated glomerulonephritis diagnosed in Shanghai Ruijin Hospital from September 1996 to March 2003 were treated with interferon-? at a dose of 3 million units every other day and the total period was 6 to 9 months.The proteinuria,serum HBV marks and bDNA were measured before and after therapy.Results At the end of the therapy urinary protein excretion significantly reduced(P

Comparison of Hybrid Capture System, Hybrid Capture II and Quantiplex HBV DNA Assay for Quantitation of Hepatitis B Virus DNA / 대한임상병리학회지

BACKGROUND: Quantitative measurement of hepatitis B virus (HBV) DNA in serum allows monitoring of HBV replication and assessing responses to antiviral treatment, which is not possible with serological markers for HBV infection. However, most assays developed for the quantitative measurement of HBV DNA have not been standardized. Therefore, we tried to compare the performance of three commercial quantitative methods for the measurement of HBV DNA. METHODS: One hundred consecutive sera with request for HBV DNA quantitation were tested with Hybrid Capture System (HCS), Hybrid Capture II (HC-II) and Quantiplex HBV DNA Assay (bDNA Assay) to evaluate the detection rate, the concordance rate and the correlation of the quantitative results measured by each method. In addition, nested polymerase chain reaction (PCR) was performed for qualitative detection of HBV DNA. RESULTs: Concordance rate was 87% for all three methods, 91% for HCS and HC-II, 94% for HCS and bDNA Assay, and 89% for HC-II and bDNA Assay. HBV DNA quantities measured by three methods showed significant correlation between HCS and HC-II (R=0.88, P<0.0001), HCS and bDNA Assay (R=0.82, P<0.0001), and HC-II and bDNA Assay (R=0.95, P<0.0001). Thirteen sera of discrepant results and 29 of 39 sera of negative results by all three methods showed PCR positivity. CONCLUSIONS: Three quantitative methods for the measurement of HBV DNA showed relatively high concordance rate and good correlation. However, the results by bDNA Assay increased more rapidly than HCS and HC-II as the amounts of HBV DNA in the sample increased that the concentrations of HBV DNA measured by bDNA Assay were two or three times higher than those measured by HCS or HC-II at high HBV DNA concentration range. Thus, further studies are necessary to develop more standardized methods.

HCV Serotypes and HCV RNA Quantitation / 대한임상병리학회지

BACKGROUND: There are several serological subtypes (serotypes) of hepatitis C virus (HCV) which can be detected by a serological enzyme immunoassay (EIA) method which detects the HCV subtype-specific antibodies against the NS4 region of the virus. We determined the HCV serotypes chronic HCV infected patients in terms of clinical applications. METHODS: Subtypes based on Simmonds' classification were detected in chronic HCV patients serologically and viral loads quantitated with branched DNA (bDNA) assay. The EIA was compared with multiplex PCR method. RESULTS: The serotype 1 (Simmonds' classification, 1a, 1b, 1c), the most prevalent form in Korea followed by serotype 2 (Simmonds' classification, 2a, 2b, 2c). The distribution of serotypes and HCV viral loads were not different according to disease severity. The patients infected with serotype 1 had higher viremic level (serotype 1, 7.4+/-15.5 MEq/mL; serotype 2, 1.1+/-2.2 MEq/mL, P=0.017). The serotype matched with the genotype in 85.7% (18/21) of cases. CONCLUSIONS: HCV serotype 1 is the most prevalent form in Korea and seemed to be able to more actively replicate than serotype 2. Both multiplex PCR and EIA can be used for detecting HCV subtypes and mutually interpretable.

Quantification of Serum Hepatitis C Virus in Patients with Chronic C Viral Liver Disease / 대한임상병리학회지

BACKGROUND: The quantification of hepatitis C virus (HCV) is useful in diagnosis and monitoring of HCV infection. We evaluated clinical usefulness of HCV quantification and two quantification methods using different assay principles. METHODS: HCV RNA quantities and liver function were measured in patients with different disease severity using bDNA assay (QuantiplexTM, Chiron, USA). HCV RNA loads were quantified at the time of pre/post-interferon treatment in some of them using RT-PCR hybridization assay (AMPLICORTM, Roche, USA). These two quantification methods were also compared. RESULTS: HCV RNA loads showed no significant difference according to disease severity (group I, 3.8 5.3 MEq/mL; group II, 3.8 7.4 MEq/mL; group III, 5.9 13.0 MEq/mL; P=0.181) or interferon response (complete responders, 1.5 105/mL; partial or non responders, 2.2 105/mL; P=0.670). But HCV viral loads decreased at 6th month after interferon treatment (P=0.063) and correlated poorly with liver function tests. The bDNA assay correlated well with the RT-PCR hybridization method (r2=0.854). CONCLUSIONS: The quantificaion of HCV RNA is useful in following up treatment effect but not in predicting therapeutic failure or assessment of disease severity. HCV RNA quantities are independent of liver function. The bDNA assay showed good correlation with the RT-PCR hybridization method.

Chain flexibility and configurational dimensions of left handed Z- DNA and right handed B-DNA helices.

The configurational behaviour of flexible helices of right handed B- and left handed Z-types have been analysed using statistical mechanical procedures. The configurationependent parameter, most importantly, the persistence length has been computed, using the heminucleotide scheme of treating polynucleotide chains under the approximation that perturbations in the backbone torsions produce sufficient flexibility in these helices. The values of persistence lengths obtained for Z-helices are very much higher than that of B-helices indicating that former is less flexible compared to the latter. These are in accordance with the results obtained recently on B- and Z-forms of poly(dG-dC) · (dG-dC) using light scattering studies. Also the persistence lengths of BII-DNA helices characterised by a skew 3'-hemiucleotide ( ε ~ 270°), and also when they coexist with B-DNA have been computed and the values lie within the range of experimentally reported values on B-helices. It is argued that the decrease in the persistence length values of B-DNA at higher salt concentration is due to additional small fluctuations in sugar residue torsions induced due to neutralisation of electrostatic repulsions between adjacent phosphates of the nucleotide. Noteworthy is that these are correlated to winding angle variations and the consequent bending of the helix.

Polymorphism in B-DNA: X-ray diffraction studies on Li-DNA fibres.

From X-ray diffraction studies it is generally believed that B-DNA has the structural parameters n = 10 and h = 3·4 Å. However, for the first time we report that polymorphism in the B-form can be observed in DNA fibres. This was achieved by the precise control of salt and humidity in fibres and by the application of the precession method of X-ray diffraction to DNA fibres. The significant result obtained is that n = 10 is not observed for crystalline fibre patterns. In fact, n = 10 and h = 3·4 Å are not found to occur simultaneously. Instead, a range of values, n = 9·6–10·0 and h = 3·35 Å–3·41 Å is observed.