Janice A. Kolberg

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Activation of tumor necrosis factor-α system in chronic hepatitis C virus infection

Tumor necrosis factor-α (TNF-α)plays a central role in the hosts immunomodulatoryresponse to infective agents. To evaluate theTNF-α system in patients with chronic hepatitis Cvirus (HCV) infection, plasma, serum, and peripheral bloodmononuclear cells (PBMC) were prospectively collectedfrom 53 patients and 33 healthy control subjects.Circulating TNF-α and TNF receptors were assayed by their respective enzyme immunoassays. Inaddition, TNF-α mRNA was quantitated in PBMC usinga branched DNA assay, and production of TNF-α byPBMC with and without lipopolysaccharide was also assessed. Patients with chronic HCV infectionhad a higher level of circulating TNF-α comparedto healthy control subjects (9.62 ± 6.01 vs 3.66± 1.23 pg/ml, P < 0.001). They also had highercirculating levels of TNF receptors compared to control(CD120a: 3323 ± 1267, pg/ml, N = 49 vs 1855± 422 pg/ml, N = 33, P < 0.001; CD120b: 1290± 650 pg/ml, N = 51, vs 863 ± 207 pg/ml,N = 33, P < 0.001). Plasma TNF-α level correlated with circulatingCD120a (r = 0.52, N = 49, P < 0.001) and weakly withCD120b (r = 0.32, N = 51, P = 0.02). Plasma TNF-αalso correlated with markers of hepatocellular injury, including ALT (r = 0.34, N = 53, P = 0.01) andα-GST (r = 0.31, N = 43, P = 0.042), but not withserum HCV RNA levels. There was no difference in theTNF-α mRNA levels in PBMC between patients with chronic HCV infection (1.4 ± 1.9units/106 cells, N = 8) and healthy control subjects(2.1 ± 1.4 units/106 cells, N = 8, P = NS). Therewas also no difference in the spontaneous production ofTNF-α by PBMC (1 × 106 cells/ml)between patients with chronic HCV infection (14.2± 36.5 pg/ml, N = 11) and healthy subjects (11.9± 14.0 pg/ml, N = 14, P = NS). However, patientswith chronic HCV infection produced more TNF-α upon stimulation withlipopolysaccharide compared to healthy control subjects(1278 ± 693 pg/ml, N = 11, vs 629 ± 689pg/ml, N = 14, P < 0.05). These data indicate thatthe TNF-α system is activated in patients with chronicHCV infection.

Development of a Type 2 Diabetes Risk Model From a Panel of Serum Biomarkers From the Inter99 Cohort

OBJECTIVE The purpose of this study was to develop a model for assessing the 5-year risk of developing type 2 diabetes from a panel of 64 circulating candidate biomarkers. RESEARCH DESIGN AND METHODS Subjects were selected from the Inter99 cohort, a longitudinal population-based study of ∼6,600 Danes in a nested case-control design with the primary outcome of 5-year conversion to type 2 diabetes. Nondiabetic subjects, aged ≥39 years, with BMI ≥25 kg/m2 at baseline were selected. Baseline fasting serum samples from 160 individuals who developed type 2 diabetes and from 472 who did not were tested. An ultrasensitive immunoassay was used to measure of 58 candidate biomarkers in multiple diabetes-associated pathways, along with six routine clinical variables. Statistical learning methods and permutation testing were used to select the most informative biomarkers. Risk model performance was estimated using a validated bootstrap bias-correction procedure. RESULTS A model using six biomarkers (adiponectin, C-reactive protein, ferritin, interleukin-2 receptor A, glucose, and insulin) was developed for assessing an individuals 5-year risk of developing type 2 diabetes. This model has a bootstrap-estimated area under the curve of 0.76, which is greater than that for A1C, fasting plasma glucose, fasting serum insulin, BMI, sex-adjusted waist circumference, a model using fasting glucose and insulin, and a noninvasive clinical model. CONCLUSIONS A model incorporating six circulating biomarkers provides an objective and quantitative estimate of the 5-year risk of developing type 2 diabetes, performs better than single risk indicators and a noninvasive clinical model, and provides better stratification than fasting plasma glucose alone.

Influence of Hepatitis G Virus Infection on the Severity of Liver Disease and Response to Interferon-α in Patients with Chronic Hepatitis C

A new RNA virus, designated hepatitis G virus (HGV), was recently identified [1-3]. Because HGV has less than 25% sequence or amino acid homology with hepatitis C virus (HCV) and other established flaviviridae, it is considered to be a new genus in this growing family of hepatotropic viruses. The clinical implications of HGV infection remain largely unresolved. It is known that HGV can be transmitted parenterally: Prospective studies on transfusion-associated infection have shown that HGV can appear in blood transfusion recipients who tested negative for the virus before transfusion [4]. Similarly, a high prevalence of HGV infection has been found in patients who have frequently been exposed to parenteral drug administration, such as intravenous drug users [5], patients undergoing hemodialysis [6, 7], and patients with hemophilia [8]. Vertical transmission [9] and a controversial association between HGV and fulminant hepatic failure [10-12] have also been described. A prospective study [13] has shown that about 75% of blood transfusion recipients infected with HGV have no biochemical evidence of liver disease. The interaction between HGV and HCV in patients with chronic hepatitis C and the influence of HGV co-infection on response to interferon- therapy are not yet well established. Tanaka and colleagues [14] found that HGV infection did not influence the response to interferon- therapy in a small group of patients with chronic hepatitis C. In that study, the method used to quantitate serum HGV RNA was not clearly defined. The objective of our study was to determine the prevalence of HGV infection in patients who have chronic hepatitis C; the influence of HGV infection on the clinical, virologic, and histologic characteristics of these patients; and the response of HCV and HGV to interferon- treatment. We also evaluated the influence of serum HGV RNA levels on the clinical, virologic, and histologic characteristics of patients and on the antiviral effect of interferon- therapy. Methods Patients We enrolled 228 patients (143 men and 85 women [mean age SD, 40 12 years]) who had previously participated in three controlled trials of interferon- at our center [15-17]. Eighty-seven patients had a history of blood transfusion, 57 were intravenous drug users, and 84 had no known source of infection. All patients received interferon- (alfa-2a [Roferon, Hoffman-LaRoche, Neuilly, France]; alfa-2b [IntronA, Schering-Plough, Levallois, France]; or alfa-n1 [Lymphoblastoid, Wellferon, Burroughs Wellcome, Issy-les-Moulineaux, France]). Three interferon- regimens were defined according to the total dose received. Dose 1 corresponded to a dosage of 3 million U three times a week for 3 months, dose 2 corresponded to a dosage of 3 million U three times a week for 6 months, and dose 3 corresponded to a dosage of 3 million U three times a week for 12 months or 5 million U three times a week for 6 or 12 months. All patients were born in France and lived in France at the time of the study. All patients tested positive for antibody to HCV on third-generation enzyme-linked immunosorbent assay. These results were confirmed by recombinant immunoblot assay [Ortho Diagnostic Systems, Roissy, France]. Diagnosis of chronic hepatitis C was based on the following criteria: 1) persistently elevated serum alanine aminotransferase levels for more than 6 months before randomization; 2) no evidence of hepatitis B virus infection [absence of detectable hepatitis B surface antigen]; 3) exclusion of other causes of chronic liver disease [alcoholism, hepatotoxic drugs, autoimmune chronic hepatitis, hemochromatosis, Wilson disease, and -1 antitrypsin deficiency]; and 4) liver histologic examination showing lesions characteristic of chronic hepatitis. No patient had a history of decompensated cirrhosis (ascites, bleeding esophageal varices, or hepatic encephalopathy), and all were negative for anti-human immunodeficiency virus (HIV) antibodies. Response to interferon- therapy was defined biochemically. Sustained response was defined as normalization of serum alanine aminotransferase levels during treatment and during the 6-month follow-up period. Response with relapse was defined as normalization of serum alanine aminotransferase levels during treatment, with elevation occurring after the end of treatment. No response was defined as elevated serum alanine aminotransferase levels at the end of the treatment. We recorded the following patient characteristics: sex; age; source and duration of infection; pretreatment serum levels of alanine aminotransferase, -glutamyltransferase, and ferritin; liver histologic characteristics; pretreatment serum levels of HCV RNA and HGV RNA; and HCV genotype. In patients who became infected as a result of blood transfusion or intravenous drug use, the duration of infection was estimated to be the interval between the date of transfusion or the date of the onset of intravenous drug use and the date of initiation of treatment. Patients with no known parenteral exposure were not considered for this variable. Liver biopsy specimens were obtained from all patients within the 6 months before initiation of treatment. The histologic preparations were examined in a blinded manner by the same pathologist and were scored for fibrosis and necroinflammatory activity according to the criteria proposed by Knodell and colleagues [18]. Activity of chronic hepatitis was considered to be low when the necroinflammatory activity score was 6 or less; activity was considered to be mild or high when the necroinflammatory activity score exceeded 6. Laboratory Studies All virologic assays were done on aliquots of the same serum samples, which were kept frozen until they were used. Measurement of Serum Hepatitis G Virus RNA and Hepatitis C Virus RNA Serum HGV RNA and serum HCV RNA were quantitated to evaluate the levels of HGV and HCV viremia before treatment. Serum HGV RNA quantitation was done with an experimental branched-DNA (bDNA) assay (Chiron Corp., Emeryville, California). In this assay, additional sensitivity was achieved by using amplimers made with iso-C and iso-G nucleotides and by using a preamplifier sequence to create an interface between target probe overhangs and the bDNA amplifier [19]. The capture and label extenders are located in the relatively conserved sequence of the 5 untranslated region of the HGV genome according to the HGV isolates described by Linnen and colleagues [3] and cover approximately 300 bases. The molecular quantitation cutoff of the assay is 0.05 106 genome equivalents/mL. All samples were run in duplicate. Serum HCV RNA quantitation was done with the improved quantitative bDNA-HCV RNA assay (Quantiplex HCV RNA 2.0, Chiron Diagnostics, Eragny, France). In this assay, refined oligonucleotide probe sets that are based on sequence variation among disparate HCV isolates are incorporated [20]. This newly developed assay is more efficient for HCV RNA quantitation of HCV genotypes 2 and 3 and leads to an equivalent quantification for HCV genotypes 1 through 6. The quantitation cutoff of the assay is 0.2 106 genome equivalents/mL. All samples were run in duplicate. To obtain comparable quantitation on bDNA assays for HGV RNA and HCV RNA, the standards for both assays were value-assigned against RNA transcripts that were synthesized to include the entire region recognized by the respective probe sets. The transcripts were independently characterized and quantified as described elsewhere [21]. To assess the quality of the transcripts, the preparations were run on agarose gels that contained formaldehyde; the gels were dried and scanned using an Ambis 4000 Radiologic Imager (Ambis, Inc., San Diego, California). The preparations of HGV RNA and HCV RNA transcripts that were used to define the quantification in the bDNA assays were greater than 80% full length and contained less than 3% free nucleotides. The analytical methods used to quantify RNA transcripts, phosphatase analysis, hyperchromicity, and absorbance at 260 nm produce values that agree within 10%. The transcripts were subsequently tested in the respective bDNA assays to determine the signal generated per attomole of RNA transcripts. In HCV RNA and HGV RNA assays, a genome equivalent is defined as the amount of RNA in a sample that generates the same signal as one molecule of the characterized transcript. Detection of Serum Hepatitis G Virus RNA and Hepatitis C Virus RNA Serum HGV RNA and serum HCV RNA were detected using a qualitative method at the end of treatment and 6 months after treatment in order to look for a correlation between the biochemical and virologic responses. Serum HGV RNA was detected by using reverse-transcription polymerase chain reaction with primers in the 5 end of the HGV genome according to the method of Linnen and colleagues [3]. The detection of serum HCV RNA was done by using reverse-transcription polymerase chain reaction with primers in the 5 noncoding region of the HCV genome [22]. Detection was done at the initiation of treatment in all patients with detectable serum HGV RNA and in all patients with detectable serum HCV RNA. Genotyping of Hepatitis C Virus Genotyping of HCV was done in the 5 untranslated region of the HCV genome by using reverse hybridization with the line probe assay [23] (InGeN, Rungis, France). In reverse hybridization, the biotinylated amplification products obtained are hybridized to oligonucleotides directed against the variable region of the 5 untranslated region and are immobilized as parallel lines on membrane strips. Incubation with streptavidin labeled with alkaline phosphatase then allows detection of the hybrids. The HCV line probe assay contains 15 probe lines that allow identification of HCV types 1 to 5 and HCV subtypes. All serum specimens were stored at 4 C immediately after collection, were centrifuged through a paraffin plug after formation of the clot within 2 hours of sampling, and were froze

Validation of a Multimarker Model for Assessing Risk of Type 2 Diabetes from a Five-Year Prospective Study of 6784 Danish People (Inter99)

Background: Improved identification of subjects at high risk for development of type 2 diabetes would allow preventive interventions to be targeted toward individuals most likely to benefit. In previous research, predictive biomarkers were identified and used to develop multivariate models to assess an individuals risk of developing diabetes. Here we describe the training and validation of the PreDx™ Diabetes Risk Score (DRS) model in a clinical laboratory setting using baseline serum samples from subjects in the Inter99 cohort, a population-based primary prevention study of cardiovascular disease. Methods: Among 6784 subjects free of diabetes at baseline, 215 subjects progressed to diabetes (converters) during five years of follow-up. A nested case-control study was performed using serum samples from 202 converters and 597 randomly selected nonconverters. Samples were randomly assigned to equally sized training and validation sets. Seven biomarkers were measured using assays developed for use in a clinical reference laboratory. Results: The PreDx DRS model performed better on the training set (area under the curve [AUC] = 0.837) than fasting plasma glucose alone (AUC = 0.779). When applied to the sequestered validation set, the PreDx DRS showed the same performance (AUC = 0.838), thus validating the model. This model had a better AUC than any other single measure from a fasting sample. Moreover, the model provided further risk stratification among high-risk subpopulations with impaired fasting glucose or metabolic syndrome. Conclusions: The PreDx DRS provides the absolute risk of diabetes conversion in five years for subjects identified to be “at risk” using the clinical factors.

Quantitation of human immunodeficiency virus plasma RNA by branched DNA and reverse transcription coupled polymerase chain reaction assay methods: A critical evaluation of accuracy and reproducibility

Abstract The present study was designed to evaluate the utility of two assays, reverse transcription coupled polymerase chain reaction (RT-PCR) and branched DNA (bDNA), to accurately and reproducibly quantitate plasma human immunodeficiency virus (HIV) RNA levels. The bDNA assay quantitated RNA transcripts, prepared from different HIV-1 subtypes (A-E), within 1.5-fold. Similarly, the bDNA assay, standardized to subtype B, was used to quantitate cultured isolates from subtypes A, C-F within 2-fold; however, the RT-PCR assay displayed a 904-fold range. Reproducibility studies demonstrated that the bDNA and RT-PCR assays could be used statistically (P

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Hepatitis G Virus and Human Immunodeficiency Virus Coinfection: Response to Interferon-α Therapy

The prevalence and consequences of hepatitis G virus (HGV) infection were determined in 180 patients with human immunodeficiency virus (HIV) infection (predominantly male homosexuals) who participated in a trial that compared treatment with zidovudine versus interferon (IFN)-alpha versus the combination. HGV RNA levels were measured by branched DNA signal amplification assay. Initially, 66 (37%) had HGV RNA. Sexual transmission was the sole risk factor for infection in all but 4 subjects. Pretreatment clinical features were similar between HGV RNA-positive and -negative patients. After 6 months, only 5% treated with zidovudine became HGV RNA negative, compared with 95% who received IFN-alpha alone and 66% on combination therapy with low-dose IFN-alpha. After therapy, HGV RNA levels returned to baseline in most subjects. Thus, HGV infection is common among HIV-infected homosexual males but does not appear to influence clinical features in early HIV infection. HGV RNA levels are suppressed by IFN but not by zidovudine.

Effect of Interferon α and Ribavirin Therapy on Serum GB Virus C/Hepatitis G Virus (GBV-C/HGV) RNA Levels in Patients Chronically Infected with Hepatitis C Virus and GBV-C/HGV

GB virus C/hepatitis G virus (GBV-C/HGV) is a newly described virus associated with hepatitis in humans, and GBV-C/HGV coinfection is common in patients chronically infected with hepatitis C virus (HCV). To determine the clinical impact of GBV-C/HGV infection in such patients and the effect of interferon-alpha and ribavirin therapy on serum GBV-C/HGV RNA levels, GBV-C/HGV RNA was detected and quantitated in serum samples from 62 chronically infected HCV patients by a combination of a qualitative nested reverse transcription-polymerase chain reaction and a newly developed quantitative branched DNA assay: 10 patients were positive for serum GBV-C/HGV RNA. There were no differences in the clinical, biochemical, and histologic features of the patients with GBV-C/HGV-HCV coinfection compared with those with HCV infection alone. Interferon-alpha treatment caused a marked but usually transient reduction in serum GBV-C/HGV RNA, and ribavirin had, at most, a modest antiviral effect.

Validation of a multi-marker model for the prediction of incident type 2 diabetes mellitus: Combined results of the Inter99 and Botnia studies

Purpose: To assess performance of a biomarker-based score that predicts the five-year risk of diabetes (Diabetes Risk Score, DRS) in an independent cohort that included 15-year follow-up. Method: DRS was developed on the Inter99 cohort, and validated on the Botnia cohort. Performance was benchmarked against other risk-assessment tools comparing calibration, time to event analysis, and net reclassification. Results: The area under the receiver-operating characteristic curve (AUC) was 0.84 for the Inter99 cohort and 0.78 for the Botnia cohort. In the Botnia cohort, DRS provided better discrimination than fasting plasma glucose (FPG), homeostasis model assessment of insulin resistance, oral glucose tolerance test or risk scores derived from Framingham or San Antonio Study cohorts. Overall reclassification with DRS was significantly better than using FPG and glucose tolerance status (p < 0.0001). In time to event analysis, rates of conversion to diabetes in low, moderate, and high DRS groups were significantly different (p < 0.001). Conclusion: This study validates DRS performance in an independent population, and provides a more accurate assessment of T2DM risk than other methods.