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Complex diseases are caused by combinatorial genetic, environmental and lifestyle factors. The emergence of multibiomarker tests to define these diseases and to identify the early, presymptomatic stages offers several advantages to the conventional use of single marker tests. The development of multibiomarker protein-based tests remains constrained by technological and operational limitations in assaying hundreds to thousands of proteins in thousands of samples. In order to develop a multibiomarker test that stratifies risk for Type 2 diabetes, we took a candidate-driven immunoassay approach utilizing a microfluidics platform to analyze 89 candidate proteins in thousands of samples, which allowed us to move from discovery to a commercial test in 2 years. Future multibiomarker test development will be enhanced by advancements in the number of proteins that can be analyzed, analytical sensitivity and throughput, and sample volume requirements, all of which depend on the further advancement of microfluidics, detection technologies and affinity-based reagents.

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.

Ten subjects received 600 to 1,200 mg of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor ritonavir per day. Following 2 weeks of therapy, plasma HIV RNA levels decreased by a mean of 1.57 (range, 0.89 to 1.96) log units. With continued therapy, HIV RNA levels began to rise in eight subjects. The initial rise in plasma RNA levels was temporally associated with the development and quantitative increase in the V82 resistance mutation. Doubling times of the V82A mutant virus were estimated to be 2.4 to 4.8 days. An L63P/A mutation was commonly present at baseline even in subjects with a durable virologic response. The concomitant acquisition of an L63P/A mutation with the V82A/F mutation at the time when plasma RNA levels rebounded suggests a role for the L63P/A mutation in improving the fitness of the V82A/F mutation. Subsequent additional genotypic changes at codons 54 and 84 were often associated with further increases in plasma RNA levels. Ongoing viral replication in the presence of drugs resulted in the appearance of additional genotypic changes, including the L90M saquinavir resistance mutation, and decreased phenotypic susceptibility. The relative fitness of the protease V82A ritonavir resistance mutation and reverse transcriptase T215Y/F zidovudine resistance mutation following drug withdrawal were estimated to be 96 to 98% that of the wild type. Durability of the virologic response was associated with plasma RNA levels at the nadir. A virologic response beyond 60 days was not observed unless plasma HIV RNA levels were suppressed below 2,000 copies/ml, consistent with estimates from V82A doubling times for selection of a single resistance mutation to dominate the replicating population.

The divergent synthesis of bDNA structures is described. This new type of branched DNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branching network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb molecules were assembled on a solid support using parameters optimized for bDNA synthesis. The chemistry was used to synthesize bDNA comb molecules containing 15 secondary sequences. The bDNA comb molecules were elaborated by enzymatic ligation into branched amplification multimers, large bDNA molecules (a total of 1068 nt) containing an average of 36 repeated DNA oligomer sequences, each capable of hybridizing specifically to an alkaline phosphatase-labeled oligonucleotide. The bDNA comb molecules were characterized by electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The branched amplification multimers have been used as signal amplifiers in nucleic acid quantification assays for detection of viral infection. It is possible to detect as few as 50 molecules with bDNA technology.

The divergent synthesis of branched DNA (bDNA) comb structures is described. This new type of bDNA contains one unique oligonucleotide, the primary sequence, covalently attached through a comb-like branch network to many identical copies of a different oligonucleotide, the secondary sequence. The bDNA comb structures were assembled on a solid support and several synthesis parameters were investigated and optimized. The bDNA comb molecules were characterized by polyacrylamide gel electrophoretic methods and by controlled cleavage at periodate-cleavable moieties incorporated during synthesis. The developed chemistry allows synthesis of bDNA comb molecules containing multiple secondary sequences. In the accompanying article we describe the synthesis and characterization of large bDNA combs containing all four deoxynucleotides for use as signal amplifiers in nucleic acid quantification assays.

Cloning of the hepatitis C virus (HCV) genome was a tremendous advance in the development of tests for diagnosis and monitoring of HCV-infected patients. Serological tests, including enzyme-linked immunoassays and RIBATM strip immunoblot assays, are primarily used to screen blood donations and to diagnose and confirm HCV infection. Tests for HCV RNA, including polymerase chain reaction (PCR)-based assays and the branched-DNA (bDNA) assay, are used for therapeutic monitoring and prognostics. Here, we present the development and future potential of these diagnostic tests. We also provide examples of how these tests are used to follow the progression of disease, select and adjust treatment protocols, and evaluate the efficacy of therapeutic regimens.

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.

Three PCR methods based on the GB virus-C/hepatitis G virus (GBV-C/HGV) 5′UTR and NS3 genomic region were used for the detection of GBV-C/HBV RNA in serum of 62 patients with chronic hepatitis C virus (HCV) infection. Ten of 62 (16%) patients were found to have GBV-C/HGV RNA, which was confirmed by sequence analysis of the 5′UTR PCR amplicon. All methods appear to be specific, but methods based on the 5′UTR appear to be more sensitive. J. Med. Virol. 52:92–96, 1997. © 1997 Wiley-Liss, Inc.

Glucose tightly regulates the synthesis and secretion of insulin by beta cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single beta cell (betaTC3 cells or mouse islets), whereas 1 million non-insulin-producing alpha cells (alphaTC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from beta cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up approximately 2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription.

The optimal method for viral quantitation and the most appropriate site for determining viral load in patients with chronic hepatitis C virus (HCV) infection are unknown. We developed a method for measuring HCV RNA in the liver with the following features: 1) efficient extraction of RNA from tissue (89% of RNA recovered); 2) accurate amplification using branched DNA with strong concordance between a single sample tested on multiple occasions either in the same or in different runs; 3) good sensitivity (95%) and specificity (100%). HCV RNA was detected in as little as 2 mg of tissue, and viral load determined in a needle biopsy was representative of viral load in other parts of the liver. Within individual livers, 68% of the samples quantitated were within 1.5-fold of the geometric mean, and 95% were within 2.2-fold of the geometric mean. The mean ratio of virus in the liver and serum was 103, range 17.4-286. A delay of 30 minutes before freezing the liver tissue resulted in a reduction in the measured viral load in some, but not all instances. A sensitive, specific and reproducible method for quantitating HCV RNA in the liver has been developed. Measurement of viral load at one site was representative of viral load at other sites. While hepatic HCV RNA levels are consistently greater than serum levels, the ratio of liver of serum viral load varies widely. The clinical use of measurement of viral load in the liver remains to be defined.

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.

Background/Aims: To investigate host- and virus-related factors predictive of early and sustained alanine aminotransferase normalization after interferon therapy for HCV-related chronic liver disease, in an area where genotype 1 is highly prevalent. Methods: We studied 100 patients with HCV-RNA positive chronic liver disease (73 chronic hepatitis and 27 cirrhosis) undergoing alpha-interferon treatment. Thirty-four patients had an early response but relapsed, 15 patients remained into sustained response for at least 12 months after therapy, and 51 patients did not respond. Serum HCV-RNA levels were assessed by bDNA (Chiron), and genotype by LiPA (Innogenetics) and by sequencing of the 5′ non-coding region. Results: Mean pre-treatment HCV-RNA level (×10 3 genome equivalents/ml±SD) was lower in sustained responders (3854±7142) than in relapsers (9587±10163) or in non-responders (5709±6618). HCV subtype 1b was highly prevalent (82%), while types 1a, 2a, 3 and 4 were rare (about 5% each). However, the prevalence of 1b was much lower (31 %) under 40 years of age. The prevalence of subtype 1b among sustained responders (74%) was similar to that observed among relapsers (82%) or non-responders (84%), but some nucleotide substitutions in the putative RNA loop of the 5′ non-coding region were seen only among relapsers or non-responders. Multiple logistic regression model showed that early response to interferon was predicted by absence of cirrhosis and a pre-treatment HCV-RNA level below 350. Sustained response to interferon was predicted by pre-treatment HCV-RNA level below 350 and a low fibrosis score. Conclusions: Among patients with hepatitis C from an area where subtype 1b is highly prevalent, absence of cirrhosis and low pre-treatment serum HCV-RNA level are the most important predictors of response to IFN. Some nucleotide substitutions found in the 5′ non-coding region of subtype 1b are associated with non-response or relapse.

The level of hepatitis C virus (HCV) in the serum of 337 blood donors infected with different viral genotypes was investigated by branched DNA assay. Viral genotype was deduced by restriction analysis of the virus 5'-noncoding region. Samples included genotypes 1a, 1b, 2a, 2b, 3,4,5, and 6. Multivariate analysis revealed that the ranges of HCV levels were similar for all viral genotypes and subtypes (P=.18), with the possible exception of genotype 4. Virus levels were significantly lower in female than in male subjects (P

Variation in the 5′ non-coding region (5′NCR) of hepatitis C virus (HCV) was investigated in detail by comparing 314 5′NCR sequences of viruses of genotypes 1 to 6. Evidence was obtained for the existence of associations between particular 5′NCR sequence motifs and virus types and subtypes. No recombination was observed between the 5′NCR and coding regions of different genotypes, implying that the sequence of subgenomic regions such as the 5′NCR can be used to deduce virus genotype. The distribution of polymorphic sites within the 5′NCR is used to propose improved oligonucleotide primers for virus detection and quantification that would be equally efficient in detecting RNA of different virus genotypes. The accuracy of two different genotyping methods (RFLP and the line probe assay) based on analysis of sequence polymorphisms in the 5′NCR is predicted from the sequences surveyed to be 97% and 83% respectively for types 1 to 6, with higher accuracies for distinguishing between subtypes 1a/1b, 2a/2b or 3a/3b. Several sites of genotype-specific polymorphism were covariant and maintained the base pairings required for a secondary structure model of the 5′NCR. Other sites of variation suggest minor modifications to this model and have implications for the probable functions of the 5′NCR

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.

RNA standards were developed for use in quantitative hybridization assays such as the Quantiplex HCV RNA Assay and Quantiplex HIV RNA Assay, which are based on branched DNA signal amplification. In vitro transcripts ranging in size from 0.5 to 9.4 kb were prepared and purified by phenol extraction following gel electrophoresis or column chromatography. Aliquots of the transcripts were digested to nucleosides and phosphate and then quantified by phosphate analysis against the U.S. National Institute of Standards and Technology phosphate standard. The quantitation was checked by OD260 and by either hyperchromicity or isotopic tracer analysis. The quantitation of each lot of RNA agreed within 20% by the three methods. The reproducibility of the methods was tested by preparing a total of 13 lots of standard RNAs. The average percentage full-length RNA of the 13 lots was 82%, with a range of 59 to 97%. The standard RNAs were used to test the ability of the branched DNA hybridization assay to quantify all target RNAs accurately regardless of size or slight variations in sequence. Standard Hepatitis C virus (HCV) RNAs of 1.3, 2.2, and 3.2 kb showed that size has no detectable effect on quantitation in the branched DNA hybridization assay. Three different lots of standard 3.2-kb HCV RNA were serially diluted and quantified over a thousand-fold range in the branched DNA hybridization assay. The average signal per attomole of target varied by less than 20% among the 3 lots. Standard HCV RNA transcripts were also prepared from clones of HCV subtypes 1b and 3a to study the effects of target sequence diversity and probe design on quantitation by hybridization. The quantitation of HCV subtype 1b RNA and 3a RNA differed by a factor of 1.6-fold in the branched DNA hybridization assay with one probe design and were indistinguishable with another probe design.

Quantification of HIV-1 RNA in human plasma has provided unique insight into AIDS pathogenesis and promises to hasten progress in antiretroviral therapy and vaccine research. However, no generally available HIV-1 RNA assay has yet been subjected to rigorous clinical testing or to comparative evaluation with research-based RNA assays using large numbers of well-characterized clinical specimens. In this study, the Chiron Quantiplex branched DNA (bDNA) signal amplification assay was used to measure viral RNA in the plasma of 152 HIV-1-positive individuals at all stages of infection and in 12 patients before and after initiating zidovudine therapy. Eighty-six percent of patients had bDNA assay results above the 10,000-RNA Eq/ml sensitivity cutoff. Branched DNA values were significantly correlated with plasma viral RNA levels determined by quantitative competitive polymerase chain reaction (QC-PCR) assay (Spearman rank correlation, r = 0.89), infectious plasma virus titers (r = 0.72), p24 antigen levels (r = 0.51), immune complex dissociated p24 antigen levels (r = 0.56), and CD4+ lymphocyte counts (r = -0.72; p < 0.0001 for all comparisons). Plasma viral RNA determinations by bDNA and QC-PCR assays were quantitatively similar in the range of 10(4) to 10(7) RNA molecules/ml [log bDNA = 0.93 + 0.80 (log QC-PCR); R2 = 0.81, p < 0.0001] and declined identically following the institution of zidovudine therapy (68-73% decrease from baseline). The close quantitative correlation between bDNA and QC-PCR results, and their significant association with other viral markers and CD4+ counts, support the use of plasma viral RNA measurement in HIV-1 clinical trials.

Serum samples from 139 US patients with chronic hepatitis C virus (HCV) infection were studied using six different genotyping systems, including both molecular and serologic methods, to determine the applicability of these approaches and the prevalence of various HCV subtypes. The concordance of genotyping results based on the various systems (except for core polymerase chain reaction genotyping) was good (93.5%). Subtypes 1a and 1b were prevalent (37.4%). Subtypes 2a (2.2%), 2b (8.6%), and 3a (5.8%) were less common. HCV genotypes could not be determined in 3.4%-16.5% of samples depending on the method used. HCV type 2 was associated with greater histologic activity but lower serum HCV RNA levels (P < .05), whereas type 3 was associated with lower serum alanine aminotransferase levels (P < .05). These data demonstrate a high concordance between HCV genotyping systems and provide a foundation for comparison of genotyping data between studies using different systems. HCV types 1a and 1b are both prevalent in the United States.

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

Isolates of hepatitis C virus (HCV) show considerable nucleotide sequence variability throughout the genome. Comparisons of complete genome sequences have been used as the basis of classification of HCV into a number of genotypes that show 67 to 77% sequence similarity. In order to investigate whether sequence relationships between genotypes are equivalent in different regions of the genome, we have carried out formal sequence analysis of variants in the 5' non-coding region (5'NCR) and in the genes encoding the core protein, an envelope protein (E1) and a non-structural protein (NS-5). In the E1 region, variants grouped into a series of six major genotypes and a series of subtypes that could be matched to the phylogenetic groupings previously observed for the NS-5 region. Furthermore, core and E1 sequences showed three non-overlapping ranges of sequence similarity corresponding to those between different genotypes, subtypes and isolates previously described in NS-5. Each major genotype could also be reliably identified by sequence comparisons in the well conserved 5'NCR, although many subtypes, such as 1a/1b, 2a/2c and some of those of type 4, could not be reliably distinguished from each other in this region. These data indicate that subgenomic regions such as E1 and NS-5 contain sufficient phylogenetic information for the identification of each of the 11 or 12 known types and subtypes of HCV. No evidence was found for variants of HCV that had sequences of one genotype in the 5'NCR but of a different one in the E1 or NS-5 region. This suggests that recombination between different HCV types is rare or non-existent and does not currently pose a problem in the use of subgenomic regions in classification.

Hepatitis C virus (HCV) infection is common in liver transplant recipients, yet the effects of immunosuppression on HCV RNA levels and the relationship of HCV RNA levels to hepatic damage have not been studied.To explore these issues, we measured HCV RNA in serum by polymerase chain reaction amplification and branched DNA assay from 100 HCV-infected patients undergoing liver transplantation.Mean posttransplant levels were 16-fold higher than pretransplant values (7,935,000 and 496,000 Eq/mL, respectively; n = 65; P0.0001). Patients with high pretransplant levels had higher mean posttransplant levels than those with low pretransplant levels (17,119,000 and 6,504,000 Eq/mL, respectively; P = 0.064). Posttransplant levels were similar in patients with recurrent and acquired infection and were independent of time of sampling. Fifty percent of patients with HCV infection had normal liver biopsy specimens, and there was no strong relationship between level of viremia and degree of hepatic damage.HCV RNA levels increase markedly following liver transplantation. The frequent finding of viremia in the absence of histological hepatitis suggests that a "carrier state" is common. Absence of allograft damage in some (despite high levels of viral RNA) suggests that in immunosuppressed patients, HCV infection may be tolerated without direct hepatic damage.

Type 2 diabetes is a chronic, debilitating and often deadly disease that has reached epidemic proportions. The onset of diabetes can be delayed or prevented in high-risk individuals by diet and lifestyle changes and medications, and hence a key element for addressing the diabetes epidemic is to identify those most at risk of developing diabetes so that preventative measures can be effectively focused. The PreDx(®) Diabetes Risk Score is a multimarker tool for assessing a patient's risk of developing diabetes within the next 5 years. Requiring a simple blood draw using standard sample collection and handling procedures, the PreDx Diabetes Risk Score is easily implemented in clinical practice and provides an assessment of diabetes risk that is superior to other measures, including fasting plasma glucose, glycated hemoglobin, measures of insulin resistance and other clinical measures. In this article, we provide an overview of the PreDx Diabetes Risk Score.

To determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma.Two HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load.In one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 10(5) copies/ml, and the branched DNA results ranged from 2.6 x 10(4) to 4.2 x 10(4) HIV RNA equivalent/ml. In the other sample the corresponding figures were 6.3 x 10(4) to 5.5 x 10(5) copies/ml and 5.7 x 10(4) to 7.5 x 10(4) HIV RNA equivalents/ml.In contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Hepatitis C virus, the major causative agent of blood-borne non-A, non-B hepatitis in the world, has been the subject of considerable nucleic acid sequence analysis. Although all reported hepatitis C sequences from the United States have been represented by the prototype hepatitis C virus type 1 sequence, two groups of variant sequences have been reported in Japan. However, we have noted five distinct, but related, genotypes (I-V) throughout the world, based on detailed sequence determination and analysis of the first 1700 nucleotides and part of the nonstructural region 5 at the C terminus of the open reading frame. The nucleotide sequence for a large number of hepatitis C virus isolates spanning six continents was obtained by direct sequence analysis of PCR products after reverse transcription. Genotype was classified by using several distinct sequence motifs. We observed that most genotypes coexist in several geographic regions, including the United States, Japan, Germany, and Italy. So far, genotype V has been found only in South Africa. Interestingly, each distinct genotype seems to be maintained throughout the genome in the segments studied. These genotype distinctions should be considered when designing specific diagnostic tests, developing potential vaccines, and studying viral transmission.

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 individual's 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.

We have reported the synthesis of comb type bDNA where a unique, primary oligonucleotide fragment is tailed with several identical copies of a secondary oligonucleotide1. The strategy for synthesis of these molecules is based on the use of a nucleoside analog branching monomer. Recent improvements in solid phase methods enable the synthesis of bDNA with as many as 50 copies of secondary sequences. Characterization of these bDNA is also discussed.

In this communication we report the synthesis of a protected abasic molecule, 1′-O-(2-nitrobenzyl)-2′-deoxyriboside, and a special N-4-(6-hydroxyhexyl)-ribocytidine derivative as light- and periodate-sensitive selectable cleavage moieties, respectively, and their use in the characterization of linear and branched single-stranded DNA molecules.

With this statement, Sherlock and Dooley have described two of the three major challenges involved in quantitatively measuring any analyte in tissue samples: the distribution of the analyte in the tissue; and the standard of reference, or denominator, with which to make comparisons between tissue samples. The third challenge for quantitative measurement of an analyte in tissue is to ensure reproducible and quantitative recovery of the analyte on extraction from tissue samples. This chapter describes a method that can be used to measure HCV RNA quantitatively in liver biopsy and tissue samples using the bDNA assay. All three of these challenges-distribution, denominator, and recovery-apply to the measurement of HCV RNA in liver biopsies.

The diagnosis and monitoring of hepatitis C virus (HCV) infection have been aided by the development of HCV RNA quantification assays A direct measure of viral load, HCV RNA quantification has the advantage of providing information on viral kinetics and provides unique insight into the disease process. Branched DNA (bDNA) signal amplification technology provides a novel approach for the direct quantification of HCV RNA in patient specimens. The bDNA assay measures HCV RNA at physiological levels by boosting the reporter signal, rather than by replicating target sequences as the means of detection, and thus avoids the errors inherent in the extraction and amplification of target sequences. Inherently quantitative and nonradioactive, the bDNA assay is amenable to routine use in a clinical research setting, and has been used by several groups to explore the natural history, pathogenesis, and treatment of HCV infection.

Adaptation of the branched DNA signal amplification technology to flow cytometry has resulted in a quantitative nucleic-acid assay with significant advantages over the microwell-based format. In this assay, microbeads, rather than microwell plates, are derivatized with nucleic-acid capture probes and the derivatized beads are used to capture single nucleic-acid targets, which then capture fluorescent reporter probes via branched DNA. The assay detects DNA or RNA targets, has a current lower sensitivity limit of 500 human immunodeficiency virus (HIV) RNA molecules and responds linearly to target level from 500 to at least 50 000 molecules. Since microbeads can easily interrogate large volumes, viral lysis and genomic RNA capture can proceed in one step from comparatively large volumes, and sample preparation is greatly simplified compared to the microwell-format bDNA assay.

Over the past decade a number of new technologies, including the polymerase chain reaction (PCR) (Mullis and Faloona, 1987), have emerged for the detection and quantification of nucleic acid molecules. Distinct among these is the branched DNA (bDNA) assay which, unlike its target amplification counterparts, uses a signal amplification scheme to enhance detection of physiologic concentrations of target nucleic acids. The basis of the bDNA assay involves the specific hybridization of bDNA and enzymelabeled oligonucleotide probes to target nucleic acids; this is described in detail in the accompanying chapter in this book, entitled “Branched DNA Technology for Direct Quantification of Nucleic Acids: Design and Performance.” The bDNA assay is inherently quantitative and nonradioactive, and has proven to be a reproducible and accurate means of quantifying nucleic acid molecules. The bDNA assay offers several advantages for research and clinical applications; it: provides direct quantification of nucleic acid molecules at physiological levels. Since bDNA assays do not require highly purified nucleic acid preparations, inhibitors of enzyme-dependent amplification techniques are of no concern. allows a wide diversity of specimen types to be used. can be used with reference standards to ensure accuracy for applications requiring absolute quantification, or without standards for applications requiring only assessment of relative changes in nucleic acid concentrations. exhibits a high level of sensitivity (moderately expressed genes can be detected using as few as 50–100 cells). The level of sensitivity can be modulated by altering the number of target-specific extenders to accommodate specific applications. is thoroughly tested for within-lot and between-lot precision to ensure reproducibility (2.2- to 3-fold changes can be discerned as statistically significant).

The branched DNA (bDNA) assay represents a significant advance in the direct quantification of nucleic acid molecules for research and clinical applications. Other methods for the detection and quantification of nucleic acid molecules, such as polymerase chain reaction (PCR) and nucleic acid sequence-based amplification (NASBA), involve molecular amplification of target nucleic acids followed by detection of amplified products. While these target amplification methods can be extremely sensitive, they suffer from two major disadvantages. First, it is difficult to obtain reproducible quantification since the target nucleic acid must be extracted and amplified in order to measure it. Second, numerous precautions must be taken to avoid false positive results caused by contamination of samples with PCR products and carryover from other specimens. A new departure from target amplification methods, the bDNA assay, directly measures nucleic acid molecules by boosting the reporter signal, rather than by replicating target sequences as the means of detection (Figure 1), and hence it is not subject to the errors inherent in the extraction and amplification of target nucleic acids. An ideal tool for nucleic acid quantification, the bDNA assay detects nucleic acid molecules at their physiological concentration, yields highly reproducible quantification, and eliminates false positives due to contamination.

The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification. Addition of a 14 site preamplifier was found to increase the signal/noise ratio 8-fold. A set of 74 oligonucleotide probes was designed to the consensus HIV POL sequence. The detection limit of this new HIV branched DNA amplifier assay was approximately 50 molecules/ml. The assay was used to measure viral load in 87 plasma samples of HIV- infected patients on triple drug therapy whose RNA titers were

IFN-gamma mRNA levels were measured in unstimulated PBMC and purified cell subpopulations, utilizing branched DNA assays, to characterize the cell type(s) that contribute to the in vivo increase in IFN-gamma gene expression seen in HIV infection. PBMC and CD8 T cells from HIV-seropositive subjects (HIV+) showed 2.5-fold increases in mean IFN-gamma mRNA levels compared to HIV-uninfected subjects (HIV-). Within individuals, CD8 T cells showed the highest IFN-gamma expression regardless of HIV status, which suggests that HIV infection enhances the IFN-gamma gene expression in CD8 T cells rather than inducing a shift to and/or increasing expression of IFN-gamma mRNA in other cell types. HIV+ subjects with increased PBMC IFN-gamma mRNA had elevated plasma levels of HIV RNA, neopterin, and beta 2-microglobulin. No differences in IFN-gamma mRNA levels were seen among HIV+ stratified by CD4 T cell number. Increased IFN-gamma may result from or be a contributing factor to increased viral load.

Quantification of hepatitis C virus RNA in liver tissue is likely to be useful in the study of the natural history, pathogenesis, progression and treatment of hepatitis C virus-associated liver disease. Quantitative measurements of hepatitis C virus RNA in liver biopsy samples using the branched DNA (bDNA) signal amplification assay were carried out. The aims of this study were threefold: first, to assess the level of hepatitis C virus RNA in biopsy samples from the right and left lobes of the liver; second, to evaluate the correlation between hepatitis C virus RNA levels in serum and liver; and third, to investigate the relationship between serum and liver hepatitis C virus RNA levels and the severity of hepatic histology in non-cirrhotic patients with chronic hepatitis C. There was a strong correlation (r = 0.92, P < 0.01) between hepatitis C virus RNA levels in the right and left lobes of the liver as well as a strong correlation between hepatitis C virus RNA levels in liver and serum (r = 0.82, P < 0.01). However, there was no significant correlation between the severity of hepatic histology and levels of hepatitis C virus RNA in serum and liver among patients with chronic active hepatitis classified according to Knodell's hepatic activity index (KI). Our results indicate that hepatitis C virus RNA quantification from a single liver biopsy is representative of both lobes in patients with chronic hepatitis, and suggest that serum hepatitis C virus RNA levels are a meaningful reflection of hepatitis C virus RNA levels in the liver.

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response.

To determine the significance of hepatic expression of hepatitis C viral (HCV) antigens, HCV core and NS4 antigens were detected by immunohistochemistry in 46 patients with chronic HCV infection. Serum HCV RNA was quantitated by branched DNA assay in 41 and HCV genotype determined in 30 patients. HCV core and NS4 antigens were detected exclusively in the cytoplasm of hepatocytes in 83% and 61% of patients, respectively. There was no correlation between the expression of HCV antigens and clinical, biochemical, histological parameters and HCV genotype. Hepatic expression of HCV antigens was positively associated with serum HCV-RNA levels (P0.02). At the end of interferon-alpha (IFN) therapy, expression of HCV antigens remained either unchanged or decreased in 11/12 patients studied (undetectable in all four patients who had complete and sustained response). We conclude that hepatic expression of HCV core and NS4 antigens parallels serum HCV-RNA levels and IFN therapy reduces hepatic expression of these viral antigens.

To determine the best method for detecting HCV infection in immunosuppressed patients, stored frozen serum from 101 liver transplant recipients was tested for hepatitis C virus. Each sample was tested by four assays. HCV RNA was detected by both polymerase chain reaction (PCR) and branched DNA signal amplification. Antibody to HCV was determined using second-generation enzyme-linked immunoassay (EIA) and recombinant immunoblot assay. Forty one transplant recipients met the working definition for true positives of HCV infection. Of these "true positives," 98% were positive by HCV RNA PCR assay, 88% by b-DNA signal amplification assay, 88% by anti-HCV EIA, and 63% demonstrated two or more reactive bands on recombinant immunoblot. Five of 57 (9%) HCV-antibody negative recipients had HCV RNA detected by both methods. Of 44 HCV enzyme-linked immunoassay (EIA) repeatedly reactive samples, the recombinant immunoblot was negative in 2 and indeterminate in 13. HCV RNA was present in 9 of 13 recombinant immunoblot indeterminate sera. Nine EIA repeatedly reactive sera were negative by both tests for HCV RNA. In liver transplant recipients, HCV infection is best determined by measurement of HCV RNA. Antibody formation may be delayed or suppressed in a minority of patients despite > 10(9) equivalents/L (> 10(6)/mL) of HCV RNA in serum. Recombinant immunoblots with a single reactive band pattern often indicate HCV infection in immunosuppressed patients.

To clarify the viral factors that may predict the therapeutic effect of interferon (IFN) in chronic hepatitis C (CHC) patients, we investigated the quantitative serum hepatitis C virus (HCV) RNA level, genotype, and liver biopsy histological features in 60 patients who were treated with 360 x 10(6) U of natural IFN-alpha for 36 to 48 weeks and for more than 12 months after therapy. A branched DNA (bDNA) assay was used to measure HCV RNA levels. All responders, defined as those individuals with normal alanine transaminase (ALT) levels at 48 weeks after therapy, had less than 2 x 10(6) HCV RNA Eq/mL before administration of IFN. Of 39 patients with RNA levels (less than 2 x 10(6) Eq/L) 23 (59.0%) were responders. The genotype was determined for each patient using type-specific polymerase chain reaction (PCR) primers. There was a significant difference in rate of response between subtype 1b and subtypes 2a and 2b (P.0002); however, all responders had less than 2 x 10(6) Eq/L independent of genotype. In a multivariate analysis, RNA level was the most statistically significant factor affecting response to IFN. Although disease severity, as defined by histological features, was not statistically correlated with nonresponse, patients that responded to IFN tended to have less severe disease.

A nonisotopic hybridization assay for human immunodeficiency virus genotypic zidovudine resistance determination is described. Biotinylated PCR product was hybridized with enzyme-labeled probes for wild-type or resistant mutant sequences and detected colorimetrically or chemiluminescently in a microplate format. Changes in mutant-to-wild-type ratios allow for the monitoring of longitudinal patient samples.

This chapter describes the quantification of viral nucleic acids using branched DNA signal amplification. Most quantitative strategies involve molecular amplification of the target nucleic acid followed by detection of the amplified product with standard, and relatively insensitive, methods. Signal amplification methods do not alter the number of target molecules and are, therefore, more amenable to quantitative analysis. In addition, viral nucleic acids are measured directly in clinical samples. Many techniques for labeling probes for Southern blots or in situ hybridization are considered signal amplification techniques, these methods usually require high concentrations of target nucleic acids. The branched oligodeoxyribonucleotides signal amplification system has been used to detect and quantify a variety of viruses in clinical samples. The assay is very reproducible compared to other nucleic acid quantification methods. The dynamic range of each assay includes at least 90% of the detectable population without dilution.

Summary. There is an increasing need for a practical assay to measure HCV RNA to assess the viral burden in chronic hepatitis C virus (HCV) infection as viral load relates to transmission and therapeutic response. This study evaluates branched DNA (bDNA) signal amplification, a technique that avoids many of the pitfalls of polymerase chain reaction (PCR). The bDNA assay uses a microtitre well format and a series of capture, target and amplification probes that bind RNA to the well and then successively bind oligonucleotides to the RNA and branched DNA molecules to the oligonucleotides. Enzyme-labelled probes are bound to the arms of the bDNA and light output from a chemiluminescent substrate is directly proportional to the amount of starting HCV RNA. Appropriate standards provide direct quantitation. Whereas PCR amplifies the HCV genome, bDNA amplifies the hybridization signal. In testing a standardized, coded panel, bDNA showed 100% specificity and detected five of six sera proven to transmit hepatitis C to the chimpanzee; PCR detected all six infectious sera. Serial samples were measured in two acute and five chronic cases of transfusion-associated hepatitis and in three commercial seroconversion panels. In acute cases, 107–108 molecular equivalents per ml (eq per ml) of HCV RNA were detected prior to peak alanine aminotransferase (ALT) activity and then rapidly declined to non-detectable levels. Similar levels of HCV RNA were observed early in the course of two patients who progressed to chronic hepatitis; the chronic course was characterized by diminished, fluctuating and sometimes non-detectable levels of HCV RNA. In two chronic cases, HCV RNA was not detected, or only transiently detected by bDNA, but was present when assayed by PCR. In one chronic case, the periodicity of HCV RNA levels closely paralleled the fluctuations of ALT suggesting a relationship between viral replication and subsequent hepatocellular injury. In testing 50 blood donors whose anti-HCV reactivity was confirmed by a recombinant immunoblot assay (RIBA), HCV RNA was detected by bDNA in 41 (81%), while PCR was positive in 45 (90%); the overall concordance between bDNA and PCR in 100 anti-HCV enzyme immunoassays (EIA) reactive donor samples was 96%. Lastly, bDNA showed the loss of HCV RNA in six out of six evaluable patients who had complete biochemical responses to interferon; five out of six non-responders also showed appreciable declines in HCV RNA level, but in only two did HCV RNA drop below the detection limit; these two cases remained PCR positive. Seventeen placebo-treated patients did not lose HCV RNA by either bDNA or PCR. Hence the bDNA assay is a practical means to measure HCV RNA in a variety of clinical settings. Although it is not as sensitive as PCR, it has greater specificity, is directly quantitative, and can be used in any routine laboratory that can perform microwell EIAs. This simplified quantitation may be of particular benefit in evaluating the probability of HCV transmission and the response to anti-viral therapy.

A branched DNA (bDNA)-based quantitation of plasma human immunodeficiency virus type 1 (HIV-1) RNA was used to monitor the virologic status of 102 patients (29-906 CD4 cells/mm3) enrolled in clinical trials of antiretroviral and immune-based therapies. Virion-associated RNA was measurable in plasma of 74% of patients tested (10,000-10,000,000 RNA equivalents/mL). Virus levels measured by the bDNA assay exceeded titers obtained by quantitative plasma culture and were inversely correlated (r = -.378; P < .05) with total CD4 cell counts. The assay was used to demonstrate a significant decline (mean, 5-fold; range, 0- to 30-fold), relative to pretreatment, in virus load after beginning antiviral therapy and a transient increase (mean, 15-fold; range, 2- to 50-fold) after treatment with interleukin-2. The decrease in RNA was more dramatic than changes in serum p24 antigen. The bDNA assay yields reproducible results, is relatively easy, and should be useful in measuring HIV-1 RNA in patients in clinical trials.

The aims of this pilot study were to evaluate the safety and efficacy of interferon-α2b for treatment of hepatitis C virus infection in liver transplant recipients, to monitor changes in hepatitis C virus RNA levels with treatment and to determine pretreatment parameters predictive of a complete response. Eighteen patients with documented hepatitis C virus viremia and histological evidence of hepatitis after liver transplantation received 3 million units of α interferon three times weekly for at least 4 mo. Pretreatment serum aminotransferase levels were at least 1.5 times the upper limit of normal and no patient had concomitant hepatitis B virus infection. Response to therapy was defined as normalization of both aspartate and alanine aminotransferase at the end of treatment. Five patients (28%) had a complete response, whereas 13 (72%) had persistent elevation of one or both aminotransferases (nonresponders). At the end of therapy, hepatitis C virus RNA levels were reduced in both responders and nonresponders (p = 0.043 and 0.039, respectively by Wilcoxon signed rank test). After cessation of treatment, aminotransferases remained normal in four of five responders but serum hepatitis C virus RNA levels returned to pretreatment levels in responders and nonresponders. There was no significant change in histological score with therapy. Responders were more likely than nonresponders to have low pretreatment hepatitis C virus RNA levels and low serum bilirubin (p = 0.0004 and 0.0077, respectively). Responders tended to have a prolonged interval between transplantation and initiation of therapy (p = 0.10 by rank logistic regression analysis). Side effects resulted in early cessation of therapy in two patients and dose reduction in six. Histological evidence of possible rejection was seen in one patient. We conclude that normalization of liver enzymes can be seen in patients with posttransplant hepatitis C virus infection on interferon therapy and that treatment is associated with fall in hepatitis C virus RNA levels, regardless of biochemical response. However, virological changes are transient. Pretreatment variables such as hepatitis C virus RNA levels and serum bilirubin may aid in the selection of patients more likely to respond to therapy and the design of future controlled clinical trials. (Hepatology 1994;20:773–779).

The branched DNA (bDNA) assay was compared with a semi-quantitative cDNA-polymerase chain reaction (cDNA-PCR) assay for monitoring HCV RNA levels in plasma in 17 haemophilia patients participating in a controlled a-interferon trial. Good correlation between the HCV RNA levels as detected by the two assays was observed, with a correlation co-efficient of 0.83 (P < 0.0001) and 0.90 (P < 0.0001) at week 0 and 24, respectively. Hepatitis C virus RNA (HCV RNA) levels could be assessed with the bDNA assay in 14/17 (82 percent) HCV cDNA-PCR positive pre-treatment samples. The bDNA assay apparently failed to detect low viral titres. (riterferon treated patients (n = 11) showed either a complete response, being a large reduction in HCV RNA level to below the detection limit of the HCV cDNA-PCR assay (6/11) or no significant reduction in HCV RNA level (5/11). A “partial” virological response was not observed. The changes in HCV RNA plasma levels in non-responders during interferon (IFN) treatment were similar to the (small) natural fluctuations in viral load observed in controls (untreated patients). Although the bDNA assay was not as sensitive as cDNA-PCR, given its user friendliness and quantitative results, it is concluded that it is a useful test for monitoring HCV RNA levels in patients treated with interferon. However, patients who are non-reactive in the bDNA assay have to be retested by cDNA-PCR because low viral titres are not detected by the bDNA assay. © 1994 Wiley-Liss, Inc.

Different isolates of hepatitis C virus (HCV) are highly polymorphic throughout the genome. Phylogenetic analysis of nucleotide sequences derived from part of the gene encoding a nonstructural protein (NS-5) has provided evidence for six major genotypes of HCV among a worldwide collection of 76 samples from HCV-infected blood donors and patients with chronic hepatitis. Many of these HCV types comprised a number of more closely related subtypes, leading to a current total of 11 genetically distinct viral populations. Analysis of other regions of the viral genome produced equivalent relationships between published sequences to those found in NS-5, apart from the more highly conserved 5′ noncoding region where only the six major HCV types, but not subtypes, could be differentiated. A new nomenclature for HCV variants is proposed in this communication that reflects the two-tiered nature of sequence differences between different viral isolates.