Saliva, diagnostics, and dentistry.

Saliva, a scientific and clinical entity familiar to every oral health researcher and dental practitioner, has emerged as a translational and clinical commodity that has reached national visibility at the National Institutes of Health and the President's Office of Science and Technology. "Detecting dozens of diseases in a sample of saliva" was issued by President Obama as one of the 14 Grand Challenges for biomedical research in the 21(st) Century (National Economic Council, 2010). In addition, NIH's 2011 Government Performance Report Act (GPRA) listed 10 initiatives in the high-risk long-term category (Collins, 2011). The mandate is to determine the efficacy of using salivary diagnostics to monitor health and diagnose at least one systemic disease by 2013. The stage is set for the scientific community to capture these national and global opportunities to advance and substantiate the scientific foundation of salivary diagnostics to meet these goals. A specific calling is to the oral, dental, and craniofacial health community. Three areas will be highlighted in this paper: the concept of high-impact diagnostics, the role of dentists in diagnostics, and, finally, an infrastructure currently being developed in the United Kingdom--The UK Biobank--which will have an impact on the translational and clinical utilizations of saliva.

Quantification of HCV RNA in Clinical Specimens by Branched DNA (bDNA) Technology.

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.

Quantification of HCV RNA in Liver Tissue by bDNA Assay.

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.

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.

Effect of interferon-alpha 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.

A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml.

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 <500 molecules/ml. In all 11 patients viral load eventually declined to below the detection limit with the new assay.

Hepatitis C--diagnosis and monitoring.

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 RIBA 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.

Circulating CD8 T cells show increased interferon-gamma mRNA expression in HIV infection.

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.

Detection of GB virus-C/hepatitis G virus RNA in serum by reverse transcription polymerase chain reaction.

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/HGV 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.

Hepatitis C virus: quantitation and distribution in liver.

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.

An improved divergent synthesis of comb-type branched oligodeoxyribonucleotides (bDNA) containing multiple secondary sequences.

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.

Chemical synthesis and characterization of branched oligodeoxyribonucleotides (bDNA) for use as signal amplifiers in nucleic acid quantification assays.

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.

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

Tumor necrosis factor-alpha (TNF-alpha) plays a central role in the host's immunomodulatory response to infective agents. To evaluate the TNF-alpha system in patients with chronic hepatitis C virus (HCV) infection, plasma, serum, and peripheral blood mononuclear cells (PBMC) were prospectively collected from 53 patients and 33 healthy control subjects. Circulating TNF-alpha and TNF receptors were assayed by their respective enzyme immunoassays. In addition, TNF-alpha mRNA was quantitated in PBMC using a branched DNA assay, and production of TNF-alpha by PBMC with and without lipopolysaccharide was also assessed. Patients with chronic HCV infection had a higher level of circulating TNF-alpha compared to healthy control subjects (9.62 +/- 6.01 vs 3.66 +/- 1.23 pg/ml, P < 0.001). They also had higher circulating 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-alpha level correlated with circulating CD120a (r = 0.52, N = 49, P < 0.001) and weakly with CD120b (r = 0.32, N = 51, P = 0.02). Plasma TNF-alpha also correlated with markers of hepatocellular injury, including ALT (r = 0.34, N = 53, P = 0.01) and alpha-GST (r = 0.31, N = 43, P = 0.042), but not with serum HCV RNA levels. There was no difference in the TNF-alpha mRNA levels in PBMC between patients with chronic HCV infection (1.4 +/- 1.9 units/10[6] cells, N = 8) and healthy control subjects (2.1 +/- 1.4 units/10[6] cells, N = 8, P = NS). There was also no difference in the spontaneous production of TNF-alpha by PBMC (1 x 10[6] 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, patients with chronic HCV infection produced more TNF-alpha upon stimulation with lipopolysaccharide compared to healthy control subjects (1278 +/- 693 pg/ml, N = 11, vs 629 +/- 689 pg/ml, N = 14, P < 0.05). These data indicate that the TNF-alpha system is activated in patients with chronic HCV infection.

HCV viraemia is more important than genotype as a predictor of response to interferon in Sicily (southern Italy).

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 (x 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.

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique.

We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n = 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.

Hepatitis C virus RNA quantification in right and left lobes of the liver in patients with chronic hepatitis C.

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.

Significance of hepatic expression of hepatitis C viral antigens in chronic hepatitis C.

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 (P < 0.02). 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.

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

Efficacy of interferon alfa therapy in chronic hepatitis C patients depends primarily on hepatitis C virus RNA level.

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.