Judith C. Wilber

Significance of serum hepatitis C virus RNA levels in chronic hepatitis C.

Hepatitis C virus (HCV) is the main cause of parenteral non-A, non-B hepatitis and serum can be tested for the virus itself by reverse-transcription polymerase chain amplification. What of the level of this viraemia? To find out if quantitative study of HCV RNA might be useful clinically we took advantage of participation in trials of interferon-alpha in patients with chronic HCV infection and applied a new assay, branched DNA (bDNA) signal amplification. Paired serum and liver biopsy specimens from 47 patients with confirmed chronic HCV infection and evidence of HCV RNA in their serum were studied. The quantitative bDNA assay (detection limit 350,000 equivalents/mL [eq/mL]) was positive in 34 sera (sensitivity 72%). Patients who acquired HCV infection by blood transfusion had a higher viraemia (median 2,701,000 eq/mL, n = 29) than health workers and intravenous drug users (635,000 eq/mL, n = 13; p < 0.01). Patients with a sustained complete response to interferon-alpha therapy had lower pre-treatment viraemia levels (median at bDNA cut-off, n = 11) than complete responders who relapsed after the drug was stopped (1,613,000 eq/mL, n = 15; p < 0.01) and non-responders (3,066,000 eq/mL, n = 20; p < 0.01). High viraemia levels were not related to the histological diagnosis but were associated with lobular inflammation, lymphoid aggregates, and bile-duct lesions. These findings indicate that mode of acquisition is an important determinant of HCV viraemia and that patients with low HCV viraemia levels are more likely to respond to interferon in a sustained fashion.

Seropositivity for HIV and the development of AIDS or AIDS related condition: three year follow up of the San Francisco General Hospital cohort

The three year actuarial progression rate to the acquired immune deficiency syndrome (AIDS) in a cohort of men in San Francisco who were seropositive for the human immuno-deficiency virus (HIV) was 22%. An additional 26 (19%) developed AIDS related conditions. β2 Microglobulin concentration, packed cell volume, HIV p24 antigenaemia, and the proportion and number of T4 lymphocytes each independently predicted progression to AIDS. β2 Microglobulin was the most powerful predictor. The 111 subjects tested who were normal by all predictors (40%) had a three year progression rate of 7%, and the 68 subjects who were abnormal by two or more predictors (24%) had a progression rate of 57%. Two thirds of all men who progressed to AIDS were in the last group. The median T4 lymphocyte count in subjects who did not progress to AIDS fell from 626 × 106 to 327 × 106/1. HIV p24 antigenaemia developed in 7% of the subjects per year. The proportion who were abnormal by two or more predictive variables rose to 41%. At three years an estimated two thirds of the seropositive subjects showed clinical AIDS, an AIDS related condition, or laboratory results that were highly predictive of AIDS. It is concluded from the observed rates and the distribution of predictive variables at three years that half of the men who were seropositive for HIV will develop AIDS by six years after the start of the study, and three quarters will develop AIDS or an AIDS related condition.

Surrogate markers for survival in patients with AIDS and AIDS related complex treated with zidovudine.

OBJECTIVE--To determine whether early effects of zidovudine treatment on CD4+ lymphocyte count and concentrations of beta 2 microglobulin, neopterin, or HIV p24 antigen or antibody are correlated with survival in patients with AIDS or AIDS related complex. DESIGN--Retrospective study of changes in laboratory markers and survival. SETTING--Multicentre trial at university hospital clinics. SUBJECTS--90 Patients with AIDS or AIDS related complex. INTERVENTION--Patients started zidovudine 200 mg orally every four hours. Fifty six of the patients died a median 17 months after starting zidovudine; the remaining 34 patients were followed up for a median 25.5 months. MAIN OUTCOME MEASURES--Changes in CD4+ lymphocyte count and serum concentrations of p24 antigen and antibody, beta 2 microglobulin, and neopterin; survival of the patient. RESULTS--The pretreatment characteristics that independently predicted poor survival were determined using a multivariate proportional hazards model: a diagnosis of AIDS (v AIDS related complex), age over 45 years, and the logarithm of serum neopterin concentration. When these baseline characteristics were controlled for the logarithm of CD4+ lymphocyte count at weeks 8-12 of treatment (p = 0.007) and an increase in serum beta 2 microglobulin concentration at weeks 8-12 (p = 0.05) also independently correlated with survival. In the 38 patients with a better pretreatment prognosis, 24 month survival estimated by the product-limit method was 88% for those with a good response on both surrogate markers during early treatment compared with only 50% for those with a poor response on either marker. In the 38 with a worse pretreatment prognosis, 24 month survival was estimated to be 49% for those with a good response on both surrogate markers compared with only 18% for those with a poor response on either. CONCLUSION--These data suggest that CD4+ lymphocyte count at 8-12 weeks and, perhaps, change in serum beta 2 microglobulin concentration could be surrogate end points for clinical outcome in trials of antiretroviral drugs for patients with HIV disease.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

AimTo determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. MethodTwo 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. ResultsIn one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 105 copies/ml, and the branched DNA results ranged from 2.6 x 104 to 4.2 x 104 HIV RNA equivalents/ml. In the other sample the corresponding figures were 6.3 x 104 to 5.5 x 105 copies/ml and 5.7 x 104 to 7.5 x 104 HIV RNA equivalents/ml. ConclusionIn 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.

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

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 to serum viral load varies widely. The clinical use of measurement of viral load in the liver remains to be defined. J. Med. Virol. 51:217–224, 1997.

Hepatitis C (HCV) genotype and viral titer distribution among Argentinean hemophilic patients in the presence or absence of human immunodeficiency virus (HIV) co‐infection

Hepatits C (HCV) infection is frequent among hemophilic patients treated with non‐inactivated factor‐concentrates. Both HCV genotype and viral load have been suggested to be important prognostic markers of disease progression and treatment outcome. In addition, co‐infection with the human immunodeficiency virus (HIV) has been associated with increased level of HCV replication and higher risk of developing liver failure. Thus, HCV genotype, viral load, and HIV co‐infection are important factors in HCV infection.

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

Branched DNA for Quantification of Viral LOAD

This is a summary of a presentation made at the 13th International Convocation on Immunology. Nucleic acids in patient samples can be quantified directly using a solid phase nucleic acid hybridization assay based on branched DNA (bDNA) signal amplification technology. For example, HIV RNA is detected in a plasma sample by hybridization of multiple specific synthetic oligonucleotides to the target, 10 of which capture the target onto the surface of a microwell plate and 39 of which mediate hybridization of branched DNA molecules to the pol region of each HIV RNA molecule. Alkaline phosphatase-labeled probes bind to each arm of the branched DNA molecules. Detection is achieved by incubating the complex with a chemiluminescent substrate and measuring the light emission. The signal is directly proportional to the level of target nucleic acid, and the quantity of HIV RNA in a sample is determined by comparison with a 4-point standard curve. In order to ensure that different subtypes of HIV-1 were detected and quantified equally, in vitro RNA transcripts of the pol region of HIV subtypes A-F were purified and quantified by OD 260, phosphate analysis, and hyperchromicity. These characterized transcripts were then quantified using the bDNA assay. Comparisons were made using a ratio of signal per attomole for each transcript. Genetic subtypes A-F quantified within a factor of 1.5, indicating that the bDNA assay can be used to measure viral load in clinical samples regardless of genotype. Accuracy is important because several studies indicate that there may be a threshold level of virus which predicts progression of HIV disease. Detection of change in viral load is important in determining the efficacy of therapy. The bDNA assay for HCV RNA can be used to determine level of virus in HCV-infected individuals and assist in establishing prognosis prior to initiation of alpha-interferon therapy. Patients with lower levels of virus are more likely to have a sustained response to therapy. Patients who respond to treatment typically have a rapid decline in virus load within one to four weeks of the start of therapy. Many patients relapse when therapy is discontinued as evidenced by a rise in virus load to near pre-treatment levels. Sustained response is most often seen with patients who have lower pre-treatment levels of RNA.