Hepatitis C Virus Polymerase Chain Reaction Research Articles

Prospective Follow‐Up of Patients with Acute Hepatitis C Virus Infection in Brazil

The natural outcome of infection with hepatitis C virus (HCV) varies substantially among individuals. However, little is known about host and viral factors associated with a self-limiting or chronic evolution of HCV infection. From 1 January 2001 through 31 December 2008, a consecutive series of 65 patients from Rio de Janeiro, Brazil, with a well-documented diagnosis of acute HCV infection, acquired via various routes, were enrolled in this study. Patients were prospectively followed up for a median of 40 months after the estimated date of HCV infection with serial measurements of serum alanine aminotransferase, HCV RNA, and anti-HCV antibodies. Spontaneous viral clearance (SVC) was defined as undetectable levels of HCV RNA in serum, in the absence of treatment, for 3 consecutive HCV polymerase chain reaction tests within the first 6 months of follow-up. Cox proportional hazards regression was used to identify host and viral predictors of SVC. The cumulative rate of SVC was 44.6% (95% confidence interval, 32.3%-57.5%). Compared with chronic HCV evolution, patients with self-limiting disease had significantly lower peak levels of anti-HCV antibodies (median, 109.0 vs 86.7 optical density-to-cutoff ratio [od/co]; P<.02), experienced disease symptoms more frequently (69.4% vs 100%; P<.001), and had lower viral load at first clinical presentation (median, 4.3 vs 0.0 log copies; P=.01). In multivariate analyses, low peak anti-HCV level (<93.5 od/co) was the only independent predictor for SVC; the hazard ratio compared with high anti-HCV levels (> or =93.5 od/co) was 2.62 (95% confidence interval, 1.11-6.19; P=.03). Our data suggest that low levels of anti-HCV antibodies during the acute phase of HCV infection are independently related to spontaneous viral clearance.

Hepatocellular carcinoma in the absence of liver cirrhosis in a treated hepatitis C virus patient.

To the Editor: A 65-year-old male patient was referred to King Faisal Hospital and Research Centre after he presented with jaundice, fever, abdominal discomfort and biochemical evidence of chronic hepatitis C virus (HCV) hepatitis with no clinical or histological evidence of cirrhosis. HCV polymerase chain reaction (PCR) was positive and HCV quantitation was 4682 copies/mL. Liver function test (LFT) results were ALT 225 IU/L (reference range, 5-60 IU/L), AST 104 IU/L (reference range, 5-43 IU/L), total bilirubin 20 μml/L (reference range, 5.1-17.0 μmol/L), INR 1.2 (reference range, 0.9-1.1), and albumin 38 g/L (reference range, 35-50 g/L). Liver biopsy showed chronic hepatitis grade II, stage II portal fibrosis and no evidence of cirrhosis. Alpha-fetoprotein was 6 μgm/L. The patient completed a full year of combination therapy of pegulated interferon 3 million units subcutaneously 3 times/week with ribavirin 1000 mg orally daily and achieved a negative viral PCR with normal LFTs. He had 3 years of follow-up with a sustained virological response (sustained loss of detectable virus in response to antiviral therapy) and was then discharged from our clinic and instructed to follow-up at his local hospital. A CT scan at discharge showed no evidence of cancer (image not available). Two years later he was referred back from his local hospital with a huge right hypochondrial mass associated with pain but no jaundice, fever or weight loss. The mass was about 10 cm in diameter, difficult to separate from the liver, ill-defined, lobulated and hard. Basic tests, including CBC and renal profile were normal. ALT 27 IU/L (reference range, 5-60 IU/L), AST 35 IU/L (reference range, 5-43 IU/L), ALP 75 IU/L (reference range, 30-120 IU/L), total bilirubin 14 μml/L (reference range, 5.1-17.0 μmol/L), INR 1.0 (reference range, 0.9-1.1), and albumin 42 g/L (reference range, 35-50 g/L). AFP was 2.3 μgm/L and carcinoembryonic antigen (CEA) was 2.1 μgm/L. Ultrasound of the abdomen showed a huge liver mass (8cm×11cm) and the right portal vein branch was not visualized distally. CT of the abdomen showed the same mass at segment 5 and 8 which was consistent with hepatocellullar carcinoma (HCC) showing the classical triphasic pattern, i.e., heterogenous enhancement in the arterial phase, but early rapid washout in the portal-venous phase leaving the HCC lesion hypo-dense compared to the surrounding normal tissue (Figure 1). The patient underwent surgical removal of both segments (5 and 8). Histopathology of the mass confirmed the diagnosis of HCC. H&E stain showed the HCC pseudoglandular and trabecular pattern (Figure 2) and extensive lymphovasculer invasion. Immunohistochemical stain for CD34 was positive and showed wrapping of the tumor cluster by endothelial cells. The rest of the non-neoplastic livers showed only mild steatosis and no cirrhosis at all (Trichrome stain) (Figure 3).

Prevalence and Incidence of Hepatitis C Virus in Hemodialysis Patients in British Columbia: Follow‐up after a Possible Breach in Hemodialysis Machines

A possible breach of the transducer protector in specific dialysis machines was reported in June 2004 in British Columbia (BC), which led to testing of hemodialysis patients for hepatitis C virus (HCV), hepatitis B virus (HBV) and HIV. This testing provided an opportunity to examine HCV incidence, prevalence and coinfection with HBV and HIV, and to compare anti-HCV and HCV polymerase chain reaction (PCR). The results of hemodialysis patients who were dialyzed on the implicated machines (65% of BC dialysis patients), and tested for HCV, HBV and HIV, between June 1, 2004, and December 31, 2004, were reviewed and compared with available previous results. Of 1286 hemodialysis patients with anti-HCV and/or HCV-PCR testing, 69 (5.4%) tested positive. Two HCV genotype 4 seroconversions were identified. HCV incidence rate on dialysis was 78.8 cases per 100,000 person-years. Younger age, history of renal transplant and past HBV infection were associated with HCV infection. No occult infection was identified using HCV-PCR. Hemodialysis patients had three times the HCV prevalence rate of the general BC population, and more than 20 times the incident rate of the general Canadian population. One of the two seroconversions occurred before the testing campaign; the patient was likely infected during hemodialysis in South Asia. The other was plausibly a late seroconversion following renal transplant in South Asia. Nosocomial transmission cannot be ruled out because both patients were dialyzed in the same centre. Baseline and annual anti-HCV testing is recommended. HCV-PCR should be considered at baseline for persons with HCV risk factors, and for returning travellers who received dialysis in HCV-endemic countries to identify HCV infection occurring outside the hemodialysis unit.

Viral Hepatitis C in a leading Brazilian hospital: epidemiological factors and genotyping

The hepatitis C virus (HCV) is classified into six different genotypes and their distribution is different throughout the world. Epidemiologic studies are important to determine several characteristics of the virus, as well as the disease. This study analysed the prevalence of HCV and its genotypes among patients from a leading hospital in Ceara, which is located in Northeast Brazil. A total of 119 anti-HCV-seropositive patients, each having previously completed a questionnaire about risk behaviours related to HCV infection were tested for HCV infection using a qualitative HCV polymerase chain reaction (PCR) assay and genotyping by restriction fragment length polymorphism (RFLP). The detection was based on amplifying of the non-coding 5' region. Of the 119 patients, 95 showed positive results in the qualitative HCV test. History of surgery was the most reported risk factor, followed by the use of drugs, having tattoos, undergoing haemodialysis and occupational exposure. Genotype 1 was the most prevalent (46.9%), followed by genotype 3 (34.4%) and 2 (8.3%). The genotype distribution was similar for all of the various risk behaviours.

The epidemiology of transfusion‐associated hepatitis C in a children's hospital

Children transfused with blood and blood products before 1992 are at risk for chronic hepatitis C virus (HCV) infection. To determine the prevalence of HCV infection and risks associated with acquisition of HCV, a single-institution lookback study was performed. A total of 5473 infants and children who received transfusions between 1982 and 1992 were identified. A control population of 600 age-, sex-, race- and zip code-matched children who did not receive transfusions with the same exclusions provided background seroprevalence data. Patients were tested for antibodies to HCV, confirmed with second generation recombinant immunoblot assay (RIBA) and when appropriate quantitative and qualitative HCV RNA by reverse transcription polymerase chain reaction (PCR). Viral persistence was assessed by serial PCR determinations for HCV RNA. Of the 5473 eligible patients, 4726 were locatable and 2758 were tested. Forty-three children (1.6%) were persistently anti-HCV enzyme immunoassay (EIA)-positive, confirmed by RIBA; 39 were positive for the presence of HCV RNA. Four cleared their virus as assessed by two negative HCV PCRs 6 months apart. There was a borderline higher number of children with HCV who received fresh whole blood than those who tested HCV-negative. Because HCV infection is generally asymptomatic, children are not identified unless they are specifically tested. We identified, enrolled, tested, and confirmed a new diagnosis of HCV infection in 43 patients. As HCV treatments become increasingly effective, it is important to identify silently infected individuals, particularly when the infection was iatrogenically induced.

Analysis of the relationship between cytokine secretion and proliferative capacity in hepatitis C virus infection

CD4(+) T-cell responses are important for the outcome of hepatitis C virus (HCV) infection. However, the functional status of HCV-specific CD4(+) T cells in persistent infection is poorly understood. It is generally recognized that proliferative capacity of HCV-specific CD4(+) T cells is weak or absent in persistent infection, but whether this results from deletion of antigen-specific cells or represents maintenance of antigen-specific but poorly proliferative populations is not defined. We used a set of ex vivo assays to evaluate the functionality of HCV specific CD4(+) T cells in persistent and resolved infection. Peripheral blood mononuclear cells (PBMC) from 24 prospectively recruited HCV polymerase chain reaction (PCR) positive individuals, 12 spontaneously resolved individuals (i.e. anti-HCV+, PCR-) and 11 healthy controls were analysed for interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) secretion by enzyme linked immunospot assays (ELISpot). HCV-specific CD4(+) proliferative responses of carboxy fluorescein succinimidyl ester-labelled PBMC were assessed using a sensitive single cell flow cytometric assay. Sustained IFN-gamma ELISpot responses were observed in the PCR+ group. However, proliferation of HCV-specific CD4(+) T cells in the PCR+ group was substantially reduced on a per cell basis, in parallel to IL-2 secretion, compared with responses in the PCR- group. In PCR- individuals, a strong relationship between cytokine secretion and proliferative capacity was seen. However, in PCR+ individuals, IFN-gamma secretion far exceeded proliferative capacity. During persistent HCV infection, some CD4(+) T-cell specificities appear to be lost, as measured using a range of techniques, but others, potentially important, are maintained as IFN-gamma secretors but with low proliferative capacity even using a highly sensitive assay. Such subsets may yet play a significant role in vivo and also provide a template for modulation in immunotherapeutic interventions.

Factors Associated with Spontaneous Clearance of Hepatitis C Virus among Illicit Drug Users

Spontaneous clearance of hepatitis C virus (HCV) occurs in approximately 25% of individuals. To better understand the characteristics associated with clearance, the present study evaluated HCV clearance in a community-based cohort study. The Community Health and Safety Evaluation project recruited 3553 individuals via community organizations and door-to-door canvassing of a random sample of single occupancy hotels in the community to monitor uptake of health services and to estimate the incidence of communicable infections. Cohort data were linked with longitudinal laboratory databases, including HCV antibody and polymerase chain reaction assay results. Overall, 762 individuals had HCV antibody and RNA testing performed between 1999 and 2005. Spontaneous HCV clearance was observed in 179 individuals (23.5%), while HCV persistence was observed in 583 individuals (76.5%). The ability to develop protective immunity against HCV, as demonstrated by viral clearance, occurred more often in individuals of Aboriginal ethnicity (adjusted OR [AOR] 2.9, 95% CI 2.0 to 4.3; P<0.001) and female individuals (AOR 1.6, 95% CI 1.1 to 2.4; P=0.01). The rate of spontaneous HCV clearance was reduced in individuals using any type of illicit drugs (AOR 0.54, 95% CI 0.29 to 1.00; P=0.05) and those with HIV coinfection (AOR 0.58, 95% CI 0.38 to 0.88; P=0.01). Of 218 HIV-infected subjects, 48 of 51 (94%) in whom the order of HCV and HIV infection was established were infected with HCV a median of 2.4 years (range 0.2 to 10 years) before becoming infected with HIV. Aboriginal ethnicity and female sex were associated with increased rates of HCV clearance, while HIV coinfection and illicit drug use were associated with increased HCV persistence.

Suppressive Anti-HCV Therapy for Prevention of Donor to Recipient Transmission in Stem Cell Transplantation

A 48-yr-old man with acute myeloid leukemia (AML) required urgent allogeneic hematopoietic stem cell transplantation because of failed attempts to induce remission via chemotherapy. He had an HLA identical donor sister who was hepatitis C virus (HCV) RNA positive. In order to prevent HCV transmission to her brother, the donor was treated with weekly injections of pegylated interferon alfa-2b (150 mug subcutaneously every week) and daily ribavirin (1 g/day) for 5 wk at which time her qualitative polymerase chain reaction (PCR) was negative. Her stem cells were successfully grafted into the recipient. The recipient remained HCV PCR negative after transplant until death from relapsed AML.

Longitudinal Long-term Follow-up Study of Children with Vertically Acquired Hepatitis C Virus Infection

Seventeen children with vertically acquired hepatitis C virus (HCV) infection were followed from birth for a mean of 104 months. Alanine aminotransferase (ALT) levels were increased significantly at 3 and 6 months of age but were stable thereafter. HCV polymerase chain reaction was positive at 3 months in 16 patients and at 12 months in one patient. Viral load remained stable during follow-up at a mean value of 5.4 +/- 0.4 log10. Mild chronic hepatitis was the most common histopathological feature on liver biopsy, occurring in six of the seven children biopsied at a mean age of 4.0 +/- 2.4 years. Genotype did not seem to be related to the type of liver involvement. The results of this study suggest that vertically acquired HCV infection has a benign course in children, despite the presence of viraemia and persistent alterations in ALT levels.

Resolution of diabetes in type 2 diabetic patient treated with IFN-?? and ribavirin for hepatitis C

We report on a patient whose type 2 diabetes mellitus resolved during IFN-alpha therapy for hepatitis C virus (HCV). A 40-year-old man was diagnosed with type II diabetes in year 2000. His body mass index (BMI) was 30.8 kg/m and glycosylated haemoglobin (HbA1c) was 10.7%. He was treated with metformin. Later, his glycaemic control deteriorated despite additional dietary and lifestyle advice and the addition of glibenclamide. He was started on subcutaneous insulin in 2002 with the continuation of metformin. In 2003 he was diagnosed with chronic hepatitis caused by HCV. In September 2003 he was started on IFN-alpha and ribavirin. After 24 weeks of treatment his HCV polymerase chain reaction remained positive and treatment was stopped as per guidelines. At the commencement of antiviral therapy, HbA1c was 7.7%. In April 2004 his BMI of 29.38 kg/m had reduced and he then stopped insulin therapy because of repeated hypoglycaemia. After stopping insulin his HbA1c was 4.7%. Fasting plasma glucose of 6.2 mmol/l and anti-glutamic acid decarboxylase antibodies were negative. Urea and creatinine levels were normal. Most of the earlier literature describes diabetes developing in the course of IFN-alpha therapy for a variety of diseases. More recent research has described a relationship between hepatitis C infection and the development of diabetes and insulin resistance. Responders to IFN-alpha treatment manifest an improvement in insulin sensitivity compared with non-responders after the completion of IFN-alpha therapy. Our case shows the resolution of pre-existing diabetes in a patient with chronic HCV infection, which did not respond to IFN-alpha therapy. Whether this occurred as a direct result of IFN-alpha on insulin sensitivity or indirectly as a result of weight loss because the therapy for HCV precipitated additional lifestyle changes in the patient is as yet unclear.

Psychological implications of hepatitis C virus diagnosis

Hepatitis C virus (HCV) diagnosis causes significant psychological stress and anxiety. We thought it would be important to illustrate the anxiety caused by HCV diagnosis in patients from the developing world. This study was conducted at the Shifa International Hospital, Islamabad, between February 2004 and April 2004. All patients who were recently diagnosed with HCV (those who tested positive to anti-HCV and HCV polymerase chain reaction) were given a questionnaire that compared stress due to HCV infection with four other variables, including death of a close family member, divorce, loss of source of income and move to another city. We also analyzed the anxiety level among these patients using the Beck Anxiety Inventory (BAI). We studied 98 patients and 100 healthy controls. Thirty-one (31.63%) patients who were diagnosed with HCV presented only with fatigue. Sixty-three (64.28%) patients admitted that diagnosis of HCV interfered with their daily life in some way. Among the newly diagnosed cases, 48 (48.97%) patients had moderate to severe anxiety (BAI Class B and C). Multi-logistic regression analysis showed that anxiety was related to HCV-related stress (P < 0.002) and self-perceived severity of disease (P < 0.001). HCV diagnosis was significantly more stressful than divorce (59.87 vs 70.95; P < 0.013), loss of source of income (50.52 vs 70.80; P < 0.001), and a move to another city (28.32 vs 70.80; P < 0.001). Diagnosis with HCV is reported to be more stressful than divorce, loss of source of income and a move to another city.

Prolonged Time until Seroconversion among Hemodialysis Patients: The Need for HCV PCR

Objective: Patients on maintenance hemodialysis are known to have an elevated risk of acquiring hepatitis C virus (HCV) infection. Therefore, a reliable diagnosis of HCV infection is essential in order to prevent the spread of the disease in dialysis units. However, whether PCR examination is dispensable in hemodialysis patients has been debated. Methods: From 1995 to 2002, serum samples from all hemodialysis patients at our hospital (n = 1,774) were screened by serological assays and by polymerase chain reaction (PCR). Results: In 25 of these patients acute HCV infection was observed and in 11 patients HCV seroconversion was delayed for 3–16 months. During this time the infection was exclusively detectable by PCR. Conclusion: Despite the growing demand for cost-effectiveness in the health system, HCV PCR examination must remain an essential part of the routine screening in hemodialysis patients.

Depressive symptoms and viral clearance in patients receiving interferon-α and ribavirin for hepatitis C

Interferon (IFN)-α plus ribavirin is an effective treatment for hepatitis C virus (HCV) infection, but is associated with a high rate of depression. Depression has been linked to a worse outcome in multiple medical disorders including viral illnesses. We examined whether increased symptoms of depression during IFN-α/ribavirin therapy were associated with a reduced treatment response as assessed by clearance of HCV. Depressive symptoms were evaluated in 102 HCV-infected patients at baseline and after 4, 8, 12, and 24 weeks of pegylated IFN-α-2b plus ribavirin therapy using the Zung self-rating depression scale (SDS). Viral clearance was determined at 24 weeks by polymerase chain reaction (PCR). Only 34% of subjects (10 out of 29) with a 20-point or greater increase in SDS Index score were HCV PCR negative at 24 weeks, compared to 59% (24 out of 41) of patients with a 10–19 point increase in SDS Index and 69% (22 out of 32) of patients with a less than 10 point increase ( χ 2=7.6, df=2, p<0.05). In addition, a 20-point or greater increase in SDS Index score during IFN-α/ribavirin therapy significantly predicted failure to clear virus when considered alone [crude odds ratio (OR), 3.2; 95% confidence interval (CI), 1.3–8.0; p<0.01] or when controlling for other factors that affected IFN-α treatment response (adjusted OR, 3.6; 95% CI, 1.3–9.5; p=0.01). These preliminary findings suggest that individuals who experience significant increases in depressive symptoms during IFN-α/ribavirin therapy may be less likely to clear virus, highlighting the importance of identifying and treating depressive symptoms in this patient population.

Diagnosis of Acute Hepatitis C: anti-HCV or HCV-RNA?

Background: In theory, the optimal method for diagnosing acute hepatitis C is nucleotide amplification. This is because of the significant delay in the emergence of hepatitis C virus (HCV) antibodies. We studied whether the use of HCV polymerase chain reaction (PCR) screening for acute HCV infection in a clinical setting would identify otherwise undetected cases. Methods: Patients clinically suspected of having acute viral hepatitis were tested over a 32-month period (n = 2023). Results: Sixty-four patients were found HCV ribonucleic acid (RNA) positive. Of these, 13 were suffering from an acute infection and 12 of these 13 patients were concomitantly anti-HCV (and HCV-RNA) positive at the time of diagnosis. One patient was HCV-RNA positive and anti-HCV negative. This symptom-free patient was tested because of known exposure to HCV 2 weeks previously. Conclusion: Anti-HCV is reliable in screening for acute hepatitis C. In cases of known/possible HCV exposure, we find that HCV PCR is the diagnostic of choice.

Development of an HCV infection risk stratification algorithm for patients on chronic hemodialysis

OBJECTIVE: The prevalence of hepatitis C virus (HCV) in patients on chronic hemodialysis (HD) is near 9%. Transaminases, which are lower in HD patients, are not effective in screening for HCV. Our aim was to design an HCV risk stratification strategy incorporating lowered aminotransferase levels and other clinical parameters. METHODS: Patient serum from 168 consecutive HD patients was analyzed for AST, ALT, ferritin, and hepatitis C antibody. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for lower transaminase values. Multivariate classification and regression tree analysis was used to determine the best combination of variables to predict risk for HCV infection. RESULTS: Median AST and ALT levels were higher in anti-HCV Ab(+) patients ( p < 0.05). Applying a lower cutoff value for ALT of 16 IU/L resulted in a sensitivity of 61.1%, a specificity of 66.7%, a positive predictive value of 33.9%, and a negative predictive value of 86.0% for detection of HCV infection. Multivariate classification and regression tree analysis derived an algorithm using patient age, months on HD, and AST, resulting in a 97.2% sensitivity and a 51.9% specificity for the detection of HCV(+) HD patients. CONCLUSIONS: A lower normal cutoff value of 18 IU/L for AST and 16 IU/L for ALT increased sensitivity and specificity for the detection of HCV infection in HD patients. An algorithm combining lower transaminases with clinical parameters improved both sensitivity and specificity in HCV detection. Prospective confirmation of this algorithm would allow more selective HCV enzyme immunoassay and polymerase chain reaction testing in dialysis units.

Development of an HCV infection risk stratification algorithm for patients on chronic hemodialysis.

The prevalence of hepatitis C virus (HCV) in patients on chronic hemodialysis (HD) is near 9%. Transaminases, which are lower in HD patients, are not effective in screening for HCV. Our aim was to design an HCV risk stratification strategy incorporating lowered aminotransferase levels and other clinical parameters. Patient serum from 168 consecutive HD patients was analyzed for AST, ALT, ferritin, and hepatitis C antibody. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated for lower transaminase values. Multivariate classification and regression tree analysis was used to determine the best combination of variables to predict risk for HCV infection. Median AST and ALT levels were higher in anti-HCV Ab(+) patients (p < 0.05). Applying a lower cutoff value for ALT of 16 IU/L resulted in a sensitivity of 61.1%, a specificity of 66.7%, a positive predictive value of 33.9%, and a negative predictive value of 86.0% for detection of HCV infection. Multivariate classification and regression tree analysis derived an algorithm using patient age, months on HD, and AST, resulting in a 97.2% sensitivity and a 51.9% specificity for the detection of HCV(+) HD patients. A lower normal cutoff value of 18 IU/L for AST and 16 IU/L for ALT increased sensitivity and specificity for the detection of HCV infection in HD patients. An algorithm combining lower transaminases with clinical parameters improved both sensitivity and specificity in HCV detection. Prospective confirmation of this algorithm would allow more selective HCV enzyme immunoassay and polymerase chain reaction testing in dialysis units.

Outcome of Infants Born to Hepatitis C Infected Women

Hepatitis C virus (HCV) may be transmitted from mother to infant either during late pregnancy or at the time of delivery. Outcomes were studied in 314 infants born to 296 HCV-positive women in the years 1994-1999. The infants were monitored prospectively from birth for as long as 52 months and for a median time of 18 months. The study infants were bom at a median gestational age of 39 weeks and had a median birth weight of nearly 3 kg; 40% were below the 10th centile for body weight. Neonatal abstinence syndrome requiring treatment developed in 46% of infants. None of the three deaths appeared to result from HCV infection. Infection was documented in 11 of 173 infants evaluated. Four infants had HCV RNA detected on a single occasion. The vertical transmission rate was 6.4%. Infection was diagnosed at a median age of 3 months. Liver transaminases were elevated in 8% of uninfected infants. Although a negative result on an HCV polymerase chain reaction (PCR) test before age 1 month did not rule out infection, all infected infants had HCV RNA detected when next examined at age 2 to 10 months. In no case was there significant exposure to blood or body fluids that made postnatal transmission unlikely. Antibody responses waned at age 3 to 6 months in a majority of infected infants, and without PCR testing, this observation could have been incorrectly ascribed to seroreversion. Three infants had an enlarged liver. All those infected had elevated liver transaminase levels but normal bilirubin levels. One infant developed acute hepatitis at age 6 months but had a favorable course. Another had liver biopsy changes of mixed inflammation, piecemeal necrosis, and mild fibrosis; HCV RNA subsequently became undetectable. None of the infants have yet received specific anti-HCV treatment. None are infected by HIV. Of the 162 uninfected infants, 77% seroconverted by age 12 months; the median age was 9 months. No late seroconversion has been noted. Thirteen of these infants had transient liver transaminase elevations unrelated to intercurrent illness; in one instance the elevation persisted at 32 months, but no cause was found. The authors propose, as a minimum monitoring schedule for HCV-exposed infants, that HCV PCR and transaminase estimates be repeated at age 6 to 8 weeks, HCV PCR and antibody testing at 6 months, and final testing at 18 months to confirm seroreversion and detect any late seroconversion.

Automation of laboratory testing for infectious diseases using the polymerase chain reaction-- our past, our present, our future.

While it is an extremely powerful and versatile assay method, polymerase chain reaction (PCR) can be a labor-intensive process. Since the advent of commercial test kits from Roche and the semi-automated microwell Amplicor system, PCR has become an increasingly useful and widespread clinical tool. However, more widespread acceptance of molecular testing will depend upon automation that allows molecular assays to enter the routine clinical laboratory. The forces driving the need for automated PCR are the requirements for diagnosis and treatment of chronic viral diseases, economic pressures to develop more automated and less expensive test procedures similar to those in the clinical chemistry laboratories, and a shortage in many areas of qualified laboratory personnel trained in the types of manual procedures used in past decades. The automated Roche COBAS AMPLICOR system has automated the amplification and detection process. Specimen preparation remains the most labor-intensive part of the PCR testing process, accounting for the majority of the hands-on-time in most of the assays. A new automated specimen preparation system, the COBAS AmpliPrep, was evaluated. The system automatically releases the target nucleic acid, captures the target with specific oligonucleotide probes, which become attached to magnetic beads via a biotin-streptavidin binding reaction. Once attached to the beads, the target is purified and concentrated automatically. Results of 298 qualitative and 57 quantitative samples representing a wide range of virus concentrations analyzed after the COBAS AmpliPrep and manual specimen preparation methods, showed that there was no significant difference in qualitative or quantitative hepatitis C virus (HCV) assay performance, respectively. The AmpliPrep instrument decreased the time required to prepare serum or plasma samples for HCV PCR to under 1 min per sample. This was a decrease of 76% compared to the manual specimen preparation method. Systems that can analyze more samples with higher throughput and that can answer more questions about the nature of the microbes that we can presently only detect and quantitate will be needed in the future.

Performance characteristics of a transcription-mediated nucleic acid amplification assay for qualitative detection of hepatitis C virus RNA.

The detection of hepatitis C virus (HCV) RNA by nucleic acid amplification techniques is the method of choice to differentiate between ongoing and past infection, and can be used to monitor the course of HCV infection. In this study, we evaluated the performance characteristics of a newly developed transcription-mediated amplification (TMA)-based assay, the VERSANT HCV RNA qualitative assay, which was designed to qualitatively detect HCV RNA. Samples tested by the TMA assay included 100 HCV antibody negative sera; serial dilutions of an HCV genotype 1a panel; the WHO HCV RNA standard 76/790; an HCV genotyping panel; and 150 clinical specimens, including sera from patients who had received alpha interferon (IFN) treatment or liver transplants. TMA test results were compared with the Cobas Amplicor HCV polymerase chain reaction (PCR) assay. The analytical specificity of the HCV TMA assay was > 98%. No carry-over contaminations were observed. The assay demonstrated an analytical sensitivity of 100% at 41 HCV RNA copies/mL (genotype 1a panel) and 5 IU/mL (WHO standard), respectively. HCV genotypes and subtypes did not affect the results. Qualitative RNA detection by diagnostic Amplicor PCR and TMA was in agreement in > 97% of all 150 clinical samples tested. In our study, the TMA-based assay proved to be a specific and sensitive method for qualitative HCV RNA detection. The test may turn out to be an attractive alternative to already established techniques for HCV RNA amplification in routine clinical laboratories.

Pretransplantation hepatitis C virus quasispecies may be predictive of outcome after liver transplantation

The evolution of hepatitis C virus (HCV) envelope variation was studied using a liver-transplant model to evaluate the role of HCV quasispecies for hepatocyte infection. Twelve HCV polymerase chain reaction (PCR)-positive liver-transplant recipients (6 with posttransplantation biochemical hepatitis and 6 without hepatitis [controls]) were prospectively evaluated and underwent detailed quasispecies analysis of pre- and postoperative serum samples. HCV amino acid sequence diversity and complexity at the first hypervariable region (HVR1) of the second envelope protein (E2) was correlated with outcome. Recurrence of HCV-induced allograft injury was defined by persistently elevated serum alanine transaminase (ALT) levels. The major variant (sequences >10% of all clones) of recipients with hepatitis accounted for a significantly smaller percent of all preoperative clones compared with controls (41% ± 6% vs. 69% ± 8%; P < .015). Recipients with hepatitis had an increased number of pretransplantation major variants (2.5 ± 0.3 vs. 1.1 ± 0.2; P < .006). Eighty-three percent of controls had a predominant variant (accounting for >50% of clones) compared with 17% of those with recurrence (P < .05). These differences did not persist postoperatively. In addition, recipients without a pretransplantation predominant variant demonstrated an increased allograft fibrosis score (2.3 ± 0.3 vs. 0.5 ± 0.3; P < .015) at 181 to 360 days posttransplantation compared with those in whom a predominant variant was present. Increased HCV envelope complexity may act as a stronger antigenic stimulus or improve hepatocyte receptor binding and lead to allograft hepatitis and fibrosis. Although pretransplantation differences in HCV quasispecies did not persist postoperatively, pretransplantation quasispecies may be a predictor of HCV-induced hepatitis and graft fibrosis after liver transplantation. (Hepatology 2000;32:375-381.)