Evaluation of a total hepatitis C virus (HCV) core antigen assay for the detection of antigenaemia in anti-HCV positive individuals

A new, sensitive enzyme immunoassay has been developed for detecting and quantifying total hepatitis C virus (HCV) core antigen in anti-HCV positive or negative sera ("trak-C", Ortho Clinical Diagnostics, Raritan, NJ). The purpose of this study was to evaluate the performance of trak-C as an additional laboratory diagnostic marker of viraemia. The performance was compared to HCV-RNA detection in the "screening" of sera from a large heterogeneous population of hospitalised patients and outpatients. Six hundred and eighteen anti-HCV negative sera, 405 anti-HCV positive/HCV-RNA negative sera, 604 anti-HCV positive/HCV-RNA positive sera and 67 anti-HCV negative sera containing antigens or antibodies potentially interfering with the performance of the assay were analysed. Supplemental HCV antibody testing was performed using a commercial strip immunoblot assay. HCV-RNA was investigated using a qualitative commercial assay. A quantitative commercial RT-PCR was used for the analysis of selected samples. Sensitivity and specificity values were 94.7 and 100%, respectively. The latter was also confirmed when anti-HCV negative samples containing potentially interfering antigens/antibodies were examined. Sensitivity below 100% was probably due to an antigenaemia below the detection limit of trak-C. Besides, because 65.6% of HCV-RNA positive/trak-C negative samples presented specific antibodies against all four RIBA antigens, the hypothesis was raised that, in some cases, the dissociation step efficiency could be sub-optimal. In conclusion, trak-C seems suitable for identifying HCV infection on large based populations. It is a rapid to perform, reliable and specific assay that can be adapted to any laboratory setting.     

丙型肝炎病毒核心抗原检测用于献血者筛查价值的探讨

目的应用酶联免疫吸附技术筛查献血员中丙型肝炎病毒核心抗原（HCV—cAg）和抗体（HCV—Ab），了解丙型肝炎病毒核心抗原筛查献血员应用价值。方法对我站2004年8-12月间的3972份献血者血清标本进行抗-HCV初、复检和HCV—cAg ELISA检测，将ELISA法阳性的25份血清标本，再做RT-PCR检测证实。结果3972份血清标本检测中，有10份仅初检抗-HCV阳性样本，经HCVRNA检测阳性有l份，12份仅复检抗-HCV阳性样本，经HCVRNA检测阳性有1份，HCV—cAg检测阳性有3份，经HCVRNA检测阳性有2份。结论HCV—cAg ELISA检测技术的敏感性与HCVRNA技术类似，但成本明显降低，可与HCV抗体联合检测应用献血员筛查。     

Evaluation of a modified commercial assay in detecting antibody to hepatitis C virus in oral fluids and dried blood spots

Oral fluid testing is an effective alternative to serum antibody testing for surveillance of human immunodeficiency virus (HIV) and hepatitis B infections, and is being extended to hepatitis C infections. The objective of this study was to determine and compare the sensitivity and specificity of a modified commercial assay for the detection of antibody to hepatitis C virus (anti-HCV) in oral fluids collected by two different oral fluid collection devices (the Epitope OraSure trade mark and Sarstedt Salivette ) and in dried fingerprick blood spots. In this study, 253 anti-HCV seropositive patients and 394 blood donors (all anti-HCV negative) were recruited between August 2000 and January 2001. Each participant provided oral fluid specimens by OraSure and Salivette, and at least one dried blood spot. Serum specimens were collected from the patients whenever possible. For those injecting drug users who did not provide a serum specimen, HCV status was established on the basis of previous testing. All the nonserum samples were tested for the presence of anti-HCV, using a modified Ortho HCV 3.0 SAVe enzyme-linked immunosorbent assay (ELISA) protocol. The recommended preliminary cutoffs for the modified ELISA were suboptimal. Further, the sensitivity, specificity, and positive and negative predictive values could be improved by varying the cutoff and taking into account the likely prevalence of HCV in the population under investigation. For instance, given a population with a 50% prevalence of anti-HCV, the optimal sensitivities of the modified assay on OraSure, Salivette, and dried blood spots were 92%, 83%, and virtually 100%, respectively, in contrast to 83%, 59%, and 99% using the preliminary cutoffs. The respective optimal specificities were 99%, 93%, and 100%. In conclusion, oral fluids collected by the OraSure device provide an extremely useful method to conduct public health surveillance of not only HIV, but also hepatitis C, among injecting drug users. In addition, dried blood spot specimens may be useful for surveillance and could be employed as a first line diagnostic specimen.     

Evaluation of Dried Blood Spots as a Feasible Alternative to Plasma for the Detection and Quantification of Hepatitis C Virus in a Tropical Setting: A Pilot Study

Introduction: Confirmatory diagnosis of hepatitis C virus (HCV) infection (HCV RNA detection) is essential before start of the therapy. HCV RNA detection is not available in many parts of India. Shipment of plasma from distant places to referral laboratories may affect HCV RNA titres. Dried blood spots (DBS) provide an easy alternative for transporting samples to centres where HCV RNA testing is done. Aim: Evaluation of DBS as a feasible alternative to plasma for HCV diagnosis. Methods: In this cross-sectional study, 40 consecutive patients’ blood samples were collected from patients referred from the Liver Clinic. Whole blood was spotted onto two Whatman 903^TM cards. One card was incubated at ≥37°C and other at 4°C for 15 days, after drying. DBS was eluted and run in Abbott RealTime HCV assay. HCV was also quantified using the Abbott ARCHITECT HCV core antigen assay for 29 of the study patients. Results were compared with normal plasma values. Results: The median log HCV RNA value (in log₁₀ IU/mL) of plasma was 5.74, with normalised DBS it was 4.92 (≥37°C) and 4.66 (4°C); difference in plasma and DBS median log values was 0.82 (≥37°C) and 1.08 (4°C) logs, respectively. Interclass correlation values were 0.943, P < 0.0001 (≥37°C) and 0.950, P < 0.0001 (4°C), showing high agreement. The median HCV core antigen value (in fmol/L) for plasma was 325.35, whereas it was 4.77 (≥37°C) and 4.64 (4°C) for DBS samples. Conclusions: DBS can be used for sampling patients from distant resource-limited settings as an alternative to plasma for HCV RNA estimation. Larger studies are required to evaluate the feasibility of DBS in the Indian subcontinent, especially for HCV core antigen estimation.     

Evaluation of a simplified sucrose gradient method for the detection of rubella-specific IgM in routine diagnostic practice

Rubella-specific IgM was measured in a single fraction of serum from a sucrose density gradient. Haemagglutination inhibition (HAI) tests were performed on paired aliquots of the fraction untreated and after treatment with 2- mercaptoethanol, dilutions of the aliquots being incubated over night with rubella antigen before the addition of red cells. Of 822 sera tested, specific IgM was found in 249, but not in 492. When first tested, the remaining 81 sera gave unsatisfactory results because of contamination of the IgM fraction with IgG (6.0%), probable aggregation of IgG (3.5%), or the persistence of chick red cell agglutinins (0.4%). Tests were performed on 134 patients with rubella confirmed by a rise of HAI antibodies. Rubella-specific IgM was found at a titre of more than eight in the sera taken from 62 of 64 patients between 10 and 29 days after the onset of the rash but in only one of the sera taken between 80 and 119 days, and in none taken later. However, specific IgM was still to be found at lower titre in the sera of 13 patients collected between 80 and 162 days after the onset of the illness. In routine diagnostic tests over three years on the serum from 479 patients with suspected acquired rubella, specific IgM was found at a titre of more than eight in 51 patients and in only 10 instances (2.1%) did a lower level pose a problem in interpretation.     

Reliability of hepatitis C virus core antigen assay for detection of viremia in HCV genotypes 1, 2, 3, and 4 infected blood donors: a collaborative study between Japan, Egypt, and Uzbekistan

Nucleic acid amplification-based methods are used for confirmation of viremia in antibody to hepatitis C virus (anti-HCV)-positive patients. However, this technology is labor intensive, time consuming, requires complex laboratory conditions, and expensive. The aim of this study was to evaluate the sensitivity and specificity of the HCV core antigen (HCVcAg) assay as an alternative approach for confirmation of viremia in HCV-infected subjects with HCV genotype 1-4. Two hundred forty-six asymptomatic HCV RNA- positive donors were enrolled in this study, consisting of 122 blood donors from Egypt (116 with genotype 4, 4 with genotype 1, and 2 with 1 + 4 genotypes), 109 from Japan (85 with genotype 1, and 24 with genotype 2), and 15 from Uzbekistan (all with genotype 3). A total of 234 (95.1%) of 246 RNA-positive specimens were detected by the HCVcAg assay; the sensitivity of HCVcAg assay consisted 93.4, 100, 100, and 94.8% for genotypes 1, 2, 3, and 4, respectively in comparison with RT-PCR assay. The specificity of the assay was confirmed in the absence of the false-positive results among 53 anti-HCV-negative, but anti-Schistosoma mansoni (anti-Sm) positive donors from Egypt. A positive correlation between HCVcAg and HCV RNA concentration levels (r = 0.671, P < 0.05) was observed among specimens with HCV genotype 4. The mean HCVcAg level was significantly lower in specimens with genotype 4 (2,935 fmol/L) comparing to genotypes 1, 2, and 3 (5,034, 4,962, and 4,740 fmol/L, respectively). No specific mutation was found in the core-encoding region of the studied specimens. In conclusion, HCVcAg is shown to be specific, sensitive, and informative qualitative index for HCV viremia in asymptomatic carriers.     

Usefulness of simultaneous screening for HIV‐specific and HCV‐specific antibodies and HBsAg by a capillary‐based multiplex rapid diagnostic test to strengthen linkage‐to‐care in sub‐Saharan patients attending sexually transmitted infection clinic

Adult outpatients attending the main sexually transmitted infection clinic of Bangui, Central African Republic, were prospectively subjected to a multiplex rapid diagnostic test for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). In group I (n = 208) of patients already followed for HIV, 6 (2.9%) were unexpectedly negative, thus corresponding to false positive for HIV by the national HIV algorithm; hepatitis B surface antigen and HCV positivities were high (18.7% and 4.3%, respectively). In group II (n = 71) of patients with unknown HIV status, at least 1 chronic viral disease was diagnosed in 26 (36.6%) patients, including 5 (7.1%) HIV, 17 (23.9%) HBV, and 3 (4.2%) HCV infections.     

Evaluation of a PCR method to determine the clinical significance of blood cultures with Staphylococcus epidermidis in patients with hematological malignancies

The aim was to investigate whether the detection and quantification of Staphylococcus epidermidis DNA in blood could distinguish S. epidermidis blood stream infections (BSIs) from blood culture contaminations in patients with hematological malignancies. The hld gene was chosen to identify S. epidermidis DNA and DNA in blood samples was detected by real‐time PCR. Blood samples were obtained simultaneously with blood cultures positive for S. epidermidis (n = 30), during blood culture‐negative episodes (n = 10) and episodes of bacteremia with other bacteria than S. epidermidis (n = 4) and from healthy blood donors (n = 10). In addition, DNA from S. epidermidis and a selection of other bacterial species were analyzed. Three different sets of criteria were used to classify episodes with positive blood cultures with S. epidermidis as BSIs or contaminations. All DNA preparations from S. epidermidis (n = 48) were hld‐positive, but other bacterial species (n = 13) were negative. Sixteen (53%) of 30 blood samples from patients with blood cultures positive for S. epidermidis were hld‐positive, but none of the controls. There was no clear association between a positive hld PCR and episodes interpreted as BSIs. In conclusion, hld PCR failed to distinguish S. epidermidis BSIs from blood culture contaminations in patients with hematological malignancies.     

Evaluation and validation of a biochip multi-array technology for the screening of 14 sulphonamide and trimethoprim residues in honey according to the European guideline for the validation of screening methods for veterinary medicines

Honey could be contaminated by antibiotic residues. There is still a great need for a cheap and single multi-residue method. The Evidence Investigator? system is a biochip and semi-automated system. The microarray kit I (AM I) for the detection of sulphonamide and trimethoprim (TMP) residues in honey was evaluated, and then validated according to the European decision EC/2002/657 and to the European guideline for the validation of screening methods for veterinary medicines (2010). The method was found to be rapid and able to screen simultaneously 14 sulphonamides and TMP in honey with a very easy sample preparation procedure. The false–positive rate of 4% maximum is very satisfactory. All detection capabilities (CCβ) were well below the recommended concentration (RC) of 50 µg kg^?1. The applicability of AM I kit with different types of honey (monofloral or multifloral, liquid or solid, different floral origins, etc.) has been proved.     

Hepatitis C virus subtyping based on sequencing of the C/E1 and NS5B genomic regions in comparison to a commercially available line probe assay

Hepatitis C virus (HCV) genotype determination is required in clinical practice to establish the dose and duration of antiviral treatment. Although subtype identification does not impact on current therapy this is changing with new specific inhibitors of HCV enzymes and functions which are becoming available worldwide. These new drugs may yield different antiviral responses and resistance profiles. Accurate classification of HCV genotype and subtype is therefore crucial. An “in‐house” method was developed for improving HCV subtyping and the results were compared with a second‐generation line probe assay (LiPA) used extensively in Portugal. Phylogenetic analysis was undertaken of the C/E1 and NS5B genomic regions of HCV isolated from 72 prisoners with chronic HCV infection and from reference samples. Although LiPA is considered to be a good method for genotyping, HCV was subtyped in only 47.2% of cases compared with 95.8% of cases by the “in‐house” method. Molecular data for both C/E1 and NS5B regions were obtained in 88.9% of the samples. Two out of 23 cases of subtype 1a were misclassified as subtype 1b by LiPA. A putative recombinant like RF1_2k/1b, two potential inter‐genotypic recombinants 1b/4a and 3a/4a, and also a potential intra‐genotypic recombinant 2q/2k in C/E1 and 2k/2a in NS5B were also identified. The “in‐house” method enabled HCV to be subtyped accurately with the detection, in some cases, of recombinant viruses or dual HCV infections. Near full‐length genomic analysis to characterize these potential recombinant viruses is planned. J. Med. Virol. 85:815–822, 2013. © 2013 Wiley Periodicals, Inc.     

Three-dimensional observations on the alterations of lobular architecture in chronic hepatitis with special reference to its angioarchitecture for a better understanding of the formal pathogenesis of liver cirrhosis

For a better understanding of the formal pathogenesis of liver cirrhosis, the angioarchitecture of the liver lobule in chronic viral hepatitis was investigated three dimensionally. The histological reconstruction method, using serial histological sections, was adopted for the three-dimensional observation. Histology of the case showed chronic active hepatitis with occasional fibrous bridging of the portal to portal tract or hepatic vein. Graphic reconstructions revealed various degrees of altered angioarchitecture from place to place. While the conducting portion of the portal vein was almost preserved, the pathological changes mostly began at the parenchymal portion, especially second step or subsequent branches of the portal vein. In general, portal vein branches showed damage such as stenoses, disappearance, an increase and decrease in number and distorted spatial arrangements. Even in less damaged portal tracts, portal veins showed such changes to some extent. In severely damaged places with bridging fibrosis, a normal lobular angioarchitecture was completely lost; instead, portal veins, arteries and hepatic veins were tangled with each other. Parenchymal nutrition was suggested to be dependent on the remaining third-step portal branches or newly formed ones. However, the hepatic vein system had a tendency to be preserved and distributed fluently in the parenchyma. The distortion of these portal vessels indicated various degrees of loss of the lobular architecture. In conclusion, it is suggested that an early histological sign of cirrhosis develops in the course of chronic hepatitis.     

Evaluation of the impact of different lengths of pre-enrichment in a nutritive broth and prolonged incubation of MRSA-ID, a chromogenic agar medium, on its performances for identifying methicillin-resistant Staphylococcus aureus in screening samples

MRSA-carrier screening is recommended to prevent MRSA dissemination in hospitals. Rapid and specific detection of MRSA in the laboratory is a key element in enabling control measures. Our objective was to evaluate the impact of different lengths of pre-incubation in a nutritive broth and prolonged incubation of MRSA-ID, a chromogenic agar medium, on its performances for identifying MRSA in screening samples. According to our results, short-length pre-enrichments only provided a weak increase of sensitivity as compared to the absence of pre-enrichment. On the contrary, the sensitivity increase provided by an overnight pre-enrichment was significant. The prolongation of incubation in the chromogenic agar medium (48 hours instead of 24 hours) did not provide any significant increase of sensitivity but was associated with a strong and significant loss of specificity. Therefore, it seems relevant to reject prolonged incubation of selective agar media and to make a choice between the absence of pre-enrichment (faster results) and an overnight pre-enrichment (higher sensitivity), according to local epidemiology and local practices implemented for prevention.     

Direct sequencing of PCR products derived from cDNAs for the proα1 and proα2 chains of type I procollagen as a screening method to detect mutations in patients with osteogenesis imperfecta

More than 150 mutations in the genes for type I procollagen have been found in unrelated patients with osteogenesis imperfecta (OI), but mutations have been difficult to define in many patients with the mildest forms of the disease. Here, we have used robotically automated sequencing of the cDNAs for type I procollagen to screen for mutations in 12 patients suspected of having nonlethal OI (types I, III, and IV). Single base mutations that changed codons for obligate glycine residues were found in seven of the patients. Altogether, we analyzed 4,379 bp of sequences of both alleles of the proα1(I) collagen (8,758 bp of allelic sequences) and 4,200 bp of sequences of both alleles of the proα2(I) collagen (8,400 bp of allelic) from each patient. © 1996 Wiley-Liss, Inc.