Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli.

Shigella causes bacillary dysentery and is classified into four species based on their antigen characteristics. This classification does not reflect genetic relatedness; in fact, Shigella species are so related to Escherichia coli , they should be classified as one distinctive species in the genus Escherichia. The differentiation of Shigella and E. coli is even more complicated with the description of enteroinvasive E. coli (EIEC). EIEC are strains that possess some of the biochemical characteristics of E. coli and have the ability to cause dysentery using the same method of invasion as Shigella does. Sequencing of multiple housekeeping genes indicates that EIEC is more related to Shigella than to non-invasive E. coli. Shigella and EIEC evolved from the same ancestor and form a single pathovar within E. coli. Shigella and EIEC could be separated from other E. coli by a PCR targeting the ipaH-gene; this is a multicopy gene exclusively found in all Shigella and EIEC. It is possible to differentiate Shigella and all E. coli, including EIEC, by using multiple tests, including ipaH-gene PCR, physiological and biochemical typing and serological typing. Based on literature study, a key is designed for daily use in diagnostic laboratories to identify Shigella and all E. coli.

Genetic variation in IL28B and treatment outcome in HBeAg-positive and -negative chronic hepatitis B patients treated with Peg interferon alfa-2a and adefovir.

In a cohort of 95 chronic hepatitis B patients, who were treated with peg-interferon and adefovir for 1 year, and who had 15% HBsAg loss (overall), no association was found between IL28B polymorphisms and HBeAg seroconversion or HBsAg clearance. These findings suggest that any association with outcome, if present, is less than that seen in chronic hepatitis C. Additional studies are needed to enlarge sample size and to refine our understanding of IL28B biology in the context of chronic hepatitis B response to immunomodulatory and direct antiviral therapy.

Validation of a sensitive and specific real-time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA in plasma of chronic hepatitis B patients.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral replication and plays a role in persistence of HBV infection. The origin and significance of cccDNA in plasma however, is not well understood. A sensitive, specific, and reproducible real-time PCR for detection and quantitation of cccDNA in plasma of chronic hepatitis B patients was developed and validated. Four HBV DNA reference panels, and 96 plasma samples of chronic hepatitis B patients were analyzed. Results were compared with total HBV DNA levels, individual ALT levels and the Histology Activity Index (HAI). This cccDNA assay had a lower limit of detection at 15 copies/PCR, a lower limit of quantitation at 91 copies/PCR and a correlation coefficient (R) of 0.98 (P < 0.0001). cccDNA was detected in two of four international panels. Significant correlation was found between cccDNA and total HBV DNA levels in both panels (R = 0.96, and R = 0.43) and in samples of the chronic hepatitis B patients (R = 0.88, P < 0.0001). In 57% of these samples cccDNA was detectable. Mean level of cccDNA was 0.16% of total HBV load. Plasma cccDNA levels were higher in HBeAg positive samples than in HBeAg negative samples (4.91 log copies/ml vs. 3.88 log copies/ml, P < 0.0001). Levels of total HBV DNA and HBV genotype did not influence cccDNA detection. ALT levels and HAI-score were not correlated with plasma cccDNA levels. These findings suggest that cccDNA levels in plasma are not the result of increased hepatocyte degeneration, but indicate that other mechanisms might be responsible.

HCV-specific T-cell responses in injecting drug users: evidence for previous exposure to HCV and a role for CD4+ T cells focussing on nonstructural proteins in viral clearance.

In order to understand the parameters associated with resolved hepatitis C virus (HCV)-infection, we analysed the HCV-specific T-cell responses longitudinally in 13 injecting drug-users (IDUs) with a prospectively identified acute HCV infection. Seven IDUs cleared HCV and six IDUs remained chronically infected. T-cell responses were followed in the period needed to resolve and a comparable time span in chronic carriers. Ex vivo T-cell responses were measured using interferon-gamma Elispot assays after stimulation with overlapping peptide pools spanning the complete HCV genome. CD4+ memory-T-cell responses were determined after 12-day stimulation with HCV proteins. The maximum response was compared between individuals. The T-cell responses measured directly ex vivo were weak but significantly higher in resolvers compared to chronic carriers, whereas the CD4+ memory-T-cell response was not different between resolvers and chronic carriers. However, HCV Core protein was targeted more often in chronic carriers compared to individuals resolving HCV infection. CD4+ T-cell responses predominantly targeting nonstructural proteins were associated with resolved HCV infection. Interestingly, observation of memory-T-cell responses present before the documented HCV-seroconversion suggests that reinfections in IDUs occur often. The presence of these responses however, were not predictive for the outcome of infection. However, a transition of the HCV-specific CD4+ memory-T-cell response from targeting Core to targeting nonstructural proteins during onset of infection was associated with a favourable outcome. Therefore, the specificity of the CD4+ memory-T-cell responses measured after 12-day expansion seems most predictive of resolved infection.

Human parechovirus infections in Dutch children and the association between serotype and disease severity.

BACKGROUND: Human parechoviruses (HPeVs) are members of the family Picornaviridae and are classified into 3 known serotypes: HPeV1, HPeV2, and the recently identified HPeV3. HPeV1 and HPeV2 infections are most commonly associated with mild respiratory or gastrointestinal symptoms and occasionally with severe disease conditions, such as flaccid paralysis and encephalitis. HPeV3 infection has been associated with transient paralysis and neonatal infection and has until now only been reported in Japan and Canada. METHODS: Culture isolates considered to be enterovirus on the basis of cell culture but that were found to be enterovirus negative by 5' untranslated region reverse-transcriptase polymerase chain reaction (5'UTR RT-PCR) during the period December 2000 through January 2005 were selected. Isolates were tested by HPeV 5'UTR RT-PCR and were genotyped by sequencing the VP1 region. Phylogenetic analysis was performed, and the association with clinical symptoms was established. RESULTS: Thirty-seven (12%) of the 303 isolates that tested positive for enterovirus by cell culture were in fact HPeV. The majority of the HPeV-positive isolates (n = 27) could be identified as HPeV1. The remaining 10 isolates, which were grown from samples obtained in 2001, 2002, and 2004, could be typed as the recently identified HPeV3. HPeV was exclusively detected in children aged < 3 years. Children infected with HPeV3 were significantly younger than children infected with HPeV1, and sepsis-like illness and central nervous system involvement were more frequently reported in children infected with HPeV3. CONCLUSIONS: We report HPeV infections in young children during the period of 2000-2005 and show an association between HPeV3 infection and sepsis-like illness and central nervous system involvement in neonates.

Development of an internally controlled real-time PCR assay for detection of Chlamydophila psittaci in the LightCycler 2.0 system.

A real-time PCR assay with a DNA purification and inhibition control (internal control; IC) was developed to detect Chlamydophila psittaci DNA in human clinical samples. Novel C. psittaci-specific primers targeting the ompA gene were developed. The IC DNA contained the same primer-binding sites and had the same length and nucleotide content as the C. psittaci DNA amplicon, but had a shuffled probe-binding region. The lower limit of detection was 80 target copies/PCR, corresponding to 6,250 copies/mL in a clinical sample. Specificity was tested using reference strains of 30 bacterial species. No amplification was observed from any of these samples. Respiratory samples from eight patients were positive with this PCR. Six of these patients were confirmed as positive for C. psittaci with serological testing. Two patients had increasing antibody titres, but did not fulfil criteria proposed previously for serologically proven Chlamydia spp. infection. The real-time PCR described in this paper is a sensitive, specific and rapid method to detect C. psittaci DNA in human clinical respiratory samples.

Occult hepatitis B virus infection before and 1 year after start of HAART in HIV type 1-positive patients.

Occult hepatitis B virus (HBV) infection is diagnosed when HBc antibodies and HBV-DNA are detectable in serum while hepatitis B surface antigen (HBsAg) is not. The clinical relevance of this phenomenon in HIV-1 patients starting highly active antiretroviral therapy (HAART) is unknown. We followed 93 therapy naive HIV-1-infected adults who were anti-HBc positive, HBsAg and HBeAg negative, during first year of HAART. At baseline, HBV-DNA was quantified, and HBV genotype was determined in the HBV-DNA-positive patients by sequencing a part of the HBV genome. Four of 93 patients (4%) were HBV DNA positive at baseline. All four patients tested negative for HBV-DNA after 1 year. They all received lamivudine as part of their HAART. They had no clinically significant liver enzyme elevations (LEE) during the first year of HAART. Two of the patients had a genotype A, one genotype E, and in the fourth patient sequencing was not possible. In one patient we found significant mutations in the a determinant region of HBsAg, at positions 142 and 144. In our population of therapy-naive HIV-1-infected adults who were anti-HBc positive, we found occult HBV infection in 4% of the patients. We did not find an increased risk for LEE in our population of patients after the start of HAART. Our results illustrate that occult HBV infection is more a diagnostic than a clinical problem. It may be caused by very low levels of HBV replication, concurrent presence of HBsAg and anti-HBs, or mutations in the HBsAg a determinant.

Hepatocellular carcinoma in Egyptians with and without a history of hepatitis B virus infection: association with hepatitis C virus (HCV) infection but not with (HCV) RNA level.

The aim of this study was to analyze the association of hepatocellular carcinoma (HCC) with hepatitis C virus (HCV) in Egypt, using hepatitis B virus (HBV) and hepatitis E virus (HEV) as virus controls. In addition, the association of HCC with HCV RNA levels among persons seropositive for HCV was analyzed. We compared 131 patients with proven HCC, 247 with bladder cancer, and 466 healthy hospital employees. Age, sex, and place of residence were recorded to study confounding factors. Among the healthy controls, 16% were seropositive for HCV, 21% for HBV, and 31% for HEV. When healthy controls were age-matched with HCC patients, the latter were significantly (P < 0.001) more often HCV seropositive (67%) than were the controls (30%). The seropositivity for HBV and HEV did not differ significantly in frequency between the two groups. The seropositivity for HCV was also significantly (P < 0.001) more often found in HCC patients (76%) than in BC patients (47%), with seroprevalences for HBV and HEV not differing significantly in these age-matched groups. In HBV-negative HCC and bladder cancer patients, seroprevalence for HCV was significantly (P = 0.002) higher in HCC patients (68%) than in bladder cancer patients (36%). This difference was even more pronounced (P < 0.001) in HBV-positive HCC and bladder cancer patients (78% versus 52%, respectively). Of HCV-seropositive individuals, 49% were HCV RNA positive by branched DNA assay, and of these, 96% were infected by HCV genotype 4. No correlation between HCV RNA load and seropositivity of HBV or age or disease state was found. Infection with HCV and HCV-HBV double infection, but not HBV or HEV infection alone, is strongly correlated with HCC in Egypt.

[Sexual transmission of hepatitis C in homosexual men]. / Seksuele overdracht van hepatitis C bij homoseksuele mannen.

An acute hepatitis C infection was diagnosed in three HIV-positive gay men, aged 43, 48 and 30 years, respectively. In all three, unprotected sexual intercourse and fisting was a universal risk factor for the infection. They all denied having used drugs intravenously, which is the most common risk factor. The third man had a documented proctitis (lymphogranuloma venereum) at the time when the HCV transmission must have taken place. No serious complications occurred during the acute HCV infection. Because the infection did not resolve spontaneously after a few months, all three men were treated with pegylated interferon and ribavirin. Recently, the number of cases of acute HCV infection has been seen to increase in The Netherlands. This may be due primarily to an increase in unprotected sexual intercourse and fisting. This hypothesis is supported by a documented increased prevalence of sexually transmissible diseases among gay men in The Netherlands. As acute infections may turn into chronic infections, treatment of an acute infection should be considered in order to prevent the chronic disease.

Validation of a sensitive and specific real-time PCR for detection and quantitation of hepatitis B virus covalently closed circular DNA in plasma of chronic hepatitis B patients.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) serves as a template for viral replication and plays a role in persistence of HBV infection. The origin and significance of cccDNA in plasma, however, are not well understood. A sensitive, specific, and reproducible real-time PCR for detection and quantitation of cccDNA in plasma of chronic hepatitis B patients was developed and validated. Four HBV DNA reference panels and 96 plasma samples of chronic hepatitis B patients are analyzed. Results are compared with total HBV DNA levels. This cccDNA assay had a lower limit of detection at 15 copies/PCR, a lower limit of quantitation at 91 copies/PCR, and a correlation coefficient (R) of 0.98 (p < 0.0001). HBV cccDNA can be detected in two of four international panels. Significant correlation is found between cccDNA and total HBV DNA levels in both panels (R = 0.96 and R = 0.43) and in samples of the chronic hepatitis B patients (R = 0.88, p < 0.0001). In 57 % of these samples cccDNA can be detected. Mean level of cccDNA is 0.16 % of total HBV load. Plasma HBV cccDNA levels are higher in HBeAg-positive samples than in HBeAg-negative samples (p < 0.0001). Total HBV DNA levels and HBV genotype do not influence cccDNA detection.

Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA.

The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (UNG) contamination control option in the HCV test for previous Roche COBAS Amplicor users was evaluated. It proved to decrease amplicon carryover by 100-fold independent of the amplicon input concentration. The protocol including UNG proved to overcome problems with false-positive negative controls. Comparison with other assays revealed only minor differences. The largest difference was observed between the Abbott HCV RealTime assay and the Roche COBAS Amplicor HCV Monitor version 2.0 assay.

Peripheral blood mononuclear cells of HIV- and HCV-antibody-positive individuals contain HCV RNA but No HCV DNA despite evidence for reverse transcription of HIV RNA into DNA.

Following reports of the finding of cDNA of RNA viruses in cells containing an endogenous retrovirus-encoded reverse transcriptase, we looked for the presence of hepatitis C virus (HCV) DNA in peripheral blood mononuclear cells (PBMC) of injecting drug users seropositive for both HCV and human immunodefiency virus (HIV). We tested serial PBMC samples from four HCV infected individuals; one was seronegative for HIV, two seroconverted for HIV during follow-up, and one was seropositive for HIV throughout the study period. HCV RNA was found in PBMC and plasma samples at all time points tested. Similarly, HIV RNA was found in all PBMC and plasma samples following HIV seroconversion. In contrast, no HCV DNA was detected in any PBMC sample, whereas HIV DNA was found in all tested PBMC samples following HIV seroconversion, indicative of active HIV reverse transcriptase in these PBMC samples. These results do not support the hypothesis that HCV viraemia is related to retrotranscription of the HCV RNA genome into DNA in peripheral blood mononuclear cells coinfected with HIV. The potential of HIV RT to retrotranscribe HCV RNA into DNA awaits studies of liver cells coinfected with HCV and HIV.

Evaluation of automated RNA-extraction technology and a qualitative HCV assay for sensitivity and detection of HCV RNA in pool-screening systems.

BACKGROUND: The objective of this study was the evaluation of NAT technology for the detection of HCV RNA in plasma pools according to the recommendations of the Paul Ehrlich Institute (5000 IU/mL/donation) and the Committee for Proprietary Medical Products (100 IU/mL/manufacturing pool). STUDY DESIGN AND METHODS: Serial dilutions of both the EUROHEP standard (3,800 genome equivalents [geq]/mL; HCV genotype 1) and the World Health Organization (WHO) international standard (100,000 IU/mL; HCV genotype 1) were made in S/D plasma (ESPEP plasma, OctaPharma), which was nonreactive in serologic tests. Serial dilutions of plasma (2 mL) were used for extraction of HCV RNA with an automated version of a nucleic acid isolation method (NucliSens Extractor, Organon Teknika). HCV RNA was co-extracted from 2 mL of plasma, together with 84 copies of an in vitro-synthesized single-strand RNA serving as internal extraction control (IC) to monitor the efficiency of extraction and PCR. Amplification and detection of both HCV RNA and IC RNA were performed with an automated PCR system and a qualitative HCV assay (COBAS Amplicor 2.0 HCV, Roche Diagnostics). RESULTS: A cutoff value of 16 geq per mL (10/10 runs [100% hit rate]) was found by using the EUROHEP standard, whereas the WHO international standard had a cutoff value of approximately 12 IU per mL (10/10 runs [100% hit rate]). The IC had a cutoff value of approximately 17.5 copies per mL (6/6 runs [100% hit rate]). Forty-two copies per mL of IC RNA were found in 282 of 284 runs (99% hit rate). The negative controls (ESDEP plasma) were negative in all experiments. Experiments with pool sizes of 12, 24, 48, and 96 using serial dilutions of the WHO international standard revealed a cutoff value of 8 IU per mL (100% hit rate). The EUROHEP standard and the WHO international standard were detected with a 50 percent detection endpoint of 5.2 geq per mL and 1.5 IU per mL, respectively. CONCLUSION: This test system (NucliSens Extractor, and the COBAS Amplicor 2.0 HCV assay) revealed a high sensitivity for HCV RNA; considering the proposed requirements for sensitivity of NAT assays for the detection of HCV RNA in donor plasma, pool sizes of about 400 donors are possible. These endpoint results indicated that 1 IU is equal to about 3.4 geq.

Improved silica-guanidiniumthiocyanate DNA isolation procedure based on selective binding of bovine alpha-casein to silica particles.

DNA purified from clinical cerebrospinal fluid and urine specimens by a silica-guanidiniumthiocyanate procedure frequently contained an inhibitor(s) of DNA-processing enzymes which may have been introduced by the purification procedure itself. Inhibition could be relieved by the use of a novel lysis buffer containing alpha-casein. When the novel lysis buffer was used, alpha-casein was bound by the silica particles in the first step of the procedure and eluted together with DNA in the last step, after which it exerted its beneficial effects for DNA-processing enzymes. In the present study we have compared the novel lysis buffer with the previously described lysis buffer with respect to double-stranded DNA yield (which was nearly 100%) and the performance of DNA-processing enzymes.

Structure, expression, chromosomal location and product of the gene encoding ADH1 in Petunia.

A genomic clone for an alcohol dehydrogenase (Adh) gene has been isolated from Petunia hybrida cv. V30 by screening a Petunia genomic library with a maize Adh1 probe. A combination of RFLP and allozyme segregation data failed to demonstrate which of two Adh loci, both of which map to chromosome 4, was the source of the cloned gene. The product of the cloned genes has been identified unequivocally by a transient expression assay in Petunia protoplasts. We have designated this gene Petunia Adh1. The expression of this gene is tightly regulated in the developing anther, where its gene product is the predominant ADH isozyme. It is anaerobically inducible in roots, stems and leaves of seedlings. The induction of enzyme activity is correlated with induction of Adh1 mRNA.

Different hepatitis C virus (HCV) RNA load profiles following seroconversion among injecting drug users without correlation with HCV genotype and serum alanine aminotransferase levels.

Hepatitis C virus (HCV) infection often persists in association with chronic hepatitis. Different factors have been proposed to determine the clinical outcome of HCV infection. The aim of this study was to examine three different factors of HCV infection among injecting drug users. Nineteen untreated HCV seroconverters were tested longitudinally for the presence of HCV RNA by reverse transcriptase (RT) PCR, and results were quantified by the branched-DNA (bDNA) assay. HCV genotypes were determined with the first sample taken after HCV seroconversion. To assess the natural course of infection, serum alanine aminotransferase (ALT) levels were measured at three stages in every individual. The concordance between bDNA and RT-PCR was 98.9%. Three distinct patterns were found, according to the HCV RNA load after seroconversion during a mean follow-up period of 5 years (range, 1 to 8 years). HCV genotype 1a was predominant (52.6%). There was a significant increase in serum ALT levels (mean 55.5 U/liter) in the early phase of HCV infection, compared with basal serum ALT levels before HCV seroconversion and at the end of the follow-up period. Three distinct HCV RNA load profiles were found, without apparent relationship to genotype and serum ALT levels in the first 5 years of HCV infection.

Molecular characterization of a nonautonomous transposable element (dTph1) of petunia.

An insertion sequence of 283 base pairs has been isolated from the DFR-C gene (dihydroflavonol-4-reductase) of petunia. This insert was found only in a line unstable for the An1 locus (anthocyanin 1, located on chromosome VI) and not in fully pigmented progenitor and revertant lines or in stable white derivative lines. This implies that the An1 locus encodes the DFR-C gene. The unstable An1 system in the line W138 is known to be a two-element system, the autonomous element being located on chromosome I. In the presence of the autonomous element, W138 flowers exhibit a characteristic pattern of red revertant spots and sectors on a white background. In the absence of the autonomous element, the W138 allele gives rise to a stable recessive (white) phenotype. Sequence analysis of progenitor, unstable, and revertant alleles revealed dTph1 to contain perfect terminal inverted repeats of 12 base pairs. In DFR-C, it is flanked by an 8-base pair target site duplication. Sequences homologous to dTph1 are present in at least 50 copies in the line W138. Sequence analysis of An1 revertant alleles indicated that excision, including removal of the target site duplication, is required for reversion to the wild-type phenotype. Derivative stable recessive alleles showed excision of dTph1 and a rearrangement of the target site duplication. dTph1 is the smallest transposable element described to date that is still capable of transposition. The use of dTph1 in tagging experiments and subsequent gene isolation is discussed.

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes.

In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5 kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays. The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with the first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively). Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.

Low levels of hepatitis C virus RNA in serum, plasma, and peripheral blood mononuclear cells of injecting drug users during long antibody-undetectable periods before seroconversion.

Screening of antibodies to hepatitis C virus (HCV) is widely used for monitoring the prevalence of HCV infections and to assess HCV infectivity. Among HCV-infected individuals in the general population, the interval between the detection of HCV RNA and the development of HCV antibodies is usually 5 to 6 weeks, but in rare cases, seroconversion may be prolonged up to 6 to 9 months. In this study, we tested for the presence of HCV RNA during the antibody-undetectable period of 19 drug-injecting HCV seroconverters to gain insight into the antibody-negative carrier status in this population. HCV seroconversion status was determined by testing the first and last serum samples obtained from each subject, using third-generation antibody screening and confirmation assays. Serial samples were tested for HCV-specific antibodies to establish the moment of seroconversion and HCV RNA by single reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA assay (bDNA) in serum. Plasma and peripheral blood mononuclear cells (PBMCs) were independently collected and tested for HCV RNA. HCV RNA-positivity was confirmed by Southern blot hybridization and sequencing of serial samples. The 19 HCV seroconverters had a mean follow-up of 5 years (range, 1 to 8 years). Of the 19, 4 were human immunodeficiency virus (HIV)-infected before HCV seroconversion. HCV RNA was detected in serum before seroconversion in 12 (63.2%) of the 19 HCV seroconverters, independent of HIV status. In 7 of these 12, the antibody-undetectable period was relatively short (2 to 10 months). The other 5, who were all HIV-negative before HCV seroconversion, had intermittent low levels of HCV RNA before seroconversion for a period of more than 12 months, with a mean of 40.8 months (range, 13 to 94 months). In all 5 individuals, independent repeats of the experiments confirmed the presence of HCV RNA in serum, and in 3 of these individuals, HCV-positivity was confirmed in independently collected plasma and PBMC samples. Low levels of HCV RNA may be present during prolonged antibody-undetectable periods before seroconversion in a number of injecting drug users. Independent of HIV status, their immune system appears to be unable to respond to these low HCV RNA levels and was sometimes only activated after reinfections with distinct HCV genotypes. These results indicate that primary HCV infection may not always elicit the rapid emergence of HCV antibodies and suggests that persistent low levels of HCV RNA (regardless of the genotype) may not elicit at all or delay antibody responses for prolonged periods of time.

Hepatitis C virus serotype-specific core and NS4 antibodies in injecting drug users participating in the Amsterdam cohort studies.

In the present study, the RIBA HCV serotyping SIA was evaluated with a cohort of injecting drug users. Serotyping may be a rapid and cost-effective method of determining genotypes in cohort studies. In this study, hepatitis C virus (HCV) antibody-positive sera from a cohort of 331 chronically infected injecting drug users, of which 167 were coinfected with human immunodeficiency virus (HIV), were serotyped by the RIBA HCV Serotyping SIA. Among the 331 specimens, serotype-specific antibodies were detected in 250 (sensitivity, 75. 5%), in which serotype 1 was predominant (57.2%), followed by serotype 3 (26.8%). Among the 331 specimens, 164 were HIV negative, and serotype-specific antibodies were detected in 151 (sensitivity, 92.1%), in which serotype 1 was predominant (59.6%), followed by serotype 3 (33.8%). For a subset of 58 samples taken from 19 chronically infected HCV seroconverters with a mean follow-up of 5 years, serotypes were compared with genotypes, which were determined by a line probe assay (HCV LiPa) and by direct sequencing of the products obtained by nested PCR of the 5' untranslated region. Among the 58 samples with known genotypes, serotype-specific antibodies were detected in 38 (total sensitivity, 65.5%), with a specificity of 78.9%. Thirty of these serotypeable samples revealed a serotype that corresponded to the genotype in the 58 samples (total positive predictive value, 51.7%). Of the 58 samples, 23 were coinfected with HIV, and when these were excluded, the total sensitivity increased to 76.5%, with a total specificity of 80.8% and a total positive predictive value of 61.8%. The serotyping assay showed a high total sensitivity (96.3%) for samples positive by HCV RIBA, version 3.0, with four bands. We conclude that the sensitivity of the RIBA HCV serotyping SIA is limited by the immunocompetence of the HCV-infected host. In general, samples from HIV-negative individuals containing genotype 1a had higher sensitivity, specificity, and concordance in the serotyping assay compared with genotyping, whereas samples containing genotype 3a were found to be more cross-reactive and untypeable. Therefore, the prevalence of genotypes other than genotype 1 could be underestimated if they are determined by serotyping, and improvements in specificity are recommended.