Zhibing Yun

Hepatitis C Infection in Patients with Primary Hypogammaglobulinemia after Treatment with Contaminated Immune Globulin

BACKGROUND In Scandinavia many patients with primary hypogammaglobulinemia contracted non-A, non-B hepatitis after intravenous treatment with an immune globulin product that was later found to contain a non-A, non-B hepatitis virus. METHODS We studied the prevalence and clinical course of hepatitis C virus (HCV) infection in a group of 55 Norwegian patients with primary hypogammaglobulinemia and investigated its association with the use of contaminated immune globulin. We used the polymerase chain reaction to detect HCV RNA and performed HCV genotyping. We also analyzed the responses to treatment with interferon. RESULTS Of 20 patients who received the contaminated immune globulin, 17 were seropositive for HCV RNA: In addition, 1 of 35 patients not exposed to the contaminated immune globulin was HCV RNA--positive. HCV genotype V was found in all 12 patients for whom genotyping was performed, but 8 patients also had genotype II or III, or both. All HCV RNA--positive patients had abnormal results on biochemical liver tests. All liver-biopsy specimens (from 15 patients) were abnormal, with portal inflammation, bile-duct damage, and focal necrosis. In six patients there was cirrhosis. Two patients died of liver failure. In 4 of the 10 patients treated with interferon there were complete, though transient, biochemical responses, but the follow-up biopsy specimens showed evidence of histologic progression. The poorest responses to interferon were among the patients with multiple HCV genotypes. All but one patient remained positive for HCV RNA: CONCLUSIONS In patients with primary hypogammaglobulinemia there was a high rate of HCV infection after treatment with contaminated immune globulin. In these immunocompromised patients HCV infection has a severe and rapidly progressive course, and responses to interferon are poor.

A real-time TaqMan PCR for routine quantitation of cytomegalovirus DNA in crude leukocyte lysates from stem cell transplant patients.

A real-time TaqMan PCR based on the cytomegalovirus (CMV) polymerase (pol) gene was developed for quantitation of CMV DNA in crude peripheral blood leukocyte (PBL) lysate from stem cell transplantation (SCT) patients. The dynamic range of the assay was between 10 and 4x10(6) copies. Both intra- and inter-assay variability were well within +/-0.25 log10 S.D. Thus, a pooled PBL sample that was used as positive control in 57 consecutive TaqMan PCR runs over 7 months showed a stable CMV quantity (4.12+/-0.13, log10 mean+/-S.D.). The sensitivity of the pol TaqMan PCR was validated by parallel analysis of 177 PBL samples with a nested PCR. The use of crude PBL lysate as PCR input did not cause PCR inhibition. We demonstrated further the clinical utility of the newly developed TaqMan PCR by monitoring changes in CMV levels in eight patients receiving antiviral therapy. This TaqMan PCR was highly sensitive, reproducible, and stable and has served a useful tool for monitoring CMV DNA levels in large number of clinical samples in a routine diagnostic setting for over 1 year.

Real-time monitoring of cytomegalovirus infections after stem cell transplantation using the TaqMan polymerase chain reaction assays.

BACKGROUND Real-time monitoring of cytomegalovirus (CMV) infections in transplant patients demands a rapid and high-throughput CMV DNA quantification method. METHODS TaqMan polymerase chain reaction assays based on CMV immediate early protein exon 4 and glycoprotein B were developed and were compared with a COBAS AMPLICOR CMV MONITOR (CMM) test for quantifying CMV DNA in peripheral blood leukocytes from seven stem cell transplant patients having received antiviral treatment. RESULTS There was a good correlation between the TaqMan assays and the CMM test for CMV DNA quantification. The throughput of the TaqMan assays was, however, about 3 times higher than that of the CMM test. The CMV DNA dynamics patterns determined by the TaqMan polymerase chain reaction were well in line with the outcome of the antiviral therapy. CONCLUSIONS The TaqMan assays may potentially serve as a useful tool for rapid quantification of CMV infections in stem cell transplant patients.

Discrepancy of hepatitis C virus genotypes as determined by phylogenetic analysis of partial NS5 and core sequences

The use of phylogenetic analyses of partial NS5 and core regions for hepatitis C virus (HCV) genotyping was evaluated by analysing seven Honduran and 24 European HCV strains. Core primers were designed with which HCV genotypes 1, 2, and 3 were readily amplified. The reliability of phylogenetic analysis of a 111‐bp core sequence was verified by comparing the typing results with those obtained using the whole core gene of 52 reference strains. Accordant genotypes (1a, 1b, 2b, and 3a) were obtained when phylogenetic analyses were undertaken on both the partial core and a 222‐bp NS5 sequence in all of the European HCV strains. Genotypes 1a, 1b, and 3a were identified among the Honduran strains by phylogenetic analysis of the partial NS 5 sequence. Interestingly, two of three Honduran type 3a strains, as determined by the NS5 sequence analysis, turned out to be type 1a by core sequence analysis. These two strains were also classified as type 1a, but not 3a, by a core type‐specific PCR. Furthermore, the R2/NS1 regions were similar to HCV‐PT, a representative strain of genotype 1a. The results indicate that chimeral HCV strains exist, although in most cases a good concordance is found when phylogenetic analysis of partial core and NS5 sequences are used for genotyping. This finding should be taken into account when HCV is genotyped by a phylogenetic analysis of a partial HCV sequence from a single genomic region.

Variation of Hepatitis C Virus Hypervariable Region 1 in Immunocompromised Patients

The viral variability of 5 hepatitis C virus (HCV)-infected immunocompromised patients was analyzed and compared with that in isolates from immunocompetent subjects. The patients were followed longitudinally with regard to changes in hypervariable region 1 (HVR1) of HCV using a direct DNA sequencing approach. For the immunocompromised patients, viral nucleotide sequence variability was markedly lower than in immunocompetent HCV-positive patients. For 1 agammaglobulinemic patient and 1 AIDS patient, no variation in the major amino acid sequence of HCV HVR1 could be observed, while another agammaglobulinemic patient exhibited transient variations and amino acid substitutions despite the lack of functioning humoral immune response. The study supports the general hypothesis of humoral immune selection as the main force of sequence variation in the HVR1 region but suggests that other selection mechanisms may contribute to modulation of the composition of the viral population.

Variation in the hepatitis C virus NS5a region in relation to hypervariable region 1 heterogeneity during interferon treatment

The putative interferon sensitivity determining region (ISDR) in the NS5a region of the hepatitis C virus (HCV) was analyzed in 13 interferon alpha (IFN‐α) treated patients representing genotypes 1a, 1b, and 2b. These patients had previously been followed longitudinally during treatment with respect to viral load and to virus heterogeneity using the hypervariable region 1 (HVR1) sequence as a marker. In the present study, the NS5a region was analyzed for nonresponders and sustained responders using direct DNA sequencing. While the previous results of analyzing viral composition and load showed evidence of selection, no corresponding selection of specific NS5a ISDR sequences was observed in the nonresponders, and identical ISDR sequences were observed among both sustained responders and nonresponders. Thus, we cannot verify a correlation between ISDR sequence and the observed selection of IFN‐α‐resistant quasispecies demonstrated as a restriction of HVR1 heterogeneity. This indicates that the potential for using ISDR as a diagnostic or prognostic marker during IFN‐α treatment is limited. J. Med. Virol. 56:33–38, 1998.

Quantification of Human Immunodeficiency Virus Type 1 Proviral DNA by the TaqMan Real-Time PCR Assay

A variety of real-time PCR assays have been applied for quantification of human immunodeficiency virus type 1 (HIV-1) (4-7). Recently, Desire et al. have reported a TaqMan PCR for quantification of HIV-1 provirus in peripheral blood mononuclear cells (PBMCs) (3). This method targets the polymerase (pol) gene of HIV-1 subtype B. Based on their data, Desire et al. have concluded that HIV-1 proviral DNA load in PBMCs does not correlate with HIV-1 RNA level in plasma and CD4+ lymphocyte counts. This conclusion is contradictory to those previously drawn by us (2) and recently drawn by others (8). In order to clarify if the use of different DNA quantification methods may have contributed to this controversy, we decided to compare the TaqMan PCR by Desire et al. with a TaqMan PCR recently developed in our laboratory. Our TaqMan PCR targeted the long terminal repeat region (LTR) of the HIV-1 major group. The sequences of the sense and antisense primers were 5′-GCCTCAATAAAGCTTGCCTTGA-3′ and 5′-GGGCGCCACTGCTAGAGA-3′. The probe sequence was 5′-CCAGAGTCACACAACAGACGGGCACA-3′. The 5′ and 3′ ends of the probe were labeled with FAM and TAMRA dyes. The sensitivity of the assay was estimated to be five copies of HIV-1 plasmid DNA (Applied Biosystems). We randomly selected 25 PBMC samples, which had been earlier confirmed to be HIV-1 DNA positive by a nested PCR (1). The HIV-1 DNA was extracted from the PBMCs with the Qiagen blood kit. Five microliters of each DNA extract (equivalent to 2 × 10 5 PBMCs) was tested with both the pol- and LTR-based TaqMan PCR assays on the same plate for 2 cycles) were obtained by the pol TaqMan PCR (Table ​(Table1).1). Furthermore, HIV-1 DNA in seven HIV-1-positive PBMC samples was not detected by the pol TaqMan assay. Among those seven HIV-1 false-negative samples, three were PBMC culture positive for HIV-1. Our results suggest that the pol TaqMan PCR exhibits an unsatisfactory low sensitivity on the selected sample panel. The fact that some of the tested samples belonged to a subtype other than B may partly contribute to the high false negativity of the HIV-1 pol TaqMan PCR assay. However, the lower sensitivity was also seen when HIV-1 subtype B SF2 strain was tested with the pol TaqMan PCR (Table ​(Table1).1). Therefore, we further examined the pol TaqMan PCR design by using PrimerExpress software (version 2.0). The melting temperature of the reverse primer P2 was significantly lower than that of the forward primer P1 (50.4 versus 60.5°C). At the reported annealing temperature (60°C), an asymmetric amplification could have been taking place during PCR due to the poor hybridization of the P2 primer with the template, resulting in decreased amplification efficiency. Considering that HIV-1 load turns out to be extremely low after efficient antiretroviral therapy (ART), we believe that a highly sensitive real-time PCR is demanded for the purpose of ART monitoring. TABLE 1. Comparison of the numbers of Ct obtained by two TaqMan PCRs for HIV-1 DNA detection in PBMCs

Longitudinal quantification of human immunodeficiency virus type 1 DNA and RNA in long-term nonprogressors.

Twenty patients with human immunodeficiency virus type 1 (HIV-1) infection for >7 years, no HIV-1-related symptoms, no treatment, and CD4+ cell counts >500/microL were included in a prospective study in 1993. Four years later, 12 patients had progressed (SPs), while 8 had not (long-term nonprogressors [LTNPs]). At inclusion, HIV-1 RNA, but not DNA, levels were higher in SPs. During follow-up, a consistent increase in HIV-1 RNA was seen in only 1 LTNP. In 2 LTNPs, plasma viremia was persistently undetectable or <110 copies/mL. Infectious virus was isolated from only 1 LTNP and from 11 SPs. In 4 LTNPs, HIV-1 DNA levels decreased spontaneously with time. The restricted viral replication and the declining HIV-1 DNA levels suggest that the HIV-1 infection can be controlled efficiently in a few LTNPs, leading to a decrease in the total virus burden with time.

Colorimetric detection of competitive PCR products for quantification of hepatities C viremia

A method based on competitive polymerase chain reaction (PCR) and colorimetric detection of the amplified products was developed to quantify hepatitis C virus (HCV) genomes. Serum samples were obtained from patients who were treated with interferon alpha (IFN-alpha). After reverse transcription of the HCV RNA, the cDNA was coamplified with a serially diluted cloned HCV competitor DNA using nested PCR. The competitor DNA consisted of the amplified region of the wild type HCV cDNA with an internal region substituted with the lac operator (lacO) sequence. The PCR products were quantitated specifically by a colorimetric solid-phase assay. The results suggest that the method is well suited for analysing the kinetics of the anti-HCV effects during IFN-alpha treatment. The quantification assay is simple, reliable and suitable for quantitating HCV genomes in a large number of clinical samples.

Parvovirus B19 infection in HIV-1 infected patients with anemia

SummarySerum samples were analysed for IgM and IgG antibodies to parvovirus by ELISA and for parvovirus B19 DNA by polymerase chain reaction (PCR) in 69 HIV-1 infected Swedish patients with anemia and in 37 HIV-1 infected subjects without anemia. In 5/69 anemic patients, parvovirus B19 DNA was detected despite the lack of IgM antibody activity to the virus. The detection of parvovirus B19 DNA was significantly correlated to the degree of anemia in the anemic patients. In two patients who had a chronic anemia, a persistent parvovirus infection was detected by PCR, but not by serology, for 1 and 1.5 years, respectively. The results suggest that persistent parvovirus infection is a rare cause of anemia, but important to identify, since the infection is potentially treatable with intravenous immunoglobulin.ZusammenfassungSerumproben von 69 HIV-infizierten schwedischen Patienten mit Anämie und von 37 HIV-Infizierten ohne Anämie wurden mittels ELISA auf IgM- und IgG-Antikörper gegen Parvovirus B19 untersucht, auch wurde mittels Polymerasekettenreaktion (PCR) das Vorliegen von Parvovirus B19 DNA geprüft. Bei 5/69 anämischen Patienten war Parvovirus B19 DNA nachzuweisen, obwohl keine IgM-Antikörperreaktion gegen das Virus vorlag. Zwischen dem Nachweis von Parvovirus B19 DNA und dem Schweregrad der Anämie bestand bei den anämischen Patienten eine signifikante Korrelation. Bei zwei Patienten mit seit 1 beziehungsweise 1 1/2 Jahren bestehender chronischer Anämie konnte unter Anwendung der PCR eine persistierende Parvovirus-Infektion festgestellt werden, für die serologisch kein Hinweis vorlag. Die Ergebnisse lassen annehmen, daß die persistierende Parvovirus-Infektion eine seltene Ursache für eine Anämie darstellt, ihr Nachweis jedoch wegen der möglichen Behandlung mit intravenösem Immunglobulin von klinischer Relevanz ist.