Hepatitis C virus transmission in a pediatric oncology ward: analysis of an outbreak and review of the literature

Hospital-related hepatitis C virus (HCV) infections continue to occur even after the introduction of blood donor screening. We report an outbreak of HCV in nine patients of a pediatric oncology ward in 1996/1997. Sequencing of the hypervariable genomic region 1 (HVR1) of the E2/NS1 region showed near identity between HCV isolates from these patients as evidence for infection with the same virus. Despite a detailed and careful investigation, the source of infection and the mode of virus transmission could not be established. Based on a review of the current literature about nosocomial HCV infection and HCV infection in children, hypotheses for possible means of transmission in this outbreak are discussed.     

Molecular epidemiology of a hepatitis C virus outbreak in a hemodialysis unit

We analyzed a hepatitis C virus (HCV) transmission case in the hemodialysis unit of a private clinic by sequencing two genome regions of virus isolates from a number of patients attending this unit and some external controls. The analysis of 337 nucleotides (nt) in the NS5B region did not provide enough resolution to ascertain which patients were actually involved in the outbreak and the potential source. Nevertheless, this region allowed the exclusion of several patients as putative sources of the transmission case based on their genotypes and phylogenetic relationships. On the other hand, the analysis of several 472-nt-long clone sequences per sample in a more rapidly evolving region of the HCV genome, coding for the envelope proteins and encompassing hypervariable region 1, allowed us to establish the existence of at least two independent transmission events involving two different source patients and three recipients. The direction of the transmissions was further corroborated by different measures of genetic variability within and among samples.     

Molecular epidemiology of a hepatitis C virus outbreak in a hemodialysis unit in Italy

Hemodialysis patients are at increased risk of hepatitis C virus (HCV) infection. The aim of this study was to investigate a HCV outbreak in a hemodialysis unit using epidemiological and molecular methods. Between April 2003 and October 2003, anti-HCV seronconversion was detected in four patients attending the unit. These cases were added to 10 patients already anti-HCV positive upon admission in the unit. All 14 anti-HCV patients were tested for HCV RNA and HCV genotype. NS5B and HVR1/ E2 genomic regions were amplified and sequenced in all HCV RNA positive patients and phylogenetic analysis was performed. Furthermore, clinical-epidemiological records obtained from all patients were examined. All four patients newly infected harbored genotype 2c. Genotype 2c was also detected in 2 of 10 patients already anti-HCV positive upon admission. Phylogenetic analysis showed that all newly HCV infected patients harbored very closely related viral isolates that clustered together with the 2c isolate found in one of the two 2c chronic infected patients. All HCV-2c infected patients had no other risk factors except hemodialysis. Three of four newly HCV-2c infected patients and the one HCV-2c chronically infected involved in the outbreak received dialysis on the same day and same shift but used different machines. The remaining HCV-2c newly infected patient and one of the above cited three received dialysis on the same day during different shifts but used the same machine. The outbreak was probably due to breaks of infection control procedures although a related-machine transmission cannot be excluded in one of the cases.     

Genetic heterogeneity and molecular epidemiology of GB virus C/hepatitis G virus in China

OBJECTIVE: The inter- and intrapatient genetic variation of GB virus C (GBV-C)/hepatitis G virus (HGV) was investigated to characterize the molecular epidemiologic profile of GBV-C/ HGV infection in China, an area endemic for viral hepatitis. The intrapatient variation of hepatitis C virus (HCV) from the same patients was compared to that of GBV-C/HGV. STUDY DESIGN/METHODS: GB virus C/HGV RNA was amplified by polymerase chain reaction in 88 patients with hepatitis C, hepatitis B or presumed non-A-E hepatitis from three cities in China. Five clones of the GBV-C/HGV NS3 region were sequenced from each GBV-C/HGV RNA-positive patient. The corresponding region of HCV was also sequenced from patients co-infected with HCV. Representative sequences of the GBV-C/HGV NS3 region from each patient and those of isolates from other continents were subjected to phylogenetic analyses. RESULTS: GB virus C/HGV was detected in 22 (25.25%) of 88 patients: 9 (21.4%) of 42 patients with presumed non-A-E hepatitis, 10 (27.7%) of 36 patients with hepatitis C, 3 (30.0%) in 10 patients with hepatitis B and C, and in none of 60 volunteer blood donors. The extent of nucleotide variation was less between Chinese isolates (2.4-17%; median, 10.4%) than between Chinese isolates and seven isolates from outside China (10.5-19.5%; median, 15.3%). Intrapatient sequence variation ranged from 0 to 1.75%, with a mean of 0.57 +/- 0.51%. Phylogenetic analysis grouped most Chinese isolates into four geographically specific clusters with a divergence of 10% to 16% from each other. The ratio of nonsynonymous to synonymous substitutions of GBV-C/HGV (Ka/Ks 0.019) was much lower than for HCV (0.071) in the same patients. CONCLUSION: Chinese isolates of GBV-C/HGV are genetically distinct. There are local strains as well as shared strains between different locales. The extent of amino acid sequence conservation suggests strong selection against nonsynonymous substitutions in the GBV-C/HGV genome.     

Molecular and epidemiological study on nosocomial transmission of HCV in hemodialysis patients in Brazil

An epidemiological and molecular study of hepatitis C virus (HCV) infection was carried out in Brazilian hemodialysis centers. A total of 1,095 patients in all 15 hemodialysis centers in the State of Goiás, Brazil, were studied. All patients were interviewed for possible risk factors to HCV infection and serum samples tested for anti-HCV by ELISA and for HCV RNA by nested RT-PCR of the 5' NC region. For sequence analysis, HCV RNA amplification for the NS5B region (nt 8,279-8,619) was performed. The phylogenetic tree was generated with MrBayes, and clusters with support values above 0.85 were considered epidemiologically related. Of the 1,095 patients, 180 were anti-HCV and/or HCV RNA positive, resulting in an overall prevalence of 16.4% (95% CI: 14.3-18.7). The prevalence of HCV infection in the dialysis centers ranged from 0% to 47.7%. Multivariate analysis of risk factors revealed that history of blood transfusion not screened for anti-HCV and length of time on hemodialysis were independently associated with HCV infection in this population. One hundred six samples could be amplified and sequenced in the NS5B region. Among them, plylogenetic tree analysis revealed that 69 sequences form 13 separated clusters, which were supported by credibility intervals ranging from 85% to 100%, indicating a very close relationship among the HCV isolates and therefore a likely transmission of the virus between patients. By combining phylogenetic analysis with epidemiological data, routes of transmission between the clustered-related-patients could be suggested. These findings provide evidence for nosocomial transmission of HCV in Brazilian hemodialysis centers.     

丙型肝炎病毒非结构基因5b(NS 5b)区一级结构的变异

目的　分析中国丙型肝炎病毒非结构基因５ ｂ 区核苷酸序列变异。方法　以逆转录套式聚合酶链反应（ＲＴｎｅｓｔｅｄＰＣＲ） 从４９ 例中国病人的血清中获得互补的ＤＮＡ片段,产物克隆后测序。结果　３３ 株系基因Ⅱ１ ｂ 型,１６ 株系Ⅲ２ ａ 型,中国ＨＣＶ株型与日本ＨＣＶ株型同属１ 个基因亚型,但在一些核苷酸保守位点上有一定的差异,对３３ 株Ⅱ１ ｂ 型和１６ 株Ⅲ２ ａ 型间的同源性进行比较,发现１６ 株Ⅲ２ ａ 型的同源性低于３３ 株Ⅱ１ ｂ 型的同源性。首次在ＨＣＶＮＳ５ ｂ 区发现１ 个新的３ 个核苷酸的缺失突变及１ 个移码突变。结论　同一基因亚型之内不同ＨＣＶ株的核苷酸序列可能具有一定的地理分布特征,每个地区有一定的流行株。     

Molecular cloning of hepatitis C virus genome from a single Japanese carrier: sequence variation within the same individual and among infected individuals.

A hepatitis C virus (HCV) genome was isolated and sequenced from a single Japanese patient with chronic non-A, non-B hepatitis. The genome (HCV-JT), which was constructed with 23 cDNA clones, consisted of 9436 nucleotides with a long open reading frame which could encode a sequence of 3010 amino acid residues. To study the sequence variation of the HCV genome in an individual, we analyzed another sequence of the HCV genome (HCV-JT') constructed with different cDNA clones derived from the same patient. The nucleotide variation between HCV-JT and -JT' was less than 1%, and was distributed throughout the genome except in the 5' non-coding region, where no variation was observed. The diversity was higher (1.6%) in the putative envelope protein region than in other regions. The nucleotide and deduced amino acid sequences of HCV-JT showed homologies of about 91 and 95%, respectively, with those of other Japanese HCV isolates. The nucleotide diversity was high in the gp 70 region (corresponding to the NS 1 region of flaviviruses) and low in the 5' non-coding and p22 (putative core protein) regions. A similar pattern of distribution of nucleotide changes was observed on comparison of HCV-JT with an American isolate HCV-US, where the homologies in nucleotide and amino acid sequences were about 79 and 85%, respectively. Base transversions contributed about 50% of the total base exchanges between the Japanese and American HCV sequences, but only 20% or less of those among Japanese HCV or among American HCV sequences. Thus, the Japanese and American HCVs are genetically distinguishable, supporting our earlier prediction that these two HCVs could be classified as different subtypes.     

Molecular epidemiology of an outbreak of fulminant hepatitis B

A nosocomial outbreak of hepatitis B occurred among the inpatients of a hematology unit. Nine of the 11 infected patients died from fulminant hepatitis. An investigation was conducted to identify the source of infection and the route of transmission. Two clusters of nosocomial hepatitis B were identified. The hepatitis B virus (HBV) genome from serum samples of all case patients, of one HBsAg-positive patient with acute reactivation of the infection, and of eight acutely infected, unrelated cases was identified by PCR amplification of viral DNA and was entirely sequenced. Transmission was probably associated with breaks in infection control practices, which occurred as single events from common sources or through a patient-to-patient route, likely the result of shared medications or supplies. Sequence analysis evidenced close homology among the strains from the case patients and that from the patient with reactivation, who was the likely source of infection. Molecular analysis of viral isolates evidenced an accumulation of mutations in the core promoter/precore region, as well as several nucleotide substitutions throughout the genome. The sequences of all patients were compared with published sequences from fulminant and nonfulminant HBV infections.     

Monitoring hepatitis C infection in a major Swedish nephrology unit and molecular resolution of a new case of nosocomial transmission

Hepatitis C virus (HCV) infection is a frequent problem in hemodialysis units. The prevalence and incidence of HCV infection over a decade were studied in a nephrology unit affected by previous nosocomial HCV transmission. The HCV non‐structural 5B protein gene was sequenced to achieve phylogenetic analysis of a new (incident) case of infection. Proportions of patients who were and were not infected with HCV remained similar over the period, as did the inflow and outflow of patients infected previously. In 1997, 12/157 (8%) of patients at the unit (treatment: hemodialysis, peritoneal dialysis, and renal transplant recipients) were positive in HCV RNA, whereas in 2007 the overall number was 9/239 (4%). One patient acquired an HCV infection, and the NS5B sequence in that case clustered with genotype 2b sequences found in patients from an earlier outbreak. Comparing the HCV from the incident patient with several stored longitudinal samples and cloned PCR products from the most likely source patient revealed close phylogenetic relationship with an HCV quasispecies member from the possible source. The source patient and the incident newly infected patient were not scheduled on the same dialysis shift, although the records showed that simultaneous treatment occurred on two occasions during the months preceding transmission. In conclusion, over the 10‐year period, the proportion of HCV‐infected patients at the unit was unchanged. Only one new infection occurred, which originated from a fellow patient's quasispecies. This establishes phylogenetic analysis as a valuable tool for tracing patient sources of HCV transmission. J. Med. Virol. 82:249–256, 2010. © 2009 Wiley‐Liss, Inc.     

Determining the source of nosocomial transmission in hemodialysis units in Tunisia by sequencing NS5B and E2 sequences of HCV

Hepatitis C virus infection is a significant problem in hemodialysis units. HCV is very variable genetically with six genotypes. Clinical and epidemiological investigation of a new infection requires the determination of both the genotype and the strain of the HCV involved. A prospective, epidemiologic study of 395 dialysis patients in Tunisia was conducted from November 2001 to November 2003 to identify the source of nosocomial transmission using phylogenetic analysis of NS5b and E2 sequences. Hepatitis C infection was diagnosed by screening for anti-HCV antibodies and HCV RNA in sera using third generation ELISA and a qualitative RT-PCR assay. HCV strains were genotyped by sequencing the NS5b region. The genetic relatedness of the HCV strains was studied by sequencing the NS5b and the HVR-1 regions of the HCV genome. Two de novo cases of HCV infection were detected during the follow-up. One of them has been described previously. The case described in this study occurred in a center in which 12 patients were already infected with HCV strains belonging to genotypes 1b (n = 8) and 1a (n = 4). Phylogenetic analysis of the NS5b region from the HCV strains circulating in this center disclosed four clusters, confirmed by analysis of the HVR-1 region, providing strong evidence for nosocomial infection. Epidemiological data showed that these patients were dialyzed during the same shift and in the same area. Phylogenetic analysis of NS5b sequences is useful for determining the HCV genotype and providing evidence of nosocomial transmission.     

Nosocomial transmission in simultaneous outbreaks of hepatitis C and B virus infections in a hemodialysis center

Reported here are details of a simultaneous outbreak of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections that occurred in a hemodialysis centre in northern Italy, with three patients seroconverting for HBsAg and four patients seroconverting for HCV antibodies. Phylogenetic analysis of the E2 region of the isolates from HCV-seroconverted patients showed the sequences were grouped in the same distinct branch as in a chronically HCV-infected patient, suggesting that the chronically infected patient was the index case. For the patients with HBV infection, phylogenetic analysis showed strong clustering among the sequences of the three patients who seroconverted to HBsAg and no relatedness between them and the sequences of patients chronically infected with HBV. For one of the patients who seroconverted to HBsAg, the last test with negative results for HBV markers had been performed 18 months prior to HBsAg seroconversion. This patient may have been previously infected with HBV and is presumed to be the source of the outbreak. This report emphasizes the importance of using universal precaution measures and HBV vaccination to prevent the transmission of viral hepatitis among chronic hemodialysis patients.     

Use of phylogenetic analysis of hepatitis C virus (HCV) hypervariable region 1 sequences to trace an outbreak of HCV in an autodialysis unit

Hemodialysis patients are at high risk of infection by hepatitis C virus (HCV). The aim of this study was to investigate an HCV outbreak that occurred in an autodialysis unit by using epidemiological and molecular methods. Seroconversion to HCV antibody (anti-HCV) was observed in two patients over an 18-month period; two other patients had previously been recorded as anti-HCV positive. All four patients involved in the outbreak were tested for HCV RNA, and hepatitis C genotype determination was accomplished by a reverse hybridization assay. Furthermore, part of hypervariable region 1 (HVR1) of the hepatitis C genome was amplified and sequenced in samples from all HCV RNA-positive patients. Phylogenetic analysis of the nucleotide sequences obtained was carried out in order to investigate any possible epidemiological linkages among patients. The nucleotide sequences of the HVR1 regions of both newly infected patients were found to be identical to sequences of samples from previously recorded anti-HCV-positive original patients, suggesting that they were infected by the same isolate. Molecular and epidemiological analysis suggested that nosocomial patient-to-patient transmission was the most likely explanation for the virus spread in the autodialysis unit under study.     

Diversity of Hepatitis C Virus Quasispecies Evaluated by Denaturing Gradient Gel Electrophoresis

Denaturing gradient gel electrophoresis (DGGE) was used to study the diversity of hepatitis C virus (HCV) quasispecies. Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the nonstructural 5b (NS5b) gene of HCV in blood of hemophilia patients, intravenous drug users, and blood donors (five specimens from each study group, ca. 40 clones studied per specimen). Clones identified by DGGE as unique were sequenced. NS5b sequence entropy and mean genetic distance in hemophiliacs did not differ significantly from those in the other groups, pointing to a lack of correlation between HCV diversity and the multiplicity of past HCV exposures. DGGE was also applied to investigate variation in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum samples serially taken from two patients during the seroconversion phase of HCV infection. E2/HVR-1 sequence entropy changes were small and not correlated with rising anti-HCV antibody levels, reflecting mutational changes not mediated by antibody selection.     

Diversity of hepatitis C virus quasispecies evaluated by denaturing gradient gel electrophoresis

Denaturing gradient gel electrophoresis (DGGE) was used to study the diversity of hepatitis C virus (HCV) quasispecies. Optimized DGGE running conditions were applied to screen for variations in sequences cloned from amplicons originating from the nonstructural 5b (NS5b) gene of HCV in blood of hemophilia patients, intravenous drug users, and blood donors (five specimens from each study group, ca. 40 clones studied per specimen). Clones identified by DGGE as unique were sequenced. NS5b sequence entropy and mean genetic distance in hemophiliacs did not differ significantly from those in the other groups, pointing to a lack of correlation between HCV diversity and the multiplicity of past HCV exposures. DGGE was also applied to investigate variation in the HCV envelope 2/hypervariable region 1 (E2/HVR-1) in serum samples serially taken from two patients during the seroconversion phase of HCV infection. E2/HVR-1 sequence entropy changes were small and not correlated with rising anti-HCV antibody levels, reflecting mutational changes not mediated by antibody selection.     

Genetic drift of parvovirus B19 is found in AIDS patients with persistent B19 infection

It is generally thought that parvovirus B19 is stable genetically. Consistently, genetic drift has not been found in patients with persistent B19 infection. In this report, longitudinal genetic changes in NS1 and VP1 gene of B19 isolates from three AIDS patients with persistent B19 infection were studied. One of the three patients was not treated with highly active anti-retroviral therapy (HAART). B19 viral DNA from these patients was amplified by polymerase chain reaction (PCR) and then sequenced directly. A single genetic change was found in the B19 isolate obtained from the patient not treated with HAART on Day 10 after intravenous immunoglobulin (IVIG) treatment. The nucleotide sequences of B19 isolated from this patient, then remained unchanged over a period of 11 months. Analysis of NS1 clones derived from his longitudinal viral isolates showed the existence of quasi-species but genetic drift was not found. One of the other two patients treated with HAART experienced treatment failure; he was later treated with mega-HAART. In contrast to the genetic stability of B19 isolates from the patient not treated with HAART, multiple genetic changes were discovered in the viral isolates from the two other patients after HAART and mega-HAART, respectively. Through analysis of B19 clones, the frequency of clones containing these mutations confirmed the genetic drift. Nucleotide substitutions seen in VP2 gene of isolates with genetic drift from both patients were all non-conserved, suggesting that they are positively selected.     

Hepatitis C virus infection among dialysis patients in Tunisia: incidence and molecular evidence for nosocomial transmission

In order to study the incidence of hepatitis C virus (HCV) infection in Tunisian haemodialysis patients and detect its nosocomial transmission, 395 patients were enrolled in a prospective study (November 2001-2003). HCV serological and virological status was determined initially using, respectively a third generation ELISA and an RT-PCR qualitative assay. The genotype of the HCV isolates was determined by sequencing NS5B region. The issue of nosocomial transmission was addressed by sequencing the HVR-1 region of the E2 gene. About 20% of the patients had anti-HCV antibodies and HCV-RNA was detected in 73% of the anti-HCV positive patients. Two cases of de novo HCV infection were identified in two dialysis centers, during virological follow-up of patients susceptible to HCV infection. The incidence of de novo HCV infection was 0.5%. Determining the genotypes in the first center disclosed that all HCV-positive patients were infected with genotype 1b; sequencing of the HVR-1 region of the E2 gene provided strong evidence that the isolate from the newly infected patient and another infected dialysis patient were closely related, confirming nosocomial contamination. The investigation of the second center is pending. Besides, one patient with negative HCV serology had detectable HCV-RNA at the beginning of the study. This case had HCV genotype 1b, two other infected dialysis patients in the same unit had HCV genotypes 4k and 3a; thus precluding nosocomial transmission. Thanks to molecular and phylogenetic methods, one case of nosocomial HCV transmission in haemodialysis was confirmed. Epidemiological investigation suggested nosocomial transmission via the medical and/or nursing staff.     

Hepatitis C virus (HCV) genotype distribution in German isolates: studies on the sequence variability in the E2 and NS5 region

Summary We report on molecular characterization of hepatitis C virus (HCV) isolates in intravenous drug abusers, as compared to non-drug using patients with posttransfusion hepatitis or sporadic hepatitis of unknown origin. Virus typing was performed by RFLP analysis of PCR products in the 5 NCR. Subtyping was done by hybridization with subtype specific probes or by sequencing in the NS4 and NS5 region, respectively. HCV subtype 1b was found most commonly among all the isolates. However, the subtype 3a had a high prevalence (about 46%) in the group of drug addicts. In these subtype 3a isolates the N-terminal part of the E2 protein was highly variable. This confirms the presence of a hypervariable region (HVR1) in this envelope protein found in all hepatitis C viruses. Each subtype 3a isolate examined had a characteristic unique hypervariable region in the E2 protein. It is noteworthy that there are four amino acids in this region which were highly conserved between all HCV sequences published. It can be assumed that such conserved amino acids are significant for structure and function of this viral protein. In our HCV subtype 3a isolates the NS5 sequences were highly conserved.     

Molecular epidemiology and evolution in an outbreak of fulminant hepatitis B virus

In order to establish the transmission pathway for two outbreak patients affected by fulminant hepatitis B (FHB) following a shared period of hospitalization, we sequenced the complete genomes of the hepatitis B viruses (HBV) isolated from them as well as from the suspected common source and 11 additional controls. Phylogenetic and statistical analyses of these sequences revealed that the two FHB patients were indeed infected by a common source and that the fatal development of the disease did not appear to be associated with any mutation previously reported to be related to FHB. These data have also allowed us to estimate the extent and distribution of genetic variability along the genomes of HBV genotype D samples from the same source population. As a result of these analyses, we provide an improved statistical method to individualize the assignment of each suspected patient and the source of an outbreak and information on which genome region to analyze in the molecular epidemiological assessment of hepatitis B virus transmission cases.