Identification of epidermal growth factor receptor-tyrosine kinase inhibitor targeting the VP1 pocket of human rhinovirus.

Screening a library of 1,200 preselected kinase inhibitors for anti-human rhinovirus 2 (HRV-2) activity in HeLa cells identified a class of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) as effective virus blockers. These were based on the 4-anilinoquinazoline-7-oxypiperidine scaffold, with the most potent representative AZ5385 inhibiting the virus with EC50 of 0.35 µM. Several structurally related analogs confirmed activity in the low µM range, while interestingly, other TKIs targeting EGFR lacked anti-HRV-2 activity. To further probe this lack of association between antiviral activity and EGFR inhibition, we stained infected cells with antibodies specific for activated EGFR (Y1068) and did not observe a dependency on EGFR-TK activity. Instead, consecutive passages of HRV-2 in HeLa cells in the presence of a compound and subsequent nucleotide sequence analysis of resistant viral variants identified the S181T and T210A alterations in the major capsid VP1 protein, with both residues located in the vicinity of a known hydrophobic pocket on the viral capsid. Further characterization of the antiviral effects of AZ5385 showed a modest virus-inactivating (virucidal) activity, while anti-HRV-2 activity was still evident when the inhibitor was added as late as 10 h post infection. The RNA copy/infectivity ratio of HRV-2 propagated in AZ5385 presence was substantially higher than that of control HRV indicating that the compound preferentially targeted HRV progeny virions during their maturation in infected cells. Besides HRV, the compound showed anti-respiratory syncytial virus activity, which warrants its further studies as a candidate compound against viral respiratory infections.

Anti-respiratory syncytial virus and anti-herpes simplex virus activity of six Tanzanian medicinal plants with extended studies of Erythrina abyssinica stem bark.

ETHNOPHARMACOLOGICAL RELEVANCE: Except for few highly pathogenic viruses, no antiviral drug has been approved for treatment of viral infections in humans. Plant extracts, selected based on their ethno-medical use, represent an important source of compounds for the development of novel candidate antiviral drugs. This especially concerns plants with ethnomedical records on their use in treatment of viral infections. AIM OF THE STUDY: To identify and document medicinal plants used by traditional health practitioners (THPs) for treatment of respiratory infections and muco-cutaneous lesions in order to study their antiviral activity including identification of active components and elucidation of mode of antiviral activity. MATERIALS AND METHODS: The ethno-medical survey was performed in the Kagera region of Tanzania. The THPs were asked for plants used for treatment of signs and symptoms of respiratory infections and watery muco-cutaneous blisters in oral and genital regions. The plants identified were successively extracted with n-hexane, ethyl acetate and water, and the extracts assayed for anti-respiratory syncytial virus (RSV), anti-herpes simplex virus 2 (HSV-2), and anti-human parainfluenza virus 2 (HPIV-2) activity in cultured cells. Antiviral components were separated by ethanol precipitation and CL-6B chromatography, and the mode of antiviral activity elucidated by the time-of-addition assay and selection for the virus variants resistant to antiviral plant extract. RESULTS: THPs identified fifteen plants used for treatment of respiratory infections and muco-cutaneous blisters. The water extract, but not n-hexane or ethyl acetate extracts, of six of these plants including Erythrina abyssinica stem bark, inhibited infectivity of two glycosaminoglycan-binding viruses i.e., RSV and HSV-2 but not the sialic acid binding HPIV-2. An activity-guided separation revealed that antiviral component(s) of water extract of E. abyssinica could be precipitated with ethanol. This sample potently and selectively inhibited RSV and HSV-2 infectivity in cultured cells with IC50 values of 2.1 µg/ml (selectivity index >476) and 0.14 µg/ml (selectivity index >7143) respectively. The sample exhibited inhibitory effect on the virus attachment to and entry into the cells by directly targeting the viral particles. Indeed, 10 consecutive virus passages in HEp-2 cells in the presence of this extract selected for a resistant RSV variant lacking the attachment, viral membrane-associated, G protein due to a stop codon at amino acid residue 33 (Leu33stop). Fractionation of the E. abyssinica extract on a CL-6B column revealed that anti-RSV and HSV-2 activity correlated with carbohydrate content. The most pronounced antiviral activity was associated with a carbohydrate containing ingredient of molecular mass of <5 kDa, which may polymerize to antiviral composites of up to 410 kDa. CONCLUSIONS: Altogether, the water extract of six medicinal plants showed anti-RSV and anti-HSV-2 activities. Extended studies of the stem bark of E. abyssinica identified antiviral components that potently and selectively inhibited infectivity of free RSV and HSV-2 particles, a feature of importance in topical treatment of these infections. This observation confirms ethno-medical information concerning the use of E. abyssinica extract for treatment of respiratory infections and herpetic lesions.

Risk factors for norovirus infection in healthcare workers during nosocomial outbreaks: a cross-sectional study.

BACKGROUND: Norovirus outbreaks cause severe medico-socio-economic problems affecting healthcare workers and patients. The aim of the study was to investigate prevalence of norovirus infection and risk factors for infection in healthcare workers during nosocomial outbreaks. METHODS: A cross-sectional study of norovirus infections in healthcare workers was performed in seven outbreak wards in a large university hospital. Packs (swab for rectal sampling, and questionnaire) were posted to healthcare workers on notification of a ward outbreak. Rectal samples were examined with norovirus-specific real-time PCR. Replies from questionnaires were analysed using logistic regression models with norovirus genogroup (G)II positive findings as dependent variable. The results are expressed as odds ratios (OR) with 95% confidence intervals (CI). Sequencing and phylogenetic analyses (1040 nucleotides) were used to characterize norovirus strains from healthcare workers. Cluster analyses included norovirus GII.4 strains detected in ward patients during the ongoing outbreaks. RESULTS: Of 308 packs issued to healthcare workers, 129 (42%) were returned. norovirus GII was detected in 26 healthcare workers (20.2%). Work in cohort care (OR 4.8, 95% CI 1.4-16.3), work in wards for patients with dementia (OR 13.2, 95% CI 1.01-170.7), and having diarrhoea, loose stools or other gastrointestinal symptoms the last week (OR 7.7, 95% CI 2.5-27.2) were associated with increased norovirus prevalence in healthcare workers. Sequencing revealed norovirus GII.4 in healthcare workers samples, and strains detected in healthcare workers and ward patients during a given ward outbreak showed ≥ 99% similarity. CONCLUSION: Norovirus positive findings in healthcare workers were strongly associated with symptomatic infection, close contact with sick patients, and dementia nursing.

Seroprevalence of Zika virus and Rubella virus IgG among blood donors in Rwanda and in Sweden.

Seroprevalence studies provide information on the susceptibility to infection of certain populations, including women of childbearing age. Such data from Central Africa are scarce regarding two viruses that cause congenital infections: Zika virus (ZIKV), an emerging mosquito-borne infection, and Rubella virus (RuV), a vaccine-preventable infection. We report on the seroprevalence of both ZIKV and RuV from Rwanda, a country without any known cases of ZIKV, but bordering Uganda where this virus was isolated in 1947. Anti-ZIKV-specific and anti-RuV-specific immunoglobulin G (IgG) antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA) in serum samples from 874 Rwandan and 215 Swedish blood donors. Samples positive for IgG antibodies against ZIKV were examined for viral RNA using real-time reverse transcription polymerase chain reaction (RT-qPCR). The seroprevalence of ZIKV IgG in Rwanda was 1.4% (12/874), of which the predominance of positive findings came from the Southeastern region. All anti-ZIKV IgG-positive samples were PCR-negative. Among 297 female blood donors of childbearing age, 295 (99.3%) were seronegative and thus susceptible to ZIKV. All Swedish blood donors were IgG-negative to ZIKV. In contrast, blood donors from both countries showed high seroprevalence of IgG to RuV: 91.2% for Rwandan and 92.1% for Swedish donors. Only 10.5% (31/294) of female donors of childbearing age from Rwanda were seronegative for RuV. In Rwanda, seroprevalence for ZIKV IgG antibodies was low, but high for RuV. Hence, women of childbearing age were susceptible to ZIKV. These data may be of value for decision-making regarding prophylactic measures.

Mechanisms downstream of reverse transcription reduce serum levels of HBV DNA but not of HBsAg in chronic hepatitis B virus infection.

BACKGROUND: Hepatitis B virus (HBV) DNA in serum of chronically infected patients declines by 3-4 log10 units at loss of HBe antigen (HBeAg) from serum. The mechanisms behind this decline, and the much smaller decline of surface antigen (HBsAg) levels, are still not well known. The aim of this study was to get a better understanding of this process by analysing both serum and intrahepatic markers of HBV replication. METHODS: Levels of HBV DNA and HBsAg in serum, and covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) and S-RNA and total intrahepatic HBV DNA (ihDNA) in liver biopsies from 84 chronically infected patients (16 positive and 68 negative for HBeAg) were analysed. RESULTS: Lower HBV DNA levels within HBeAg-positive stage reflected lower levels of cccDNA and pgRNA with strong correlation. In HBeAg-negative patients, ihDNA levels were greater and HBV DNA levels in serum lower than expected from pgRNA levels. A lower HBV DNA/HBsAg ratio corresponded with lower pgRNA/cccDNA (p < 0.01) and higher S-RNA/cccDNA (p < 0.0001) ratios, suggesting that in HBeAg-negative patients transcription of pgRNA, but not of S-RNA, becomes suppressed. CONCLUSIONS: The marked reduction of HBV DNA in serum after loss of HBeAg appears to be due to combined reduction of cccDNA, pgRNA and yet unidentified mechanisms downstream of reverse transcription. Such mechanisms include faster clearance of circulating virus or blocked secretion of virions, the latter supported by the observed relative increase of ihDNA in HBeAg-negative patients. The smaller reduction of S-RNA than of pgRNA partly explains why HBsAg remain high in the HBeAg-negative stage, supporting the possibility of HBsAg synthesis from integrated HBV DNA.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Norovirus GII.4 detection in environmental samples from patient rooms during nosocomial outbreaks.

Norovirus (NoV) is an important cause of nosocomial gastroenteric outbreaks. This 5-month study was designed to characterize NoV contamination and airborne dispersal in patient rooms during hospital outbreaks. Air vents, overbed tables, washbasins, dust, and virus traps designed to collect charged particles from the air were swabbed to investigate the possibility of NoV contamination in patient rooms during outbreaks in seven wards and in an outbreak-free ward. Symptomatic inpatients were also sampled. Nucleic acid extracts of the samples were examined for NoV RNA using genogroup I (GI) and GII real-time reverse transcription-PCR (RT-PCR). The NoV strains were characterized by RT-PCR, sequencing, and phylogenetic analysis of the RNA-dependent RNA-polymerase-N/S capsid-coding region (1,040 nucleotides [nt]). Patient strains from two outbreaks in one ward were sequenced across the RNA-dependent-RNA-polymerase major capsid-coding region (2.5 kb), including the hypervariable P2 domain. In the outbreak wards, NoV GII was detected in 48 of 101 (47%) environmental swabs and 63 of 108 patients (58%); NoV genotype II.4 was sequenced from 18 environmental samples, dust (n = 8), virus traps (n = 4), surfaces (n = 6), and 56 patients. In contrast, NoV GII was detected in 2 (GII.4) of 28 (7%) environmental samples and in 2 (GII.6 and GII.4) of 17 patients in the outbreak-free ward. Sequence analyses revealed a high degree of similarity (>99.5%, 1,040 nt) between NoV GII.4 environmental and patient strains from a given ward at a given time. The strains clustered on 11 subbranches of the phylogenetic tree, with strong correlations to time and place. The high nucleotide similarity between the NoV GII.4 strains from patients and their hospital room environment provided molecular evidence of GII.4 dispersal in the air and dust; therefore, interventional cleaning studies are justified.

HBsAg quantification for identification of liver disease in chronic hepatitis B virus carriers.

BACKGROUND & AIMS: Quantification of hepatitis B surface antigen (HBsAg) has been proposed as a useful diagnostic marker for clinical staging (identification of inactive carrier state) and prognosis of chronic hepatitis B virus (HBV) infection. The aim of this study was to investigate the correlation between HBsAg levels in serum and histological liver damage in patients with chronic infection. METHODS: HBsAg levels in serum (by Abbott Architect) were related to HBV DNA, ALT and histological score (n=160) and covalently closed circular DNA (cccDNA) (n=84). RESULTS: HBsAg levels correlated with cccDNA, serum HBV DNA, ALT and high inflammation scores (P<0.001). Among HBeAg-negative patients, an HBsAg level below 3.0 log10 IU/ml identified minimal liver damage (normal ALT and mild inflammation) with a predictive value of 92% (alone) or 96% (in combination with HBV DNA<4.0 log10 copies/ml), whereas an HBsAg level above 3.5 log10 IU/ml identified severe inflammation with a predictive value of 16% (alone) or 33% (in combination with HBV DNA>5.0 log10 copies/ml). CONCLUSIONS: HBsAg levels reflect clinical stage and liver disease, and a combined quantification of HBsAg and HBV DNA may improve clinical staging.

Comparison between terminal-restriction fragment length polymorphism (T-RFLP) and quantitative culture for analysis of infants' gut microbiota.

The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism (T-RFLP). A database was constructed for direct identification of T-RFLP peaks by analysis of pure-culture bacteria and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at >106 CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts. In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be identified using the database. T-RFLP readily identified bacteria to the genus level and also provided some sub-genus discrimination. Both T-RFLP and culture identified Bifidobacterium, Clostridium and Bacteroides spp. among the most common colonizers of the infantile microbiota throughout the first year of life. T-RFLP analysis showed that microbiota complexity was high in the first weeks of life, declined to a minimum at 1-2 months of age, and thereafter increased again. Principal component analysis revealed that early samples (1 week-6 months) chiefly differed between individual infants, while 12-month samples were similar between children, but different from the early samples. Our results indicate that T-RFLP has high sensitivity and adequate taxonomic discrimination capacity for analysis of gut microbiota composition, but that both culture and molecular based analysis have limitations and both approaches may be needed to obtain a full picture of the complex gut microbiota.

Hepatitis B viral DNA decline at loss of HBeAg is mainly explained by reduced cccDNA load--down-regulated transcription of PgRNA has limited impact.

BACKGROUND: Quantification of hepatitis B virus (HBV) DNA and surface antigen (HBsAg) serum levels have become increasingly important for the assessment of clinical stage and response to treatment for chronic hepatitis B. Effective immune clearance results in reduction of viremia by 4-5 log units and HBsAg levels by 2 log, but these processes are not well understood. Thus, it is uncertain to what extent mechanisms that inhibit transcription of the pregenomic RNA (pgRNA), an RNA intermediate, contribute to suppression of viremia. Likewise, it is unclear if transcriptional regulation is important for the excessive production of surface antigen (HBsAg) that is a hallmark of HBV infection. METHODS: HBV RNA and cccDNA were quantified in 19 liver biopsies from patients with chronic HBV infection, as well as in transfected Huh7.5 cells and in PLC/PRF/5 cells carrying integrated HBV genome. RESULTS: Patients negative for HBeAg had 2.15 log lower levels of cccDNA in liver tissue, 4.84 log lower serum levels of HBV DNA and 1.45 log lower serum levels of HBsAg, than HBeAg-positive patients. The pgRNA in liver tissue correlated strongly with cccDNA (R(2) = 0.87, p<0.0001) and HBV DNA levels in serum (R(2) = 0.81, p<0.0001), whereas S-RNA correlated strongly with cccDNA (R(2) = 0.65, p<0.0001) and HBsAg levels (R(2) = 0.57, p = 0.0003). The S-RNA/pgRNA ratio was higher in HBeAg-negative patients (ratio 40 vs. 3, p = 0.01) and in PLC/PRF/5 cells, and was in transfected Huh7.5 cells not influenced by mutations in the HBV core promoter. CONCLUSION: The reduction of viremia that is observed after loss of HBeAg was mainly explained by reduced cccDNA load in the liver, whereas the contribution of down-regulation of pgRNA transcription was relatively small. Enhanced transcription of S-RNA does not explain excessive production of HBsAg.

Genotype impact on long-term virological outcome of chronic hepatitis B virus infection.

BACKGROUND: The importance of hepatitis B virus (HBV) genotype on the clinical course of chronic HBV infection is not yet clarified. OBJECTIVES: To investigate genotype impact on long-term virological outcome of chronic HBV infection. STUDY DESIGN: HBsAg, HBeAg, ALT and HBV DNA levels were determined after a median of 9.2 years of follow-up of 124 adults with chronic HBV infection, of whom 33 were HBeAg-positive at inclusion. RESULTS: HBV DNA levels decreased significantly in patients carrying genotype A (n=28), B (n=21) or D (n=63), but not in those with genotype C infection (n=12). Loss of HBeAg was seen in 44% (4/9) of patients with genotype C, as compared with 92% (22/24) with non-C genotypes. Loss of HBsAg was seen in 36% (10/28) patients with genotype A, 5% (1/21) with B, 0% (0/12) with C, and 11% (7/63) with genotype D. CONCLUSIONS: HBV DNA levels decreased over time in patients infected with genotypes A, B or D. However, highly active genotype C or D infection often remained highly active, implying a risk for progressive liver damage.

Marked genomic diversity of norovirus genogroup I strains in a waterborne outbreak.

Marked norovirus (NoV) diversity was detected in patient samples from a large community outbreak of gastroenteritis with waterborne epidemiology affecting approximately 2,400 people. NoV was detected in 33 of 50 patient samples examined by group-specific real-time reverse transcription-PCR. NoV genotype I (GI) strains predominated in 31 patients, with mixed GI infections occurring in 5 of these patients. Sequence analysis of RNA-dependent polymerase-N/S capsid-coding regions (∼900 nucleotides in length) confirmed the dominance of the GI strains (n = 36). Strains of NoV GI.4 (n = 21) and GI.7 (n = 9) were identified, but six strains required full capsid amino acid analyses (530 to 550 amino acids) based on control sequencing of cloned amplicons before the virus genotype could be determined. Three strains were assigned to a new NoV GI genotype, proposed as GI.9, based on capsid amino acid analyses showing 26% dissimilarity from the established genotypes GI.1 to GI.8. Three other strains grouped in a sub-branch of GI.3 with 13 to 15% amino acid dissimilarity to GI.3 GenBank reference strains. Phylogenetic analysis (2.1 kb) of 10 representative strains confirmed these genotype clusters. Strains of NoV GII.4 (n = 1), NoV GII.6 (n = 2), sapovirus GII.2 (n = 1), rotavirus (n = 3), adenovirus (n = 1), and Campylobacter spp. (n = 2) were detected as single infections or as mixtures with NoV GI. Marked NoV GI diversity detected in patients was consistent with epidemiologic evidence of waterborne NoV infections, suggesting human fecal contamination of the water supply. Recognition of NoV diversity in a cluster of patients provided a useful warning marker of waterborne contamination in the Lilla Edet outbreak.

Genotype X/C recombinant (putative genotype I) of hepatitis B virus is rare in Hanoi, Vietnam--genotypes B4 and C1 predominate.

There are eight known genotypes of hepatitis B virus, A-H, and several subgenotypes, with rather well-defined geographic distributions. HBV genotypes were evaluated in 153 serum samples from Hanoi, Vietnam. Of the 87 samples that could be genotyped, genotype B was found in 67 (77%) and genotype C in 19 (22%). All genotype C strains were of subgenotype C1, and the majority of genotype B strains were B4, while a few were B2. The genotype X/C recombinant strain, identified previously in Swedish patients of indigenous Vietnamese origin, was found in one sample. This variant, proposed to be classified as genotype I, has been found recently also by others in Vietnam and Laos. The current study indicates that the genotype X/C recombinant may represent approximately 1% of the HBV strains circulating in Vietnam.

Molecular analysis of an oyster-related norovirus outbreak.

BACKGROUND: Contaminated raw oysters were implicated in a severe outbreak of norovirus (NoV) gastroenteritis affecting 30 restaurant guests. OBJECTIVES: To define the outbreak source by using molecular methods to characterize NoV strains detected in patient and oyster samples. STUDY DESIGN: Molecular epidemiological studies based on nucleotide sequencing and phylogenetic analyses of patient and oyster NoV strains, and comparison to background dataset. RESULTS: NoV genotype (G) I.1 was detected in the one patient stool analyzed by in-house TaqMan real time RT-PCR and classical nested RT-PCR targeting NoV RNA-dependent polymerase (RdRp, 285 nt), and by nested RT-PCR targeting RdRp-capsid-poly(A)-3' (3085 nt). Patient strain showed >or=99% similarity (285 nt) with three NoV strains detected in two of five oysters examined by classical nested RT-PCR (RdRp). A third oyster tested positive for NoV GII.3. Phylogenetic analysis showed clustering of patient and oyster strains related to this outbreak with GI.1 strains from previous local outbreaks, and mussel studies. CONCLUSIONS: Sequence data revealed >or=99% similarity (285 nt) between NoV GI.1 strains detected in patient stool and suspect oysters, linking the contaminated oysters to the outbreak. Identification of human NoV GI and GII strains in oysters indicated contamination of human fecal origin, presumably from inappropriate storage in the harbor. Comparative long-fragment analysis of the patient strain revealed 99% similarity (3085 nt) with NoV GI.1 strains detected in previous outbreaks and environmental mussel studies from West Sweden, 87% with M87661 (Norwalk68) and 96% with L23828 (SRSV-KY-89/89/J). These results indicated considerable genomic stability of NoV GI.1 strains over time.

Colonization dynamics of ampicillin-resistant Escherichia coli in the infantile colonic microbiota.

OBJECTIVES: To compare the colonization dynamics of ampicillin-resistant and ampicillin-susceptible Escherichia coli strains in the infantile intestinal microbiota. METHODS: We followed 128 infants over the first year of life with regular quantitative faecal cultures and recordings of antibiotic treatment. E. coli strains were quantified, and their resistance pattern and carriage of beta-lactamase genes (TEM, SHV and OXA), phylogenetic group (A, B1, B2 or D), virulence gene profile (fimA, papC, sfaD/E, kfiC neuB, hlyA and iutA) and time of persistence in the microbiota were determined. RESULTS: Twelve percent (n = 32) of the E. coli strains were resistant to ampicillin, as they carried the bla(TEM) (84%) or bla(SHV) genes. Ampicillin-resistant strains belonged mostly to phylogenetic group D and carried pap genes (P = 0.023) significantly more often than ampicillin-susceptible strains due to a strong association between carriage of pap and bla(SHV). In 31 of 32 cases, colonization by ampicillin-resistant strains occurred in infants not previously treated with beta-lactam antibiotics. Ampicillin-resistant strains were equally capable as susceptible ones of persisting in the intestinal microbiota and did not have lower faecal population counts. Genes encoding beta-lactamases were in most cases retained during the entire colonization period. CONCLUSIONS: The results suggest that ampicillin-resistant E. coli strains are not hampered in their colonizing capacity, and beta-lactamase genes, therefore, may only slowly be eliminated from the commensal E. coli strain pool.

Tracing of norovirus outbreak strains in mussels collected near sewage effluents.

Noroviruses from mussels collected near sewage effluents were compared with local patient outbreak strains. Sequence analyses of RNA polymerase-capsid-poly(A)-3' (3.1-kilobase) regions confirmed the 99.9% similarity between genotype I.1 strains from mussels and patient strains from recreational-bathing outbreaks, indicating the potential usefulness of sentinel norovirus mussel studies in tracing human norovirus contamination of coastal waters.

Mutation analysis of lamivudine resistant hepatitis B virus strains by TaqMan PCR.

Hepatitis B virus infected patients on long-term lamivudine treatment are exposed to a 15-32% risk compounded annually of developing resistance mutations. Such resistance results in a progression of the liver damage caused by chronic hepatitis B, and may also impair the effect of other antivirals through cross-resistance. At present lamivudine is used frequently as monotherapy because of its relatively low price and negligible side effects. Thus, simple methods for identifying resistance mutations are required. A method based on real-time polymerase chain reaction with TaqMan chemistry is described. The method combines both primer specificity, in order to target wild type and mutant viral strains at codon 180, and a mixture of three minor groove binding probes distinguishing the YMDD wild type and the YVDD and YIDD variants at codon 204. The accuracy of the method was verified by concordance with results of direct sequencing and restriction fragment length polymorphism when examining 27 samples from five patients, in whom lamivudine resistance was known to have developed. This method is rapid, cost effective, and should prove useful for monitoring patients treated with lamivudine.

Lamivudine resistance of hepatitis B virus masked by coemergence of mutations in probe region of the COBAS AMPLICOR assay.

The COBAS AMPLICOR hepatitis B virus assay targets a conserved region of the genome and is widely used to monitor treatment of hepatitis B in order to identify emerging resistance. However, the assay failed to recognize increasing viremia levels when YMDD mutations were paralleled by mutations in the segment targeted by the COBAS AMPLICOR probe.

Detection of hepatitis A virus genotype IB variants in clams from Maputo Bay, Mozambique.

Clams provide an important source of food and income for the population of Maputo, Mozambique, where conditions of poor water supply and inadequate sanitation favor endemic infection with hepatitis A virus (HAV). To determine the role of bivalves in an endemic area, clams gathered from Maputo Bay were bought from market and examined for HAV. Four batches, total 150 clams, were sampled over the year. RNA extracted from individual digestive glands was assayed by nested RT-PCR and sequencing of HAV 5' noncoding region (5' NCR). Specific HAV signals were detected in one batch, 23 of 34 clams (67%) testing positive. Phylogenetic analyses of VP3/VP1, VP1/P2A, and 5' NCR determined clustering of clam strains as genotype I, subtype B. In addition to identifying HAV IB strains with predicted conserved amino acid sequence, IB variants exhibiting novel amino acid substitutions at the VP1/P2A junction were detected. HAV strains from clams showed 93%-99% homology with wild-type IB strains from South African outbreaks and from a panel of HAV IgM positive Swedish patients. DNA from enteric human adenovirus 40/41 was found in a limited number of clams from two batches, 6/34 (17%) and 4/35 (11%). Detection of HAV subgenotype IB in bivalves provided indirect evidence of the strains circulating in a densely populated coastal region where HAV is presumed to be hyperendemic. The results suggest that clams may be an important source of HAV in Maputo region, and indicate the need for further molecular study of strains circulating in the indigenous population.

Genotyping of hepatitis C virus by Taqman real-time PCR.

BACKGROUND: Genotype of hepatitis C virus (HCV) is of major importance for the outcome of treatment. The response rate is considerably lower for genotype 1, the predominant genotype in western countries. OBJECTIVES: To develop and evaluate a new, simple method for genotyping of HCV based on real-time polymerase chain reaction (PCR) and Taqman probes targeting the 5' non-coding region. STUDY DESIGN: The method was compared with Innolipa on 220 serum samples representing genotypes 1-4, and was applied on a further 614 clinical samples. RESULTS: Taqman typing of the 220 samples showed genotype 1 in 69, genotype 2 in 58, genotype 3 in 57 and genotype 4 in 19, while 17 were non-reactive. There was a complete concordance with Innolipa with the exception of seven samples, which were of genotype 1 by Taqman, but genotype 4 by Innolipa. Sequencing of these samples showed a subtype 4 variant which differed at two positions compared with subtypes 4b/c/d, which are targeted by the probe. By adding a modified probe, these genotype 4 variants could also be identified. Out of 614 consecutive clinical samples, 524 could be typed by the Taqman assay; 45.2% were genotype 1, 19.3% genotype 2, 33.8% genotype 3 and 1.7%, genotype 4. CONCLUSION: The method was overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced. Inclusion of probes targeting genotypes 5 and 6 is required for the method to be useful in areas where these genotypes are present.