Journal of Clinical Virology - Articles in Press

Identification of G8 rotavirus strains determined as G12 by rotavirus genotyping PCR: Updating the current genotyping methods - Corrected Proof

Farah Aladin, Sameena Nawaz, Miren Iturriza-Gómara, Jim Gray — 2010-02-08

Abstract: Background: Rotaviruses are classified into G- and P-types, which are determined by the reactivity with antibodies to the outer viral proteins, VP7 and VP4, respectively, or sequence variation in the genes encoding these proteins. There are presently a number of different rotavirus strains co-circulating within the UK, with the common human strains G1P[8], G2P[4] and G9P[8] being the most prevalent. As part of strain surveillance for the European Rotavirus Network (EuroRotaNet) a cluster (n=29) of G8 strains was detected in the UK between February and May 2009.Objectives: G8 strains were initially mistyped as G12 through multiplex RT-PCR, therefore further investigation was performed to ascertain the reasons behind this mistyping.Study design: The genes encoding the VP7 of these G8 strains were sequenced and aligned with the existing G8- and G12-specific oligonucleotide primers.Results: Multiple alignment of sequences derived from these strains and the G8- and G12-specific oligonucleotide primers revealed a series of point mutations which resulted in mismatches at the 3′ end of the G8-specific primer binding site that prevented amplification with the G8-specific primer, whilst a close homology with the G12-specific primer allowed mis-priming. Both the G8 and G12 primers were redesigned and their ability to correctly identify G8 and G12 strains was evaluated and confirmed.Conclusion: These findings highlight the importance of monitoring the specificity and sensitivity of the genotyping methods in order to detect changes in the genotype distribution and changes associated with genetic drift of common or uncommon genotypes.

Viral load assay sensitivity and low level viremia in HAART treated HIV patients - Corrected Proof

2010-02-08

Abstract: Background: The recent introduction of highly sensitive viral load assays resulted in a significant increase in number of treated HIV-infected patients with a detectable viral load. The significance of a viral load between 20 and 50copies/mL remains unclear.Objectives: To compare the performance of three viral load assays, with special attention for specificity and sensitivity at the lowest level of quantification.Study design: Samples (n=181) were selected from 62 HIV-positive individuals that experience viral blips or episodes of low but detectable viremia under antiretroviral treatment, and from 216 HIV-negative individuals. Each sample was tested in at least two of three assays: the Cobas Amplicor HIV-1 Monitor (CAP/CA), the Cobas Ampliprep/Cobas TaqMan HIV-1 version 1 (CAP/CTM1) and the Cobas Ampliprep/Cobas TaqMan HIV-1 version 2 (CAP/CTM2).Results: No false positive results were recorded. Kappa statistics revealed fair to moderate agreement between the results of the three assays, but important differences in sensitivity were observed, with the highest sensitivity reported for CAP/CTM2 followed by CAP/CTM1 and CAP/CA. The differences in sensitivity remained after equalization of the detection limit for all assays at 50copies/mL. Analysis of samples collected over time showed that patients with single blips in CAP/CA present with recurrent blips in CAP/CTM1 and persistent detectable viremia in CAP/CTM2.Conclusions: Viral load results between 20 and 50copies/mL in either CAP/CTM1 or CAP/CTM2, indicate true viremia. The availability of highly sensitive assays force reconsideration of the terms ‘undetectable’ viral load and ‘virological success’ of antiretroviral treatment.

Incidence and characterization of cytomegalovirus resistance mutations among pediatric solid organ transplant patients who received valganciclovir prophylaxis - Corrected Proof

Mélanie Martin, Nathalie Goyette, Jane Ives, Guy Boivin — 2010-02-08

Abstract: Background: Drug-resistant cytomegalovirus (CMV) infections can cause significant morbidity among high-risk transplant recipients.Objectives: The aims of this study were to determine the incidence and clinical consequences of CMV mutations conferring ganciclovir resistance in pediatric solid organ transplant (SOT) patients who received valganciclovir oral solution or tablets for prophylaxis of CMV disease. Recombinant CMV mutants were also generated to assess the role of two UL97 mutations of unknown significance.Study design: Genotypic resistance mutations and CMV viral load were sought in blood samples from pediatric SOT recipients who received valganciclovir prophylaxis for 100 days. Recombinant viruses containing novel CMV UL97 mutations were generated using a bacterial artificial chromosome containing the CMV genome to assess ganciclovir susceptibility.Results: Overall, four known resistance UL97 mutations were observed in blood samples from 2 of 46 patients during the study with no development of CMV disease. Two UL97 changes (M615V and V466G) of unknown significance and one UL97 mutation (C603R) associated with ganciclovir resistance, but not yet confirmed by marker transfer, were also detected. Recombinant viruses containing these novel mutations were generated to assess ganciclovir susceptibility. The M615V recombinant virus was susceptible to ganciclovir while the V466G and C603R mutant viruses displayed 3.5-fold and 3.6-fold decreases in susceptibility, respectively.Conclusions: The low incidence of ganciclovir resistance-associated mutations and the absence of clinical consequences associated with drug-resistant viruses observed in this pilot study should encourage the design of larger clinical trials aimed at evaluating the efficacy of valganciclovir prophylaxis and treatment in the pediatric setting.

Phylogenetic analysis of human P[8]G9 rotavirus strains circulating in Brazil reveals the presence of a novel genetic variant - Corrected Proof

2010-02-05

Abstract: Background: Group A rotavirus (RV-A) genotype P[8]G9 has emerged as one of the leading causes of gastroenteritis in children worldwide and currently is recognized as one of the five most common genotypes detected in humans. High intragenotype diversity in G9 RV-A has been observed, and nowadays, based on the genetic variability of the VP7 gene, six different phylogenetic lineages and eleven sublineages were described.Objectives: To study the degree of genetic variation and evolution of Brazilian P[8]G9 RV-A strains.Study design: Phylogenetic analysis of 19 P[8]G9 RV-A strains isolated from 2004 to 2007 in five different Brazilian states was conducted using the NSP1, NSP3, NSP5, VP4 and VP7 genes. For the VP4 and VP7 genes, 3D protein structure predictions were generated to analyze the spatial distribution of amino acid substitutions observed in Brazilian strains.Results: Based on the phylogenetic analyses, all Brazilian strains clustered within lineage G9-III and P[8]-3 for VP7 and VP4, respectively, and were classified as genotype A1, T1 and H1 for the NSP1, NSP3 and NSP5 genes, respectively. Interestingly, all the strains isolated in Acre State (Northern Brazil) formed a closely related cluster clearly separated from the other Brazilian and prototype strains with regard to the five genes studied. Unique amino acid substitutions were observed in Acre strains in comparison with the prototype and Brazilian strains.Conclusion: Inclusion of Acre strains in the phylogenetic analysis revealed the presence of a novel genetic variant and demonstrated a diversification of P[8]G9 rotaviruses in Brazil.

Relative lymphopenia and monocytosis may be considered as a surrogate marker of pandemic influenza a (H1N1) - Corrected Proof

2010-02-04

A novel influenza A (H1N1) [pandemic influenza A (PI)] virus has spread rapidly across the globe. In March and April 2009, an outbreak of respiratory illness was first noted in Mexico, which was eventually identified as being related to PI. The outbreak disseminated rapidly to the other countries. Turkey is among the mostly affected countries.

Antigenic and physicochemical characterization of the 2nd International Standard for hepatitis B virus surface antigen (HBsAg) - Corrected Proof

2010-02-01

Abstract: Background: Standard preparations for HBsAg are required for quality control of test kits and clinical studies on HBsAg quantitation. WHO provides purified heat inactivated HBsAg diluted in negative defribinated plasma as 2nd International Standard (IS) for quality control of tests.Objective: Study of possible alterations of antigenicity, protein composition, size and density of the heat inactivated source material (SM) for the 2nd IS.Study design: Native HBsAg and SM were examined by quantitative immune electrophoresis (QIE), SDS-PAGE, ultracentrifugation and gel chromatography. HBV DNA was sequenced and the HBsAg geno/subtype derived.Results: The SM contained 97,600 International Units HBsAg/ml in QIE which agreed very well with the previous evaluations by WHO using 10 different assays. In SDS-PAGE, SM showed on a strong background the small HBs proteins but no preS proteins. SM had a more heterogeneous density than native HBsAg and contained particle aggregates. The HBsAg geno/subtype of SM was A2/adw2.Conclusions: The IS has very good HBs antigenicity, but it lacks the preS domains, has modified HBs proteins and is partially aggregated. While it has been proven very useful for quality control of tests, certain inconsistencies due to the altered structure of its HBsAg cannot be excluded.

First report of a human autochthonous hepatitis E virus infection in Brazil - Corrected Proof

2010-01-29

Abstract: Background: Sporadic acute hepatitis E cases occurring in non-endemic areas have been associated to genotypes 3 and 4 of hepatitis E virus. Several studies have demonstrated the relationship among human and animals strains, mostly pigs and deers, from respective areas characterizing zoonosis. Circulation of genotype 3 of HEV in Brazilian swine herds have already been demonstrated. Nevertheless, no confirmed human cases have been reported to date in Brazil.Objectives: A study was developed to attempt the identification of hepatitis E acute cases in Brazil.Study design: A retrospective study carried out with 64 serum samples from patients with acute non-A–C hepatitis was performed to identify human cases of acute hepatitis E.Results: We could identify a confirmed case of acute hepatitis E. The patient seroconverted to hepatitis E virus-specific IgM and IgG antibody, HEV-RNA was amplified from serum, and the analysis of the sequence of a 242 nucleotide fragment from the ORF1 genome region classified the strain within genotype 3 and subgenotype 3b. Investigation of risk factors and results from phylogenetic analysis suggested a likely zoonotic origin for the infection.Conclusions: The first report of a human autochthonous in Brazil contributes with new information for hepatitis E epidemiology in Latin America and to considerate further broadly epidemiological studies.

Transient reappearance of serum hepatitis C virus RNA observed by real-time PCR during antiviral therapy with peginterferon and ribavirin in patients with HCV genotype 1b - Corrected Proof

2010-01-29

Abstract: Background: The “response-guided therapy” based on response of hepatitis C virus (HCV) during antiviral combination therapy with peginterferon and ribavirin is important for patients with HCV genotype 1. However, the sensitivity of previous assays for serum HCV RNA is limited.Objectives: We evaluated the changes in serum HCV RNA during the combination therapy using a novel method for measurement based on real-time PCR.Study design: Changes in serum HCV RNA during the combination therapy were reanalyzed using TaqMan PCR assay in 144 patients with chronic HCV genotype 1b infection who underwent the therapy under HCV RNA monitoring with the Amplicor Monitor assay. Treatment duration was elongated from 48 weeks to 72 weeks in 17 patients based on the time when serum HCV RNA became negative.Results: In 9 of 144 (6.3%) patients, serum HCV RNA transiently appeared again on the TaqMan PCR assay after having previously become negative. At the point of reappearance, the Amplicor Monitor assay gave a negative result in all patients, and no flare of alanine aminotransferase activity was observed. Each of the 9 patients achieved an end-of-treatment response but relapsed after the end of treatment, including 3 patients in whom the treatment duration was elongated to 72 weeks.Conclusions: Attention should be paid to this phenomenon in the antiviral treatment for patients with HCV infection. The transient reappearance of HCV RNA in the serum indicates a high likelihood of relapse, and is likely to be missed without frequent measurements by a sensitive detection method.

High prevalence of primary Enfuvirtide (ENF) resistance-associated mutations in HIV-1-infected patients in Hong Kong - Corrected Proof

2010-01-29

Abstract: Background: Enfuvirtide (ENF) is a viral fusion inhibitor used in patients failing highly active antiretroviral therapy (HAART). Mutations associated with ENF resistance have been identified within amino acid positions 36–45 of gp41. As ENF will be introduced to Hong Kong, an understanding of the prevalence of naturally occurred ENF resistance mutations is important before implementation of ENF treatment.Objectives: To investigate the prevalence of ENF resistance-associated mutations in the HR1 and HR2 of HIV-1 strains obtained from ENF-naïve patients.Study design: HIV-1 strains isolated from 185 patients (156 antiretroviral treatment [ART]-naïve and 29 HAART-experienced) were screened for ENF resistance-associated mutations using RT-PCR and DNA sequencing.Results: Primary mutations were detected in 19.4% of HARRT-experienced patients and 20.5% of ART-naïve patients. G36D was encountered most frequently and more prevalent in non-B subtypes. N42S, L54M and V69I were the major polymorphisms detected. N42S and L54M were predominant in CRF01_AE and subtype B, respectively. V69I was found in all samples harboring G36D. In three longitudinal samples from an ENF-treated patient, G36D was detected after ENF treatment for 6 months and the mutation persisted after termination of ENF for 6 months.Conclusions: The high prevalence of ENF resistance-associated mutations in HARRT-experienced and ART-naïve patients identified in this study highlights the importance of mutation screening before ENF therapy in Hong Kong. Our findings from the ENF-treated patient showed that G36D mutation persisted as long as 6 months after ENF withdrawal. Phenotypic assays will be necessary to confirm the influence of this mutation to ENF susceptibility.

Molecular characterization and distinguishing features of a novel human rhinovirus (HRV) C, HRVC-QCE, detected in children with fever, cough and wheeze during 2003 - Corrected Proof

2010-01-27

Abstract: Background: Human rhinoviruses (HRVs) are associated with more acute respiratory tract infections than any other viral group yet we know little about viral diversity, epidemiology or clinical outcome resulting from infection by strains, in particular the recently identified HRVs.Objectives: To determine whether HRVC-QCE was a distinct HRV-C strain, by determining its genome and prevalence, by cataloguing genomic features for strain discrimination and by observing clinical features in positive patients.Study design: Novel real-time RT-PCRs and retrospective chart reviews were used to investigate a well-defined population of 1247 specimen extracts to observe the prevalence and the clinical features of each HRV-QCE positive case from an in- and out-patient pediatric, hospital-based population during 2003. An objective illness severity score was determined for each HRVC-QCE positive patient.Results: Differences in overall polyprotein and VP1 binding pocket residues and the predicted presence of a cis-acting replication element in 1B defined HRVC-QCE as a novel HRV-C strain. Twelve additional HRVC-QCE detections (1.0% prevalence) occurred among infants and toddlers (1–24 months) suffering mild to moderate illness, including fever and cough, who were often hospitalized. HRVC-QCE was frequently detected in the absence of another virus and was the only virus detected in three (23% of HRVC-QCE positives) children with asthma exacerbation and in two (15%) toddlers with febrile convulsion.Conclusions: HRVC-QCE is a newly identified, genetically distinct HRV strain detected in hospitalized children with a range of clinical features. HRV strains should be independently considered to ensure we do not overestimate the HRVs in asymptomatic illness.

Evaluation of immunohistochemistry and in situ hybridization methods for the detection of enteroviruses using infected cell culture samples - Corrected Proof

2010-01-25

Abstract: Background: Enterovirus infections are frequent in all age groups. In addition to acute infections, they have been connected to chronic diseases such as cardiomyopathies and type 1 diabetes. Based on this there is an increasing need for the reliable detection of enteroviruses in different kinds of tissue samples.Objectives: The aim of this study was to set up a test panel which can detect a wide range of different enteroviruses in paraffin-embedded samples and fresh frozen samples using immunohistochemical and in situ hybridization methods.Study design: A panel of nine enterovirus antibodies was optimized for the detection of different enterovirus types in both paraffin-embedded and frozen cell culture samples. In addition, an oligonucleotide probe detecting all human enteroviruses was evaluated for ISH in formalin-fixed paraffin-embedded cell culture samples.Results: Most antibodies worked well in both sample types. Some antibodies detected only one of the tested serotypes, whereas others detected several serotypes. ISH was able to detect all tested enterovirus types.Conclusions: This test panel makes it possible to detect a wide range of different enterovirus types in both formalin-fixed paraffin-embedded and frozen samples. The same methods can also be applied for tissue sections, but may need further optimization for each tissue type.

Factors predictive of successful darunavir/ritonavir-based therapy in highly antiretroviral-experienced HIV-1-infected patients (the DARWEST study) - Corrected Proof

2010-01-25

Abstract: Background: Darunavir (DRV) is the latest protease inhibitor (PI) to be approved for antiretroviral-naive and -experienced HIV-infected patients.Objectives: We examined virologic and immunologic outcomes of highly antiretroviral-experienced patients with triple-class drug resistance receiving DRV/r-based regimens, and attempted to identify factors predictive of virologic success.Study design: We studied patients beginning a ritonavir-boosted DRV (DRV/r 600/100mg twice daily)-containing regimen. Virologic success was defined as plasma viral load (pVL)<50copies/ml at week 36.Results: We studied 62 patients with very severe immunodeficiency (CDC stage C in 69% of cases; median CD4 cell nadir 12/mm3). They had previously received a median of four PI and had extensive PI resistance, with a median of three major PI and two DRV resistance mutations. The baseline median pVL and CD4 cell count values were 4.6log10 and 150/mm3. At week 36, pVL had fallen by 2.6log10 and the CD4 cell count had risen by 123cells/mm3. The virologic success rate was 55% overall, and was improved by concomitant first use of enfuvirtide (67%), raltegravir (69%) or etravirine (75%). Virologic success was independently associated with fewer major PI mutations, previous tipranavir exposure, and concomitant first use of enfuvirtide or raltegravir.Conclusions: In these highly antiretroviral-experienced patients with triple-class drug resistance, virologic success of DRV-containing regimens was mainly associated with the use of new drug classes and/or fully active drugs. Interestingly, previous tipranavir failure did not undermine the efficacy of DRV, confirming the low level of cross-resistance and, probably, distinct resistance profiles between DRV and tipranavir.

Corrigendum to “The clinical performance of Invader® technology and SurePath® when detecting the presence of high-risk HPV cervical infection” [J. Clin. Virol. 45 (Suppl. 1) (2009) S79–S83] - Corrected Proof

2010-01-22

The authors regret that in the above paper, the method for DNA extraction for high risk HPV testing of SurePath collected Pap specimens was incorrectly referenced as being the same method used in a previous study, Johnson et al., Am. J. Clin. Pathol. 130 (3) (2008) 401–408. The protocol described below was used for the most recent study published in J. Clin. Virol.

Corrigendum to “History of the use of HPV testing in cervical screening and in the management of abnormal cervical screening results” [J. Clin. Virol. 45 (1) (2009) S3–S12] - Corrected Proof

J. Thomas Cox — 2010-01-22

In the article above I identified the 1989 article by Tidy et al. as the first study on the utility of HPV testing in a clinical setting. However, a December 1988 letter published in Lancet by Morris and colleagues described the evaluation of the first use of a new polymerase chain reaction (PCR) HPV test in 107 women attending a sexually transmitted disease clinic in Sydney. All women received cytology, colposcopy and cervicography in addition to the collection of exfoliated cells for HPV testing. The study, subsequently published as an original article in early 1989 in the J Clin Virol, compared PCR testing for HPVs 6, 11, 16, 18 and 33 with dot blot hybridization techniques to determine the suitability of the PCR test for implementation in routine clinical settings. The authors reported that, to their knowledge, this was the first report to describe the use of PCR for HPV detection in routine cervical specimens, and predicted that it was possible that such tests might gain widespread use in cervical cancer screening. The authors also pointed out in this 1989 paper that as early as 1984 a number of prior studies had looked at the use of filter hybridization techniques for detection of HPV DNA in cervical scrape specimens collected in parallel with samples for routine cytology. Hence, all of these investigators should receive due credit for pioneering the earliest studies on the clinical utility of HPV testing.

Corrigendum to “Not polymorphism but methylation of class II transactivator gene promoter IV associated with persistent HBV infection” [J. Clin. Virol. 37 (2006) 282–286] - Corrected Proof

2010-01-20

The authors fully accept that they inadvertently reproduced some results that were also published in a previous article at an earlier stage of research and did not cite the work clearly (reference below). They understand that, strictly, this contravenes publishing policy and apologize for any transgression they have committed but hope that their peers will accept this regrettable oversight.

A summary of the 25th International Papillomavirus Conference 2009: Vaccines, screening, epidemiology and therapeutics - Corrected Proof

Alcina F. Nicol, Gerard J. Nuovo, Joakim Dillner — 2010-01-18

Abstract: The 25th International Papillomavirus Conference was held in Malmo, Sweden, on May 8–14, 2009. The conference encompassed all areas of papillomavirus (PV) research, from clinical vaccinology to molecular biology. This review highlights some of the 237 presentations and 887 abstracts which were presented and summarizes sessions on prophylactic vaccines, screening, epidemiology and therapeutics. Important advances included identification of variants in four genes associated with HPV persistence, new HPV detection are likely new infections and not latency reactivation, and development of effective DNA vaccines that targets E6/E7 genes of HPV11. Also, many studies from different countries demonstrated that HPV vaccination provided sustained protection and substantial reduction of disease burden in both women and men, and in HIV-infected neonates. All the references cited are from the abstract book of the IPV Conference. See http://www.hpv2009.org/.

Prospective evaluation of a novel multiplex real-time PCR assay for detection of fifteen respiratory pathogens—Duration of symptoms significantly affects detection rate - Corrected Proof

2010-01-18

Abstract: Background: Nucleic acid amplification techniques have improved the diagnostic possibilities in respiratory tract infections, although their clinical applicability is not yet fully defined. We have evaluated a multiplex real-time PCR method for the detection of 13 respiratory viruses and 2 bacteria (Mycoplasma and Chlamydophila) in a clinical setting.Objectives: The aim of the present study was to evaluate the diagnostic performance and clinical use of a novel multiplex PCR method in adults with community-acquired respiratory viral infection, and the impact of duration of symptoms on detection rates.Study design: Nasopharyngeal swab samples were prospectively collected from 209 adult outpatients with respiratory infections and 100 asymptomatic controls.Results: An infectious agent was identified in 43% of samples from patients and 2% of asymptomatic controls. The detection rate was significantly higher in samples from patients with a duration of symptoms of 6 days or less (51%) than in samples from patients with a duration of symptoms of 7 days or more (30%, p<0.01). For human corona viruses, and influenza virus A and B there was a correlation between the amount of virus in each patient sample as measured Ct values and duration of symptoms.Conclusions: Duration of symptoms significantly affects the detection rate of respiratory pathogens by multiplex real-time PCR in nasopharyngeal swab samples from adult patients with respiratory infections. Our finding should be taken into account when using these tests in clinical practise.

Features and clinical implications of hepatitis B virus genotypes and mutations in basal core promoter/precore region in 507 Chinese patients with acute and chronic hepatitis B - Corrected Proof

2010-01-18

Abstract: Background: The association of hepatitis B virus (HBV) genotypes and basal core promoter (BCP) and precore (PC) mutations with the clinical characteristics is increasingly recognized.Objective: To investigate virologic features and clinical implications of HBV genotypes, BCP and PC mutations between large-size patients with acute hepatitis B (AHB) and chronic hepatitis B (CHB).Study design: One hundred and eighty-two AHB patients and 325 CHB patients were investigated. HBV genotypes and BCP/PC mutations were determined by direct sequencing. Mutations at 10 interested sites of the BCP/PC region were compared between the two groups of patients.Results: AHB patients had a significantly higher ratio of genotype B to C than CHB patients (37.4–62.6% vs. 16.6–83.4%, P<0.001). The prevalence of BCP/PC wild-type virus was 60.4% in AHB patients in contrast to 28.9% in CHB patients. Significantly lower prevalence of A1762T, G1764A, G1896A, and G1899A but higher prevalence of T1758C was found in AHB patients. Interestingly, T1758C and A1762T/G1764A appeared mutual restraint. Genotype B virus had lower BCP mutation frequency and similar PC mutation frequency compared to genotype C virus. AHB patients with BCP/PC mutant virus had higher viral load, whereas CHB patients with BCP/PC mutant virus had lower viral load and elevated alanine aminotransferase, in comparison with those with the wild-type virus.Conclusion: Patients with genotype B virus, BCP/PC wild-type virus or T1758C mutant virus were more susceptible to develop AHB, whereas high prevalence of the BCP/PC mutations was associated with CHB development.

Use of rapid influenza diagnostic tests under field conditions as a screening tool during an outbreak of the 2009 novel influenza virus: Practical considerations - Corrected Proof

2010-01-18

Abstract: Background: Rapid influenza diagnostic tests (RIDTs) are used in various settings as a first-line screen of patient specimens. During the initial outbreak of the 2009 novel influenza A/H1N1 virus, the Nebraska Public Health Laboratory (NPHL) adopted a testing algorithm, attempting to maximize the usefulness of RIDTs. However, it became apparent that a high percentage of the positive specimens received from off-site facilities were negative for influenza viruses by the confirmatory test, the Luminex xTAG Respiratory Viral Panel (RVP) molecular assay.Objectives: To explore the cause of discrepancies between RIDTs results obtained from on-site facility testing versus confirmatory testing performed at NPHL.Study design: Specimens (n=336) tested with RIDTs at off-site facilities and screened for high-probability of containing H1N1 were sent to the NPHL for confirmatory testing by RVP.Results: Of 336 specimens analyzed, 104 were negative for influenza A or B by both RIDT and RVP; 127 were positive by both tests; 102 were positive by RIDT only; and 3 were positive by RVP only. Using the RVP assay as the gold standard, overall RIDT characteristics in this screened population were: sensitivity=97.7% (95%CI: 92.5, 99.3); specificity=48.1% (95%CI: 40.4, 55.8); positive predictive value=54.3% (95%CI: 47.0, 61.4); and negative predicative value=97.1% (95%CI: 90.6, 99.1).Conclusions: The results show that the confirmation of RIDT-positive results varied widely by testing site. Possible explanations for the discrepancies in performance characteristics include testing a narrowly defined sample population, test facility characteristics, facility work load, and seasonal timing.

A staining control for the HCMV pp65 antigen test - Corrected Proof

2010-01-18

The HCMV phosphoprotein 65 (pp65) antigen test represents a fast, cheap and sensitive method for the routine diagnosis of an active HCMV infection. However, the lack of economic and easy accessible (immuno-)staining control slides for the validation of this test has been considered as one problem for its standardization and quality assurance. Approaches to manufacture suitable staining control slides were manifold, including their preparation out of pp65 positive patient peripheral blood leukocyte (PBL) samples or their in vitro-generation by co-cultivation of donor PBLs with HCMV infected endothelial cells. Commercially available HCMV pp65 antigen test providers supply staining control slides that are based on HCMV infected and uninfected fibroblasts (ARGENE, France), or based on donor PBLs preparations mixed with defined ratios of recombinant, pp65 expressing SF9 insect cells (CMV Brite™ Kit, IQ Products, The Netherlands). The disadvantages of these existing rather sophisticated, labor intensive and thus expensive methods are the lack of unlimited amounts of biological material, the dependency on infectious HCMV and differences in staining intensity of recombinant pp65, respectively.

Viral agents responsible for febrile respiratory illnesses among military recruits training in tropical Singapore - Corrected Proof

2010-01-15

Abstract: Background: Military personnel are highly susceptible to febrile respiratory illnesses (FRI), likely due to crowding, stress and other risk factors present in the military environment.Objective: Our objective was to investigate the viral etiological agents responsible for FRI among military recruits training in a tropical climate in Singapore.Study design: From March 2006 through April 2007, a total of 1354 oropharyngeal (throat) swabs were collected from military recruits who reported sick with an oral temperature of ≥38°C and a cough and/or sore throat. Real-time polymerase chain reaction (PCR) was used to assay for the presence of influenza A and B viruses and adenoviruses (H-AdV), and conventional PCR used for the remaining respiratory viruses in all specimens.Results: Influenza A virus was the dominant infection with a laboratory-confirmed incidence of 24% (326/1354) and a predominance of the H3N2 subtype. The temporal pattern for influenza A virus infections coincided with the nation-wide pattern in the civilian community. Detection rates of 12% (159/1354) and 2.7% (5/1354) were obtained for influenza B virus and other respiratory viruses, respectively.Conclusions: The laboratory findings identified influenza A virus as the primary causative viral agent for FRI in the Singapore military, in strong contrast to findings from temperate countries and countries where recruits are often vaccinated for influenza. Our results suggest that influenza vaccination should be considered as a requirement to reduce the incidence of influenza infections. This is the first report describing respiratory infections in a tropical military setting, in a developed country in Asia.

Response to Stegmann S, et al. [J. Clin. Virol. 47 (1) (2010) 79–81] - Corrected Proof

Darrell H.S. Tan, Sharon L. Walmsley — 2010-01-15

Stegmann et al. report an interesting case in whom foscarnet and zidovudine were added to a failing regimen as salvage therapy for a patient with highly drug-resistant HIV-2 infection, and were associated with a 1.48log10copies/mL decrease in viral load to undetectable levels (<100copies/mL). The authors primarily attribute this improvement to foscarnet since the patient had previously failed zidovudine-containing regimens and because zidovudine alone is unlikely to decrease plasma viral load to this degree even in the absence of thymidine-associated mutations (TAMs). The paper builds on previous reports from their group and others in which foscarnet was successfully used as salvage therapy for HIV-1-infected patients, particularly in the presence of TAMs.

Resequencing microarray for detection of human adenoviruses in patients with conjunctivitis - Corrected Proof

2010-01-13

Abstract: Background: Although high-density resequencing microarray is useful for detection and tracking the evolution of viruses associated with respiratory tract infections, no report on using this technology for the detection of viruses in patients with conjunctivitis is available.Objectives: To test if high-density resequencing microarray can be applied to detection of viruses in conjunctival swabs for patients with conjunctivitis.Study design: In this prospective proof-of-concept study, every 4 or 5 bacterial culture-negative conjunctival swab samples were pooled and subject to viral detection using TessArray™ Resequencing Pathogen Microarrays-Flu 3.1 (RPM-Flu-3.1). Results were compared with human adenovirus (HAdV) hexon gene PCR sequencing and viral culture.Results: Thirty-two of the 38 conjunctival swab samples were bacterial culture-negative. Four of the 7 pooled samples were positive for HAdV using RPM-Flu-3.1. Hexon gene PCR sequencing on the 38 original individual samples showed that 3 and 4 samples contained HAdVs species D and B respectively. All the 6 samples that were positive for hexon gene PCR but negative for bacterial culture were also positive by the resequencing microarray. Viral culture was positive for HAdV type 3 in 1 sample, which was also positive by PCR and resequencing microarray.Conclusions: Resequencing microarray is as sensitive as PCR for detection of HAdV in conjunctival swabs. Unlike viral culture and hexon gene PCR sequencing, resequencing microarray was not able to differentiate the type and species of HAdV. Development of microarrays for conjunctivitis can be performed for rapid diagnosis of the viral cause of conjunctivitis.

Acute hepatitis B virus infection with simultaneous high HBsAg and high anti-HBs signals in a previously HBV vaccinated HIV-1 positive patient - Corrected Proof

Laura van Dommelen, Annelies Verbon, H. Rogier van Doorn, Valère J. Goossens — 2010-01-11

Abstract: We present a case of a clinical manifest hepatitis B virus infection and a potentially misleading HBV serological profile in an HIV-1 positive patient despite previous HBV vaccination. The patient presented with an acute hepatitis B and there was no indication of chronic HBV infection or the presence of a mutation in the ‘a’ determinant. Remarkably, simultaneously with high HBV surface antigen and HBV viral load, high anti-HBs antibodies were present. If, due to previous HBV vaccination only anti-HBs was tested in this patient, the result of the high anti-HBs antibodies could be very misleading and offering a false sense of security. Our findings contribute to the ongoing discussion on how to assess HBV specific immunological memory and determining the role of HBV booster vaccinations in immunocompromised individuals.

Clinical presentation and response to treatment of novel influenza A H1N1 in a university-based summer camp population - Corrected Proof

2010-01-11

Abstract: Background: Little is known about the clinical presentation and course of novel H1N1 influenza in summer camps.Objectives: To describe the clinical course and evaluate the effect of influenza treatment in a summer camp population.Study design: Two large influenza outbreaks occurred in university-based residential camps between May 21 and August 2, 2009. Through active daily surveillance, medical evaluation at symptom onset, and data collection during isolation, we describe the clinical course of a large outbreak of novel H1N1 influenza.Results: Influenza-like illness (ILI) was documented in 119 individuals. Influenza A was confirmed in 66 (79%) of 84 samples tested. Three early samples were identified as novel H1N1. ILI cases had an average age of 15.7 years and 52% were male. Sixty-three were treated with oseltamivir or zanamivir, which was initiated within 24h of diagnosis. Cough, myalgia and sore throat occurred in 69, 64 and 63% of cases, respectively. The highest temperature over the course of illness (Tmax) occurred within 48h after symptom onset in 87.5% of individuals. Average Tmax was 38.4°C (range 36.1–40.2°C). Among confirmed influenza cases, 69% defervesced by 72h and 95% defervesced by 96h. Defervescence at 72h was not different in the treated and untreated groups (p=0.12).Conclusions: Novel H1N1 generally has a mild, self-limited course in healthy adolescent campers. Defervescence occurred within 72h and was unaffected by treatment.

Human polyoma viruses and disease with emphasis on clinical BK and JC - Corrected Proof

Raghavender Boothpur, Daniel C. Brennan — 2010-01-08

Abstract: Polyoma viruses are ubiquitous infecting many different mammalian species including humans. There are five known human polyoma viruses. JC virus and BK virus are two polyoma viruses identified nearly three decades ago. Recently WU, KI and Merkel cell polyoma viruses have been isolated from humans. The exact role of these three newly discovered viruses in human disease is not known. Most human polyoma disease is caused by BK and JC viruses which are usually acquired in childhood. Approximately 50–80% of humans have seropositivity to these viruses. Clinically apparent diseases in immunocompetent hosts are extremely rare. These viruses remain latent possibly in the lymphoid organs, neuronal tissue, and kidney and under the circumstances of severe immunosuppression both these viruses reactivate. Neurotropic JC virus reaches the brain and causes progressive multifocal leukoencephalopathy, a demyelinating disease of the central nervous system with a high mortality rate. BK virus is urotheliotropic and its reactivation causes a form of interstitial nephritis, known as BK or polyoma virus associated nephropathy which is associated with high graft loss if not recognized early. There are no known effective antiviral agents for any of the polyoma viruses.

Respiratory viruses, a common microbiological finding in neutropenic children with fever - Corrected Proof

2010-01-07

Abstract: Background: Febrile neutropenia is a common complication in children undergoing chemotherapy for malignancies. A microbial agent is only identified in 15–30% of the fever episodes and corresponds mostly to bacterial findings.Objective: To investigate viral infections as possible etiologic agents in episodes of febrile neutropenia.Study design: Nasopharyngeal aspirates (NPAs) from patients presenting with neutropenic fever at two pediatric oncology wards in Sweden and Australia were analyzed with a conventional virus-diagnostic approach and RT-PCR. Coupled blood samples were analyzed for the detection of CMV, EBV, adenovirus and erythrovirus. Bacterial blood culture was performed routinely.Results: Conventional virus-diagnostic approach coupled to routinely performed bacterial analyzes revealed an infectious agent in 29% compared to 60% when using PCR. By adding PCR, a viral pathogen was detected in 46% of the NPAs and in 4% of the blood samples collected. In half of the patients with bacteremia, respiratory tract viruses were co-detected.Conclusion: Respiratory viruses were frequently detected in NPAs suggesting a significant role of viral infections in children presenting with neutropenic fever. The meaning of these findings needs to be further evaluated but has the potential to individualize infection treatment and to reduce the extensive use of antibiotics in immunocompromised children with neutropenia.

Waterborne gastroenteritis outbreak at a scouting camp caused by two norovirus genogroups: GI and GII - Corrected Proof

2010-01-07

Abstract: Background: A cross-border gastroenteritis outbreak at a scouting camp was associated with drinking water from a farmer's well.Objectives: A retrospective cohort study was performed to identify size and source of the outbreak, as well as other characteristics.Study design: Epidemiological investigation included standardized questionnaires about sex, age, risk exposures, illness and family members. Stool and water (100mL) samples were analyzed for bacteria, viruses and parasites.Results: Questionnaires were returned by 84 scouts (response rate 82%), mean age of 13 years. The primary attack rate was 85% (diarrhoea and/or vomiting). Drinking water was the strongest independent risk factor showing a dose–response effect with 50%, 75%, 75%, 93% and 96% case prevalence for 0, 1, 2–3, 4–5 and >5 glasses consumed, respectively. Norovirus (GI.2 Southampton and GII.7 Leeds) was detected in 51 stool specimens (75%) from ill scouts. Water analysis showed fecal contamination, but no norovirus. The secondary attack rate was 20%.Conclusions: This remarkable outbreak was caused by a point-source infection with two genogroups of noroviruses most likely transmitted by drinking water from a well. Finding a dose–response relationship was striking. Specific measures to reduce the risk of waterborne diseases, outbreak investigation and a good international public health network are important.

Viro-immunological dynamics in HIV-1-infected subjects receiving once-a-week emtricitabine to delay treatment change after failure: A pilot randomised trial - Corrected Proof

2010-01-07

Abstract: Background: In HIV-1-infected patients harbouring the M184V mutation (M184V), lamivudine monotherapy leads to a smaller decrease in CD4 percentages (CD4%) than treatment interruption, possibly due to the reduced fitness of the mutated virus.Objective: We assessed whether a minimal dose of a cytidine analogue that is theoretically sufficient to maintain M184V (one emtricitabine tablet once-weekly) may be as effective.Study design: In a proof-of-concept, randomised clinical trial, HIV-1-infected patients with CD4 cells >400/mm3, failing on lamivudine- or emtricitabine-containing combination antiretroviral therapy (cART), received emtricitabine once-a-week (A), or emtricitabine once-a-day (B), or lamivudine once-a-day (C). The primary endpoint was the proportion of subjects without a 12-week loss in CD4%. The patients resumed cART after 24 weeks or in the case of CD4 cells <350/mm3.Results: The 38 enrolled patients had similar baseline characteristics across groups. The primary endpoint was reached by 5/13 patients (38.5%) in arm A, 3/13 (23.1%) in arm B, and 3/12 (25%) in arm C (P=0.644), and respectively 4/13 (30.8%), 4/13 (30.8%) and 5/12 (41.7%) had to resume cART within 24 weeks (P=0.805). The immunological changes over 24 weeks were similar in the three groups, but there was a higher median viral rebound in once-weekly treatment recipients (A) than in once-daily (B+C): 0.97 versus 0.52log10copies/ml (P=0.033). M184V was maintained in all the participants.Conclusions: Once-weekly emtricitabine led to a higher viral rebound than once-daily monotherapy, but similar immunological changes, thus suggesting a role of M184V in slowing the decrease in CD4% in treatment failing subjects.

Transmission of betapapillomaviruses between domestic partners in an Australian community - Corrected Proof

2010-01-06

Abstract: Background: Betapapillomaviruses may be associated with the development of cutaneous squamous cell carcinoma but little is known about their transmission. One suggestion is that they are transmitted through close skin contact.Objectives: To test this hypothesis we assessed whether co-habiting opposite-sex couples were more or less likely to share betaPV types than each member of the couple and an age-matched, opposite-sex control.Study design: Betapapillomavirus was measured in eyebrow hairs of 57 couples and 114 age- and sex-matched controls. We compared the proportion of partners who shared at least one betaPV type with the proportion of control partnerships sharing a betaPV type. We further subdivided those who shared at least one type into those who shared only one and those who shared more than one. We tested the significance of differences in these proportions using Chi-squared tests. A case-wise concordance index was used to calculate the overall concordance of the partners and the control pairings.Results: At least one betaPV type was shared by 39% of the co-habiting couples and 26% of the control pairs (p=0.10). When restricted to all people with at least one virus infection (26 couples) 74% of the partners and 46% of the control pairs shared at least one type (p=0.02). The case-wise concordance index for partners was 0.28 (95% CI 0.21–0.35) and for the matched control pairs 0.16 (95% CI 0.12–0.20) (p<0.001).Conclusions: Our results support the hypothesis that skin-to-skin contact is the primary means of betapapillomavirus transmission.

Unsupervised self-initiation of antiretroviral drugs in a newly diagnosed HIV-1 infected haemodialysis patient - Corrected Proof

S.E. Moses, M. Zuckerman, P. Donohoe, M. Sudhanva, M. Poulton — 2009-12-09

A 50-year-old Afro-Caribbean male with underlying hypertension and chronic kidney disease stage 5 was found to be HIV-1 antibody positive after annual screening for HIV in the renal unit. He had been expecting this result as his new female partner had been found to be HIV-1 infected having been admitted to hospital recently from the accident and emergency department with tuberculosis. He had been on haemodialysis for the preceding 4 years and was active on the national cadaveric kidney transplant list, from which he was suspended pending control of his HIV infection. His baseline HIV-1 RNA load was 92,400copies/ml (5.0log10) with a CD4 count of 251cells/mm3 (19%). No antiretroviral resistance mutations were found on baseline HIV-1 drug resistance testing. The partner also had wild-type virus on baseline testing. He was booked in to the HIV clinic where he was to be started on combination antiretroviral therapy (cART) but defaulted his first clinic appointment and was seen 3 months after diagnosis. It was then discovered that he had purchased Truvada (combination of tenofovir 300mg and emtricitabine 200mg) and Viramune (nevirapine 200mg) over the Internet and had been self-medicating for about 10 weeks. Laboratory investigations at that point showed an HIV-1 RNA level of 57,100copies/ml (4.8log10), CD4 count of 329cells/mm3 (21%) with normal liver enzyme levels. He reported that he had not been taking the antiretroviral drugs (ARVs) regularly and had not always been taking them in combination. He had omitted the ARVs for the preceding few days when he was seen in the clinic. This clinic scenario posed the following questions.

Febrile illness in a returned traveller from Thailand - Corrected Proof

Bjørn Waagsbø, Anders Sundøy, Liv Ragnhild Høyvoll — 2009-12-09

A previously healthy female in her fifties, returned from Phuket, Thailand after a 3-week holiday stay. She was an experienced traveller with multiple earlier journeys to the south-east Asia. On day 3 after returning home to Norway, she fell ill and contacted her primary health care physician by telephone complaining over headache, myalgia, joint pain, vomiting and fever of 38.7°C. Over the next days she experienced transient improvement, although high-peeking fever reaching 39°C persisted. On day 7 she once more phoned her physician and was advised to stay home in town, instead of spending the weekend at her cabin as planned. She went anyway.

WITHDRAWN: Viral Hepatitis: global goals for vaccination - Corrected Proof

Daniel Lavanchy — 2009-07-06

This article has been withdrawn at the request of the author. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.