Journal of Clinical Virology - Articles in Press

Detection of ganciclovir resistance mutations by pyrosequencing in HCMV-infected pediatric patients - Corrected Proof

2012-02-03

Abstract: Background: Human cytomegalovirus (HCMV) is an opportunistic pathogen especially for immuno-suppressed subjects that might develop pharmacological resistance in patients undergoing prolonged antiviral treatment. Ganciclovir (GCV) is the drug used as first choice therapy in affected children and a GCV-resistant phenotype is mainly linked to mutations of the viral protein kinase UL97.Objectives: Here a new quantitative pyrosequence (PSQ) method is presented that allows detection and quantification of the viral species carrying the more frequent UL97 mutations responsible for GCV resistance in clinical samples (>80% of known cases).Study design: The system has been validated using two independent approaches (cloning and sequencing of UL-97 gene fragments and real-time PCR) and clinical samples derived from 3 pediatric patients.Results: The UL97 pyrosequencing analysis has indicated a significant increase of mutant viruses carrying the H520Q and C592G mutations.In particular, the H520Q viral mutation, known to increase GCV resistance (IC50=10) increased around 5 times during hospitalization. In addition, C592G (known to have IC50=2.9) also increased 3 times.Conclusions: PSQ is a quick, cheap, high throughput and sensitive analysis method to detect GCV-associated resistance mutation useful to follow antiviral therapy in perinatal CMV-infection as well as in immune-suppressed patients.

Comparison of two broadly multiplexed PCR systems for viral detection in clinical respiratory tract specimens from immunocompromised children - Corrected Proof

2012-02-01

Abstract: Background: The detection of viral respiratory tract infections has evolved greatly with the development of PCR based commercial systems capable of simultaneously detecting a wide variety of pathogens.Objectives: Evaluate the relative performance of two commercial broad range systems for the detection of viral agents in clinical respiratory tract specimens from immunocompromised children.Study design: A total of 176 patient samples were included in the analysis, representing only the first sample collected for each patient, and excluding failed reactions. Samples were de-identified and assayed in parallel using two different, broadly multiplexed PCR systems: ResPlex™ II Panel v2.0 (ResPlex), Qiagen, Hilden, Germany and FilmArray® Respiratory Panel (FilmArray), Idaho Technology Inc., Salt Lake City, UT. Method comparison was based upon pair-wise concordance of results according to patient age, viral target and number of targets detected.Results: The two systems showed an overall concordance, by patient, of 83.8% (p=0.0001). FilmArray detected at least one target in 68.8% of samples, while ResPlex detected at least one target in 56.8%. ResPlex failed to detect 20.7% of FilmArray positives, and FilmArray failed to detect 4% of ResPlex positives. The relative performance of each system (including which system detected a higher number of positive samples) varied when stratified by target viral pathogen.Conclusions: Broadly multiplexed PCR is an effective means of detecting large numbers of clinically relevant respiratory viral pathogens.

A new highly automated extraction system for quantitative real-time PCRs from whole blood samples: Routine monitoring of opportunistic infections in immunosuppressed patients - Corrected Proof

C. Mengelle, J.-M. Mansuy, K. Sauné, C. Barthe, J. Boineau, J. Izopet — 2012-02-01

Abstract: Background: Rapid, high throughput extraction systems are needed to monitor viral infections in immunosuppressed patients.Objectives: Evaluate the performance of the MagNA Pure 96™ extraction system, and compare it to the COBAS Ampliprep™ for quantitative real-time PCR from whole blood samples.Study design: Compare the MagNA Pure LC™, COBAS Ampliprep™ and MagNA Pure 96™ using ten-fold dilutions of blood samples containing cytomegalovirus. Evaluate analytical performances of the MagNA Pure 96™ from test samples containing cytomegalovirus. Evaluate clinical performances from 209 blood samples collected prospectively, extracted with the COBAS Ampliprep™ and the MagNA Pure 96™ systems and tested for cytomegalovirus, Epstein–Barr, BK and JC viruses.Results: All three extraction systems gave similar results with dilutions of a cytomegalovirus-positive sample. Analytical tests showed that the limit of detection was 500copies/ml, specificity was 100%, with no cross-contamination. Quantification was linear from 3.0 to 6.0log10copies/ml. Intra-assay variation was 8.3–0.9% and inter-assay variation 8.8–5.2%. Clinical specimens extracted with the MagNA Pure 96™ and COBAS Ampliprep™ instruments agreed well for cytomegalovirus (r=0.54; p=0.07), Epstein–Barr virus (0.69; p=0.0005) and BK virus (0.85; p=0.01). All 55 samples were negative for JC virus. Mean loads were similar for cytomegalovirus (0.17log10copies/ml) and BK virus (−0.24log10copies/ml) while that of Epstein–Barr virus was slightly lower (1.02log10copies/ml).Conclusions: The MagNA Pure 96™ instrument is an easy-to-use, reliable high throughput platform for extracting nucleic acid from clinical whole blood specimens.

Variable capacity of 13 hepatitis B virus surface antigen assays for the detection of HBsAg mutants in blood samples - Corrected Proof

2012-02-01

Abstract: Background: Natural variation and mutations in the envelope protein (S) of hepatitis B virus can translate into HBsAg variants no longer detectable by conventional HBsAg assays.Objectives: The aim of the study was to assess the performance of 13 commercial assays currently used for screening and clinical analysis of HBsAg variants.Study design: The limit of detection (LOD) for each assay was established using two reference standards (WHO HBsAg 00/588 and the SFTS French reference). Sensitivity was evaluated using different panels. Panel 1 included 25 recombinant HBs variants at three concentrations, panels 2 and 4 included 8 recombinant HBsAg variants and 9 wild-type proteins (genotypes A–F), respectively, panel 3 included 16 natural HBsAg variants.Results: LODs ranged from 0.011 to 0.095IU/ml with the WHO standard, and from 0.021 to 0.326ng/ml with the French reference. The overall percentage of positive signals using HBsAg variants ranged from 62.9% to 97.9%. Three substitutions: T123, D144A and G145, were negative at all concentrations with at least one assay.Discussion: Our findings show that, although they fulfil CE requirements for analytical sensitivity (LODs below 0.13IU/ml), HBsAg assays may vary in their capacity to detect HBsAg variants. This limit in diagnosis performance should encourage the health regulatory agencies to include HBsAg variant panels in the evaluation process.

Pacific region influenza surveillance for oseltamivir resistance - Corrected Proof

2012-02-01

Abstract: Background: Hawaii and the United States-affiliated Pacific islands (USAPI) host over 8 million travelers annually, most of whom originate in Asia, Australia, and the Americas where prevalence of oseltamivir resistance in 2009 pandemic influenza A (H1N1) has been reported to be 2.5–3.5%.Objective: To survey a collection of samples from Hawaii and the USAPI that had tested positive for the 2009 pandemic influenza A (H1N1) virus by RTI-PCR to assess whether antiviral resistance emerged in these island communities during the 2009 H1N1 pandemic.Study design: We examined RNA extracted from Hawaiian and USAPI cases for the neuraminidase H275Y mutation associated with oseltamivir resistance by pyrosequencing.Results: Two hundred and sixty-three (263) 2009 pandemic influenza A (H1N1) positive specimens were tested and 263/263 (100%) were shown to lack the mutation most commonly associated with oseltamivir resistance.Conclusions: There was no evidence of oseltamivir resistant A(H1N1)pdm09 virus during the 2009 pandemic in the Pacific islands despite considerable travel exposure. Geographic isolation, the lack of a “second wave” of pandemic influenza, judicious antiviral use, aggressive vaccination, and below average tourism due to the global economic crisis may have been contributing factors. Continued surveillance and vigilance is necessary to monitor unpredictable influenza activity.

Prevalence of antibodies and RNA genome of hepatitis E virus in a cohort of French immunocompromised - Corrected Proof

2012-01-31

Abstract: Background: Recently, cases of chronic hepatitis E have been identified in immunocompromised patients.Objectives: To evaluate the prevalence of anti-HEV IgG antibodies and the persistence of HEV-RNA in sera of immunocompromised patients with regular follow-up at Saint-Louis Hospital in Paris, France.Study design: 307 samples collected from 261 HIV-infected patients and 46 kidney transplant (KT)-patients were retrospectively tested for the presence of the following hepatitis E virus (HEV) infection markers: anti-HEV IgM antibodies, anti-HEV IgG antibodies, anti-HEV IgG avidity index, and HEV-RNA.Results: Anti-HEV IgG positive serology was found in 4 HIV-infected patients (1.5%) and 3 KT-patients (6.5%), leading to an overall seroprevalence of 2.3%. HEV-RNA detection was not observed among 55 HIV-patients at higher risk of chronic HEV (<200CD4 cells/mm3, elevated alanine aminotransferase (ALT) levels, and/or positive anti-HEV antibodies) and among 44 KT-patients. None of the seven patients had anti-HEV IgM antibodies, thereby excluding any acute infection. The IgG avidity index confirmed past HEV infection among tested patients.Conclusions: The low seroprevalence observed in the Paris region does not warrant a systematic evaluation of HEV infection in immunocompromised patients. However, HEV infection must be examined as a possibility if unexplained increases in ALT should occur and after more common viral hepatitis infections are excluded.

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings - Corrected Proof

2012-01-31

Abstract: Background: In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner.Objectives: Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS.Study design: 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells.Results: The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS.Conclusions: Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay.

Lack of seasonality of primary human cytomegalovirus infection in pregnancy - Corrected Proof

2012-01-25

Human cytomegalovirus (HCMV) is an opportunistic pathogen belonging to the herpesviridae family and, like all herpesviruses, undergoes latency and reactivation in its human host. HCMV has developed very efficient ways for infecting human beings and horizontal transmission occurs very efficiently through contacts with infected bodily fluids, such as saliva, blood, urine and genital secretions. In immunocompetent individuals, primary infection is generally asymptomatic, or, when symptoms are present, they are nonspecific, like asthenia, low grade fever, headache or upper respiratory symptoms.

Prospective genotyping of human rhinoviruses in children and adults during the winter of 2009–2010 - Corrected Proof

2012-01-24

Abstract: Background: About 100 serotypes of human rhinovirus (HRV), classified into two species, have been identified by 1990. Uncultivable HRV variants have recently been identified and designated a new species. Recent improved diagnosis has led to a re-appraisal of the clinical impact of HRV infections in lower respiratory diseases.Objectives: To characterise clinical features in hospitalised patients with positive HRV RNA detection and to determine the distribution of HRV species in respiratory infections diagnosed during the winter of 2009–2010.Study design: Prospective virus typing was conducted by sequencing the VP4/VP2 genomic regions, and clinical data were collected.Results: Fifty-eight patients (for 63 respiratory specimens) were included. Phylogenetic analysis identified 52% of HRV species A, 6% of species B and 40% of species C, and revealed the co-circulation of 34 different HRV types during the study period. Three infants had successive infections with two or three different types. Five patients were admitted to an intensive care unit, four of them on arrival. Bronchiolitis, pneumonia and exacerbation of asthma were observed in 34/45 children. Pneumonia and severe exacerbation of chronic lung disease were observed in 8/13 adults, of whom 1, with immunocompromised status, died of multivisceral failure.Conclusions: This study underlines the diversity of co-circulating strains and the potential severity of clinical presentations associated with HRV infections.

Association of age and gender with alphaherpesvirus infections of the central nervous system in the immunocompetent host - Corrected Proof

Elisabeth Puchhammer-Stöckl, Stephan W. Aberle, Harald Heinzl — 2012-01-24

Abstract: Background: The alphaherpesviruses Varicella-zoster virus (VZV) and human herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) can cause severe infections of the central nervous system (CNS).Objectives: To analyze whether age and gender of immunocompetent individuals are associated with the incidence of herpesvirus CNS diseases.Study design: A total of 241 patients with virologically confirmed HSV-1, HSV-2 or VZV-infection of the CNS (excluding neonatal infection and varicella), diagnosed at the Department of Virology, Medical University Vienna, from 2001 to 2009 were analyzed retrospectively. The relative incidence of disease was evaluated statistically with respect to gender and age.Results: The relative incidence of VZV CNS disease increased with age (p<0.0001), and nonlinear age dependences were observed for HSV-1 (p=0.005) and HSV-2 disease (p=0.002). These effects were influenced significantly by the patient's gender in VZV (p=0.0003) and HSV-1 disease (p=0.008). Overall, 50.7% of VZV infections in males, but only 23.5% of those in females, occurred before age 45, and 28.9% of HSV-1 infections in males and 8.8% of those in females occurred before age 30. Women represented 71.9% of HSV-2 CNS infections (p=0.02).Conclusions: The patient's gender is clearly associated with the incidence of CNS disease caused by VZV, HSV-1 and HSV-2, and its influence varies over one's lifetime.

Seroepidemiology of the recent mumps virus outbreaks in Ireland - Corrected Proof

2012-01-24

Abstract: Background: Two recent mumps outbreaks have occurred in Ireland in 2004/2005 and 2008/2009.Objectives: To retrospectively investigate any potential shifts in the gender bias and age profile and to identify cohorts who are maintaining mumps virus in circulation.Study design: 2600 cases of acute mumps infection, as determined by the presence of mumps-specific IgM in sera and oral fluids, were confirmed at the National Virus Reference Laboratory.Results: Acute mumps infection occurred more frequently in males with a ratio of approximately 2:1 in the 1–9 and 10–19 years old age groups. A 3:2 ratio was observed in the 20–29 years old cohort and the 30+ age group did not show a gender bias. Serological evidence of prior immunological exposure to mumps virus, as determined by the presence of mumps-specific IgG, was high and similar in males and females of all age cohorts (93.1–100%). A significant increase in the number of acute mumps cases in the ≥30 years old age group was observed. This increase was most striking in the periods between the outbreaks (71.1% in 2007 and 56.2% in 2010).Conclusions: Acute mumps infection showed a male gender bias. The consistent and significant increase of mumps infection in the ≥30 years old age group which is also evident in the periods between outbreaks suggests that this may be the cohort maintaining the mumps virus in circulation.

Seroepidemiology of Enterovirus 71 infection prior to the 2011 season in children in Shanghai - Corrected Proof

2012-01-24

Abstract: Background: In 2010, China experienced the largest outbreak on record of Enterovirus 71 (EV71)-associated Hand Foot and Mouth Disease (HFMD) with more than 1.7 million cases, 27,000 patients with severe neurological complications and 905 deaths. Understanding of the seroprevalence of neutralizing antibodies (NAb) against EV71 and their protective role against HFMD in children is crucial for the implementation of future therapeutic and prophylactic intervention.Objectives: To correlate the prevalence of NAb against EV71 genotype C4a in children prior to the 2011 epidemic season with severe EV71-associated HFMD disease during the subsequent 2011 epidemic season.Study design: 614 sera samples were collected from children without HFMD. EV71 NAb were tested by a quantitative PCR assay. Samples with NAb ≥1:8 were scored as positive.Results: 122 (19.9%) of 614 sera were EV71-seropositive. The NAb seroprevalence was highest in infants 0–5 months of age (28.6%) and lowest in children 1–1.9 years of age (13.4%). 64.1% of severe EV71-associated HFMD occurred in children 1–2.9 years.Conclusions: Despite the large 2010 outbreak, the overall seroprevalence of EV71 in children is relatively low. The seropositive rate of EV71 NAb prior to the 2011 season was inversely correlated with the number of EV71-infected severe cases in 2011. Loss of maternal antibodies in infants and lack of acquired anti-EV71 immunity are responsible for increased proportion of severe HFMD in the 1–2 years age group. Our data suggest that future vaccination campaigns should be initiated as early as 6 months.

HIV-1 subtypes D and F are prevalent in Guinea Conakry - Corrected Proof

G.L. Freimanis, A. Loua, J.P. Allain — 2012-01-24

Abstract: Background: Limited data is available upon the distribution of different HIV-1/2 genotypes in the blood donor population from Guinea Conakry.Objectives: To investigate the prevalence of HIV-1/2 subtypes in asymptomatic blood donors in Guinea Conakry, in order to update knowledge of HIV-1/2 epidemiology within this country.Study design: Samples from 104 blood donors seropositive for HIV-1/2 were tested for HIV-1 by real-time RT-PCR. Those negative for HIV-1 were tested with HIV-2 nested RT-PCR. Positive samples were further amplified in the HIV-1 gag and pol regions and sequenced. Subtypes were determined by phylogenetic analysis on amplicon sequences.Results: 61 samples were positive by HIV-1 real-time RT-PCR. Of the 43 negative, 2 (4.6%) were positive for HIV-2. 52/61 (85.3%) samples were positive by nested RT-PCR. Of the 52, 43 (70.5%) and 31(59.6%) sequences were obtained in the gag and pol regions, respectively; 23 for both regions. HIV-1 subtype distribution was 1 B (2.1%), 8 F (17%), 8 D (17%) and 28 CRF02_AG (59.6%) with 2 unclassified recombinants (4.3%). Unique clusters for subtype D and F distinguished Guinea from HIV-1 subtype distribution in neighboring countries.Conclusions: Subtype F and subtype D strains, uncommon in West Africa, are a substantial part of HIV-1 epidemiology in Guinea.

Multiple cranial neuropathies after a rash in two patients - Corrected Proof

Peng-Soon Ng, Cheng-Chuan Lee, Kevin Tan — 2012-01-24

A previously healthy 31-year-old male presented with acute left facial weakness and horizontal binocular diplopia on looking to the left. Three weeks prior to that, he had fever, myalgia and a generalized papulovesicular rash 10 days following exposure to his 7-month-old son who had similar skin lesions. His constitutional symptoms and rash resolved completely over 2 weeks. Neurological examination revealed complete left facial paralysis with incomplete eye closure and abduction failure of the left eye with consequent horizontal diplopia. The reminder of the cranial nerve examination was normal. There were no skin rashes, meningism or long tract signs. These findings were consistent with acute left lower motor neuron facial and left abducens neuropathies. Cranial magnetic resonance imaging (MRI) did not detect abnormal parenchymal, leptomeningeal or ependymal lesions or enhancement. Cerebrospinal fluid (CSF) examination revealed a white cell count of 8/mm3 (99% lymphocytes, 1% monocytes) and protein of 0.56g/L.

Genetic diversity of human adenovirus in hospitalized children with severe acute lower respiratory infections in Paraguay - Corrected Proof

2012-01-24

Adenoviruses (AdVs), members of the family Adenoviridae, belong to a complex group of viral pathogens that infect many species of vertebrates and cause a broad range of clinical manifestations in humans, compromising the respiratory, gastrointestinal, and neurological systems, with billions of people infected worldwide. They are non-enveloped viruses, with an icosahedral capsid that surrounds the genome, which is composed of a linear, double-stranded DNA of ∼35,000 base pairs.

Awareness of cytomegalovirus infection among pregnant women in France - Corrected Proof

2012-01-24

Abstract: Background: Cytomegalovirus (CMV) is the most frequent cause of congenital virus infection. Approximately 1% of newborns are infected by CMV at birth with severe consequences among 10% of them. Efficacy of hygienic counselling is nowadays established and should be spread.Objective: To evaluate pregnant women's awareness of cytomegalovirus infection in France.Study design: Pregnant women receiving prenatal care, at any moment of their pregnancy, in two different obstetrics clinics with different information policies, were asked to complete a written questionnaire about CMV infection.Results: More than half (217/362, 60%) of the pregnant women had heard of congenital CMV infection, and most of them (72%) knew the hygiene measures to use to prevent infection. Nevertheless, most could not correctly identify the symptoms associated with congenital CMV disease. Awareness was associated with hospital's policy concerning CMV infection information, the mother's educational level, parity, and employment in health care. Indeed, when information is supposed to be given (hospital A), 74% (vs 34%) know congenital CMV infection and among them the knowledge is more precise.Conclusions: This study tends to confirm that there is a large gap between knowledge of CMV and the burden of this disease. To bridge this gap, women should receive education about congenital CMV. Hospital-based prenatal education increases awareness and knowledge about CMV and CMV prevention.

Human papillomavirus oncogene mRNA testing for the detection of anal dysplasia in HIV-positive men who have sex with men - Corrected Proof

2012-01-20

Abstract: Background: Anal human papillomavirus (HPV) infection and anal dysplasia are frequent in HIV-positive men who have sex with men (HIV+MSM), and progression of low-grade (LSIL) to high-grade squamous intraepithelial lesions (HSIL) or anal cancer (AC) occurs faster than in HIV-negative individuals. High-risk (HR)-HPV-E6/E7 oncogene mRNA testing has a higher specificity and a higher positive predictive value (PPV) than HR-HPV-DNA testing for detecting high-grade cervical lesions.Objective: To evaluate the diagnostic accuracy of the NucliSENS-EasyQ HPV1.1 E6/E7-mRNA-assay for the detection of anal dysplasia in HIV+MSM.Study design: 289 intraanal swabs from HIV+MSM participating in a screening program that included anal cytology, high-resolution anoscopy and histology were analyzed. HR-HPV-DNA detection was performed by PCR and hybridization using a bead-based multiplex genotyping assay. E6/E7-mRNA detection of HR-HPV-types 16, 18, 31, 33 and 45 was performed using the NucliSENS-EasyQ assay.Results: 269 swabs had valid results in both test formats (111 normal, 10 ASCUS, 105 LSIL, 42 HSIL, 1 AC). For the detection of LSIL+(LSIL+HSIL+cancer) sensitivity, specificity, negative predictive value (NPV) and PPV were 80.4%, 26.4%, 52.5%, and 57.2% for HR-HPV-DNA testing, respectively, compared to 75.7%, 57.9%, 66.0% and 68.7% for E6/E7-mRNA testing. The respective values for the detection of HSIL/cancer were 95.3%, 26.1%, 96.7%, 19.7% for HR-HPV-DNA and 95.3%, 46.0%, 98.1%, 25.2% for E6/E7-mRNA detection.Conclusion: Compared to HR-HPV-DNA detection, E6/E7-mRNA testing has an increased specificity (approximately two-fold), similar sensitivity and higher NPV and PPV for the detection of low- and high-grade anal dysplasia in HIV+MSM.

Comparison of a commercial Varicella Zoster glycoprotein IgG enzyme immunoassay with a reference time resolved fluorescence immunoassay (VZV TRFIA) for measuring VZV IgG in sera from pregnant women, sera sent for confirmatory testing and pre and post vOka vaccination sera from healthcare workers - Corrected Proof

P.A.C. Maple, J. Breuer, M. Quinlivan, G. Kafatos, K.E. Brown — 2012-01-19

Abstract: Background: Recently, a commercial, standardised VZV IgG glycoprotein EIA, Binding Site VaccZyme™VZV glycoprotein IgG low level EIA (VaccZyme™EIA) has become available. The VaccZyme™EIA is more robust and user friendly than the reference VZV time-resolved fluorescence immunoassay (VZV TRFIA).Objectives: To assess the usefulness of the VaccZyme™EIA in the diagnostic laboratory by comparing VZV IgG levels generated by both assays on serum panels representing, non-vaccinated, and vOka vaccinated populations.Study design: Sera from non-vaccinated individuals were tested; 248 from pregnant women, 117 from various patient groups referred to the Virus Reference Department for confirmatory VZV IgG testing and 102 from healthcare workers enrolled in a study (ROVE) of antibody/IgG response to vOka. From the ROVE study, 282 post vaccination sera were tested; 108 and 101 collected at six weeks post first and second doses of vOka, respectively, and 73 collected at 18month follow-up.Results: Sensitivities and specificities (equivocals treated as negatives) of the VaccZyme™EIA for sera from pregnant women were 97.8% (95% CI: [94.6%, 99.4%]) and 96.8% (95% CI: [89.0%, 99.6%]), respectively, and for sera referred for confirmatory testing were 81.2% (95% CI: [71.2%, 88.8%]) and 96.9% (95% CI: [83.8%, 99.9%]), respectively, and for ROVE baseline sera were 54.2% (95% CI: [32.8%, 74.4%]) and 100% (95% CI: [95.4%, 100.0%]), respectively. For the post vOka serum panels sensitivities of the VaccZyme™EIA ranged from 65.3% (95% CI: [50.4%, 78.3%]) to 80.4% (95% CI: [71.1%, 87.8%]). Specificities were all 100%. Correlation with VZV TRFIA was high and agreement varied between the serum panels tested.Conclusions: VaccZyme™EIA is recommended for detecting VZV IgG in sera from non-vaccinated populations; however, caution is advised when measuring post vOka VZV IgG levels.

Human papillomavirus subtypes in oral lesions compared to healthy oral mucosa - Corrected Proof

2012-01-19

Abstract: Background: Human papillomaviruses (HPV) are involved in the etiology of cervix cancer, but it is still unclear whether they play a role in related oral lesions.Objectives: The presence of HPV in oral leukoplakia biopsies (n=50) and oral squamous carcinoma biopsies (n=50) was compared to normal oral mucosa swabs (n=50) for the purpose of indicating a possible etiological role for the virus.Study design: DNA was extracted from tissue biopsies and from mucosa swabs of control samples. Nested PCR was performed with primers targeting conserved sequences within the capsid gene L1. PCR products were sequenced to identify the HPV genotype.Result: The results reveal a profile of low-risk HPV genotypes in oral leukoplakia similar to that in healthy controls, while HPV was less frequently observed in oral squamous carcinoma.Conclusions: HPV does not seem to represent an important causal factor for the development of oral leukoplakia or oral squamous carcinoma.

Comparison of the rate and size of HIV-1 viral load blips with Roche COBAS TaqMan HIV-1 versions 1.0 and 2.0 and implications for patient management - Corrected Proof

2012-01-19

Abstract: Background: The Roche COBAS TaqMan HIV-1 version 1.0 (v1.0) real-time PCR test detects more low level viral loads (VL) compared to the previous Roche Amplicor version 1.5 assay. Due to under-quantification issues, the Roche TaqMan HIV-1 version 2.0 (v2.0) was introduced in 2009. Controversy remains on differences at the low VL end, where clinical decisions regarding possible viral escape are based.Objectives: To compare the rate and size of VL blips with v1.0 and v2.0 in virologically suppressed patients and describe the impact of v2.0 on patient management.Study design: A cohort study of HIV-positive patients on antiretroviral therapy with a VL <50 copies/ml at the beginning and end of the study period (July 2008–February 2010). VL blips were compared during two consecutive 9-month periods, initially measured by v1.0, then v2.0. Genotypic resistance testing and treatment switches were described.Results: 1037 of 2584 patients (73.1% male) with median age 43 years were included. 2465 VL samples were measured on v1.0 and 2206 on v2.0. 108 (10.4%) patients had blips on v1.0 (4.4% of samples) compared to 99 (9.5%) patients (4.5% of samples) on v2.0. Median log VL was 1.89 (78 copies/ml) for v1.0 and 2.06 (116 copies/ml) for v2.0 (p=0.002). Further characterisation of 11 samples detected no resistance and no treatment modifications were identified.Conclusions: TaqMan v1.0 and v2.0 have similar blip rates, while blips are higher with v2.0. This study supports the strategy to increase the threshold of concern for VL blips on v2.0.

High TNF-alpha and IL-8 levels predict low blood dendritic cell counts in primary cytomegalovirus infection - Corrected Proof

2012-01-18

Abstract: Background: In vitro studies suggest that human cytomegalovirus (CMV) modulates the functions of dendritic cells (DCs). However, there are limited data on DC homeostasis in CMV-infected patients.Objectives: The aim of this study was to characterize circulating DCs and plasma cytokine levels in immunocompetent patients with primary, symptomatic CMV infections.Study design: The study population consisted of 14 patients suffering of CMV mononucleosis and 14 healthy volunteers (11 CMV-seropositive and 3 CMV-seronegative subjects) included as controls. Peripheral blood mononuclear cells were isolated and used to characterize DCs and to quantify CMV in the blood. Plasma levels of pro-inflammatory and anti-inflammatory cytokines were also measured.Results: We observed that patients who were developing CMV mononucleosis presented lower myeloid and plasmacytoid DC counts in peripheral blood compared with healthy controls. We also noted elevated levels of inflammatory mediators, of which tumor necrosis factor-α (TNF-α)—which activates DCs and endothelial cells—was the highest. Notably, the decrease in blood DCs correlated with high TNF-α and IL-8 levels by a hyperbolic function.Conclusions: Our results suggest that increased levels of inflammatory factors facilitate alterations in DC homeostasis during primary CMV infection, which may contribute to viral-induced modulation of host immunity.

Pet dogs—A transmission route for human noroviruses? - Corrected Proof

Maija Summa, Carl-Henrik von Bonsdorff, Leena Maunula — 2012-01-13

Abstract: Background: Human noroviruses (HuNoVs) are one of the leading causes of diarrhoeal diseases worldwide in all age groups. Virus transmission can occur via the faecal-oral route from person to person or via contaminated food, water, or surfaces. The most common NoV strains circulating among humans belong to genogroup GII. Thus far, to our knowledge, no HuNoVs have been detected in pets.Objectives: We investigated whether pet dogs could serve as carriers for HuNoVs and thereby transmit the infection to humans.Study design: Ninety-two faecal samples of indoor pet dogs were obtained. The main criteria for sample collection were that the dog or humans in the household had suffered from diarrhoea or vomiting. All samples were screened for HuNoV genogroups GI, GII, and GIV by real-time one-step RT-PCR.Results: We detected HuNoV in four faecal samples from pet dogs that had been in direct contact with symptomatic persons. Three of the positive samples contained genotype GII.4 variant 2006b or 2008 and one GII.12. All NoV-positive dogs lived in households with small children and two dogs showed mild symptoms.Conclusions: Our results suggest that HuNoVs can survive in the canine gastrointestinal tract. Whether these viruses can replicate in dogs remains unresolved, but an association of pet dogs playing a role in transmission of NoVs that infect humans is obvious.

Divergent KSHV/HHV-8 subtype D strains in New Caledonia and Solomon Islands, Melanesia - Corrected Proof

2012-01-13

Abstract: Background: KSHV/HHV-8 is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma and most multicentric Castleman's disease cases. KSHV exhibits a high genetic variability comprising five genotypes (A–E). Few data are yet available concerning the situation of KSHV, its genetic variability and the associated diseases in Melanesia.Objectives: We performed a study on 626 natives Melanesians from New Caledonia and Vanikoro Island to evaluate KSHV seroprevalence and characterize molecularly the viral strains.Study design: Plasma from 343 males and 283 females (age range: 15–86 years, mean age: 60) were tested for KSHV latent antibodies by an immunofluorescence assay (IFA) using BC-3 cells. DNAs extracted from peripheral blood buffy-coat of KSHV seropositive individuals were amplified to obtain a 737-bp fragment of the ORF-K1 gene. Phylogenetic analyses were then performed.Results: Among 626 samples, 148 were IFA positive (dilution≥1:80). The overall seroprevalence was 23.6% (25.2% in New Caledonia, 17.5% in Vanikoro). Fifteen (8 men and 7 women, mean age 69 years) out of 148 DNA samples were found PCR positive. All ORF-K1 sequences belonged to KSHV genotype D. A geographic clustering according to the island of origin of KSHV infected persons was clearly observed with sequences from New Caledonia clustering with most Vanuatu strains.Conclusions: New Caledonia and Vanikoro are endemic for KSHV with a high diversity of genotype D variants. These strains were probably introduced into New Caledonia during multiple waves of migrations of Melanesian and Polynesian individuals that have colonized this archipelago.

Assessment of immunovirological features in HIV related non-Hodgkin lymphoma patients and their impact on outcome - Corrected Proof

2012-01-13

Abstract: Background: Despite the era of highly active antiretroviral therapy, non-Hodgkin lymphoma (NHL) remains one of the main causes of death in HIV-infected patients, with a wide variation on the outcome.Objectives: We investigated immunological status and EBV, HHV8, HIV viral load in a group of HIV-infected patients at diagnosis of NHL to evaluate their prognostic significance.Study design: Eighty-one consecutive HIV+ NHL patients were studied. CD4 and CD8 cell counts, HHV8 DNA, EBV DNA, HIV RNA and HIV DNA were assessed at diagnosis and at 3 months after chemotherapy initiation. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of disease free survival (DFS) and overall survival (OS) were computed according to CD4 and CD8 cell counts, EBV DNA, HIV RNA and HIV DNA. HRs were, thereafter, computed also for continuous variation of CD4, CD8 cell counts and EBV DNA.Results: In the multivariate analysis, CD4<160 and CD8<590cell/μl and EBV DNA≥300c/ml were independently associated to DFS (HR=2.98; 95%CI: 1.26–7.03; HR=2.65, 95%CI: 1.13–6.19; HR=4.01; 95%CI: 1.81–8.91) and OS (HR=3.32; 95%CI: 1.41–7.83; HR=4.62, 95%CI: 1.91–11.19; HR=3.11, 95%CI: 1.42–6.80). HRs for DFS and OS decreased continuously with increasing CD4 and CD8 cell counts, while they increased continuously with increasing EBV DNA levels.Conclusions: The association with survival of low CD4 and CD8 cell counts and detectable EBV viremia, measured at lymphoma's diagnosis, identified three independent prognostic biomarkers that might help in the management of NHL HIV+ patients, offering complementary information in the ascertainment of their outcome.

Sequential changes in pathophysiology of systemic inflammatory response in a disseminated neonatal herpes simplex virus (HSV) infection - Corrected Proof

2012-01-11

Abstract: Background: Disseminated neonatal herpes simplex virus (HSV) infection causes a typical systemic inflammatory response syndrome and has a high mortality rate. However, the validity of anti-inflammatory intervention against this condition remains unknown.Objectives: We sought to demonstrate the sequential changes in the pathophysiology of disseminated neonatal HSV infections.Study design: The HSV serum copy number as well as high-mobility group box 1 (HMGB1) and cytochrome c concentrations, which predict the severity and mortality rate of sepsis, were sequentially evaluated in a patient with disseminated neonatal HSV infection caused by HSV-2.Results: As the patient presented with evidence of hyper-inflammation and severe illness, we empirically undertook anti-inflammatory intervention that included the administration of prednisolone, high-dose immunoglobulin, and blood exchange therapy in addition to high-dose acyclovir (ACV) therapy. The patient survived without significant neurological sequela. We found that (1) the serum concentrations of both HMGB1 and cytochrome c were extremely high, (2) temporal increases in these biomarkers were observed after admission, and (3) interestingly, the increase in HMGB1 level preceded that of cytochrome c. These results suggested that the pathophysiology of this condition changed sequentially in a dramatic manner, and the timing of our anti-inflammatory intervention was prior to the transition of pathological status from hyper-inflammation to massive apoptosis.Conclusions: Anti-inflammatory intervention may only be effective if it is undertaken during the early phase of disseminated neonatal HSV infections.

Helminthic infection and the risk of neurologic disease progression in HTLV-1 - Corrected Proof

2012-01-11

Abstract: Background: Infection with the human T-cell lymphotropic virus, type 1 (HTLV-1) has been associated with an increased Th1 response. Interestingly, a higher prevalence of helminthic coinfection has been observed among infected individuals, and subsequent modulation of the immune response typically associated with helminths may influence clinical outcomes among HTLV-1 coinfected individuals.Objective: This study was conducted to elucidate the association between helminthic coinfection and the development of clinically characterized neurologic disease that occurs in HTLV-1 infection.Study design: In a cohort analysis, incidence of HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP) was recorded. Incidence of clinical outcomes and disease-free survival of several neurologic outcomes associated with HTLV-1 were estimated using the Kaplan–Meier method with log-rank tests. The relationships between helminthic infection and risk of HTLV-1 neurologic outcomes were assessed by Cox proportional hazard modeling.Results: Seventy-four coinfected and 79 non-coinfected patients were followed, with 92 helminthic infections observed in the coinfected group. One patient per group developed HAM/TSP and the risk of progression to neurologic disease outcomes did not differ among those with and without helminthic coinfection (p>0.45). A significant difference was noted in the prevalence of neurologic disease outcomes among all patients at the conclusion of the study (p<0.01).Conclusions: These data suggest that treated helminthic infection does not affect risk of development of neurologic disease in HTLV-1 infection, and reinforce that treatment of helminths does not adversely affect patients with HTLV-1. Importantly, among all patients, an overall progression of neurologic disease was observed.

Incidence of cytomegalovirus UL97 and UL54 amino acid substitutions detected after 100 or 200 days of valganciclovir prophylaxis - Corrected Proof

Guy Boivin, Nathalie Goyette, Mahdi Farhan, Jane Ives, Robert Elston — 2012-01-11

Abstract: Background: The IMPACT study was a randomized, double-blind study comparing 100 to 200 days of VGCV prophylaxis (900mg once daily) in D+/R− kidney transplant recipients. Although extending the duration of prophylaxis resulted in a significant reduction in confirmed cytomegalovirus (CMV) disease (100-day: 36.8% vs 200-day: 16.1%), the consequence of extending the duration of prophylaxis on the development of viral resistance remains unknown.Objective: To determine whether extending valganciclovir prophylaxis from 100 days to 200 days increased the incidence of ganciclovir resistance.Study design: Genotypic analysis of CMV UL97 and UL54 was conducted on virus isolated from patients meeting the predefined resistance analysis criteria (RAC).Results: A greater number of patients met the RAC in the 100 day prophylaxis arm (50/163; 31%) compared to the 200 day prophylaxis arm (22/155; 14%). Sequence data were successfully generated for all 200-day patients and 48/50 100-day patients. Three patients in each treatment arm (100 day: 3/163 (1.8%) vs 200 day: 3/155 (1.9%)) had a single known valganciclovir resistance mutation detected (100 day: UL97 gene: M460V, C592G twice; 200 day: UL97 gene: C603W, M460V and UL54 gene: P522S). Overall, a resistance mutation was more likely to be detected if the patient met the RAC during prophylaxis (5/12 (42%)) compared to post-prophylaxis (1/58 (2%)). All six patients with known ganciclovir resistance mutations cleared the virus; three cleared virus without treatment and three cleared virus following treatment.Conclusions: Extending valganciclovir prophylaxis from 100 days to 200 days did not significantly affect the incidence of ganciclovir resistance.

Evaluation of the RIDAQuick norovirus immunochromatographic test kit - Corrected Proof

2012-01-09

Abstract: Background: Norovirus infections occur frequently and are widespread throughout the US population causing greater than half of all foodborne gastroenteritis cases. A rapid norovirus assay would be a useful clinical tool for identification of this common virus in gastroenteritis patient samples, thereby identifying outbreaks and facilitating rapid implementation of control measures.Objectives: To determine the suitability of the RIDAQuick norovirus kit as a clinical tool by determining the specificity and sensitivity of the assay, and its cross-reactivity with other enteric viruses.Study design: Archived stool specimens containing norovirus genogroup I or II or other viruses were tested using the RIDAQuick norovirus assay and results compared to those obtained with real-time RT-PCR.Results: We tested 62 samples: 19 norovirus genogroup I, 25 genogroup II samples, and 18 norovirus negative samples. Compared to PCR results, RIDAQuick assay sensitivity was 61.4%, and specificity was 100%. The low sensitivity was mainly due to poor results with genogroup I specimens; only 11 of 19 were detected. Additionally, samples of four other common enteric viruses all tested negative with the RIDAQuick assay.Conclusions: The RIDAQuick kit effectively detects norovirus genogroup II strains, but not genogroup I strains. We found no cross-reactivity with several common enteric viruses. As most norovirus cases are currently genogroup II strains, positive results with RIDAQuick can be used for rapid detection of norovirus in a large percentage of cases, thus also aiding in identification of outbreaks. However, final confirmation and negative results require further testing with more sensitive methods.

Hepatitis C virus viral kinetics during α-2a or α-2b pegylated interferon plus ribavirin therapy in liver transplant recipients with different immunosuppression regimes - Corrected Proof

2012-01-06

Abstract: Background: Predictors of sustained virological response (SVR) to antiviral therapy post-liver transplantation (LT) for chronic hepatitis C are needed. In non-transplanted patients, viral kinetics can predict SVR.Objectives: To determine the early viral kinetics in LT recipients with different immunosuppression (tacrolimus – Tac- vs. cyclosporine – CsA-) during treatment with peg-IFN+RBV.Study design: Prospective pilot study in HCV-1b infected patients: (LT CsA n=8; Tac n=8; non-LT n=4), treated with IFN α-2a vs. α-2b (180μg or 1.5μg/kg, respectively) once weekly plus weight-based RBV. Median CsA or Tac baseline trough levels were 141 and 7.70ng/mL, respectively. HCV-RNA was quantified before treatment and after 3, 6, 12h; days 1–6; and weeks 4, 12, 24, 48 and 78 (follow-up).Results: Different kinetics were observed: early viral load declines with shoulder phase (n=12), delayed monophasic without first phase (n=5, all CsA), and biphasic (n=1) or flat (n=1), without influence of IL28B rs12979860 donor/recipient alleles. In LT, median declines (log10UI/mL) at week 4 were −3.62 and −1.49 for Tac vs. CsA; and −2.10 vs.−1.50 for IFN α-2a vs. α-2b (NS), with a trend for faster declines in Tac patients. Generalized additive models suggested a cut-off for predicting response in LT patients of 30 days for Tac, but beyond day 40 for CsA.Conclusion: In LT, the viral kinetics during peg-IFN+RBV treatment is delayed. HCV-RNA at 48h. may not be predictive of response, and CsA-immunosupressed patients with delayed monophasic declines may potentially achieve ETVR and SVR despite unfavourable or absent early viral load declines.

Phylogenetic analysis of human metapneumovirus from New York State patients during February through April 2010 - Corrected Proof

Daryl M. Lamson, Sara Griesemer, Meghan Fuschino, Kirsten St. George — 2012-01-05

Abstract: Background: Human metapneumovirus (hMPV) is the second leading cause of lower respiratory infection (LRI) in children around the world and has been linked to LRI in multiple studies. Currently, hMPV is classified into 2 major subtypes (A and B), each with 2 subgroups (1 and 2).Objective: To determine which hMPV genotypes were present in NYS patients with influenza-like illness (ILI) from February through April 2010, during a period of unusually heightened activity.Study design: Specimens were collected from February through April of 2010 from patients presenting with ILI who were previously confirmed as positive for hMPV by real-time RT-PCR. A 700 base pair region of the hMPV fusion (F) gene was amplified, sequenced and resulting sequences aligned. A phylogenic tree was constructed based on prototype strains, and the partial F gene sequences obtained in this study.Results: Bi-directional sequence was obtained from 30 patient samples and included in the phylogenic analysis. Specimen sequences grouped into hMPV genotype A2a (16), A2b (9), B2 (4) and B1 (1). No A1 genotypes were found.Conclusion: Previously, reports have demonstrated that genotypes A1, A2, B1 and B2 circulate every season, usually with one dominant strain. In contrast, late in the 2009–2010 respiratory season, 4 of the 5 recognized genotypes of hMPV were isolated from NYS ILI patients, and by sequencing a larger portion of the fusion gene, we were able to identify the A2a and A2b genotypes.

Broad reactivity of the Luminex xTAG Respiratory Virus Panel (RVP) assay for the detection of human rhinoviruses - Corrected Proof

2012-01-05

Human rhinoviruses (HRV) are a major cause of the common cold and a variety of both upper and lower respiratory tract infections including sinusitis, otitis media, bronchitis, primary pneumonia and have been associated with the worsening of asthma and chronic obstructive pulmonary disease. With the advent of new antivirals directed at the treatment of serious HRV infections it is becoming increasingly important to detect a broad range of HRV strains so that targeted antiviral therapy can be appropriated instituted.

Prevalence of HIV, HBV, HCV, HTLV and Treponema pallidum among patients attending a rural hospital in Southern Ethiopia - Corrected Proof

2012-01-04

Human immunodeficiency virus type 1 (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell lymphotropic virus type 1 (HTLV) and Treponema pallidum represent major public health problems in sub-Saharan countries. Ethiopia is among the countries where HIV and HBV infections are highly prevalent. Knowing the prevalence of HIV, HBV, HCV, HTLV and T. pallidum infection among persons seeking medical care in rural areas of the country may provide important clues to reduce the spread of these diseases and could be of help in evaluating the effectiveness of prevention and control programs. The aim of this study was to determine the seroprevalence of HIV, HBV, HCV, HTLV and T. pallidum among patients attending a rural hospital in Ethiopia.

Performance of an immunofiltration assay detecting IgM antibodies against ZEBRA and viral capsid p18 proteins (Immunoquick® filtration EBV M) for the diagnosis of heterophile antibody-negative primary Epstein-Barr virus infection in children - Corrected Proof

2012-01-04

The detection of heterophile antibodies (HA) allows for a rapid and reliable diagnosis of primary Epstein-Barr virus (EBV) infection. Nevertheless, HA are frequently absent in young children. We previously evaluated the performance of an immunofiltration assay (IMFA) detecting IgM antibodies to EBV ZEBRA (BamHI Z EBV replication activator) protein (Immunoquick® filtration EBV M assay; BioSynex Immunodiagnostic, Strasbourg Cedex, France) for the diagnosis of acute EBV infectious mononucleosis (IM). Overall, the sensitivity and the specificity of the assay were found to be 92.5%, and 97.3%, respectively. In the current study we evaluated the performance of this IMFA assay for the diagnosis of HA-negative primary EBV infection in previously healthy children. A total of 177 sera testing negative for HA by a differential agglutination assay (I.M. Kit; Microgen, Surrey, GB) from unique patients (101 males and 76 females; median age of 3 years, range 0–14 years) attended at the Pediatric emergency service of the Hospital Clínico Universitario of Valencia between January and September 2011 were included in the study. EBV-serology testing was requested because of the presence of fever (n=91), mononucleosis syndrome (n=31), fever and exanthema (n=16), pneumonia (n=5), fever and bi or pancytopenia (n=4), or hiperbilirubinemia (n=3). The underlying condition was not reported for 27 patients. The IMFA assay was performed following the instructions of the manufacturer. It must be noted that the new version of the IMFA assay includes the viral capsid antigen (VCA) p18 antigen in the testing line in addition to the ZEBRA protein. The sera were later analyzed for the presence of VCA (p18) IgM and IgGs and Epstein-Barr nuclear antigen-1 (EBNA-1) IgGs by chemiluminiscent immunoassays (CLIAs) in the Liaison instrument (CLIA-L; DiaSorin, S.p.A., Italy), as recommended by the manufacturer. The results obtained by these CLIAs have been shown to closely correlate with those obtained by indirect immunofluoescence (VCA IgGs and IgMs) or by anticomplement immunofluorescence (EBNA IgGs), the reference assays for EBV antibody testing. The criteria used to define EBV serostatus were based on consensus EBV-specific antibody profiles. Data are shown in . Forty sera tested positive by the IMFA assay. Thirty-three out of these 40 sera displayed and EBV-serological profile compatible with a primary EBV infection, as determined by CLIAs. Of the remaining 7 sera, 4 had a serological profile of past EBV infection and 3 tested negative for all EBV-antibody especificities by CLIAs. A total of 137 sera tested negative by the IMFA assay, of which 130 displayed an EBV-serological profile compatible either with a past infection (n=70) or with no previous infection (n=60), as determined by CLIAs. The remaining 7 sera had an EBV-antibody profile compatible with a primary EBV infection. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the assay for the diagnosis of primary EBV infection were 82.5%, 94.8%, 82.5% and 94.8%, respectively. Our data support the use of the IMFA evaluated herein as a first line EBV-antibody specific assay in children not developing HA, particularly for point-of-care diagnostic testing.

Evidence of immune memory 8.5 years following administration of a prophylactic human papillomavirus type 16 vaccine - Corrected Proof

2012-01-04

Abstract: Background: The duration of protection conferred by prophylactic human papillomavirus (HPV) L1 virus-like particle vaccines is a critical determinant of their public health impact. A feature of vaccines that confer long-term immunity is their ability to induce immune memory.Objectives: We evaluated antibody responses against HPV types 6, 11, 16 and 18 following administration of the quadrivalent HPV-6/11/16/18 vaccine to women who had previously received a monovalent HPV-16 vaccine.Study design: As part of an extended follow-up study conducted between 2006 and 2009 in Seattle, Washington, we administered the quadrivalent HPV-6/11/16/18 vaccine to 52 women (19 vaccine and 33 placebo recipients) who had participated in a monovalent HPV-16 vaccine trial 8.5 years earlier. Serum samples were tested for anti-HPV antibodies using competitive Luminex immunoassay.Results: Following administration of the first dose of the quadrivalent HPV-6/11/16/18 vaccine, the anti-HPV-16 geometric mean titer among monovalent HPV-16 vaccine recipients (GMT=5024.0milli-Merckunitspermilliliter [mMU/mL]; 95% confidence interval [CI]: 2710.1, 9313.6mMU/mL) substantially exceeded that among the placebo recipients (GMT=136.1; 95% CI: 78.5, 235.8mMU/mL; p<0.01) and their own highest anti-HPV-16 response observed during the original trial (GMT at month 7 of the original trial=1552.7mMU/mL; 95% CI: 1072.6, 2247.7mMU/mL; p<0.01).Conclusions: The findings suggest that the administration of the three-dose regimen of the monovalent HPV-16 vaccine had produced memory lymphocytes, characterized by a heightened immune response following administration of the quadrivalent HPV-6/11/16/18 vaccine that effectively served as an antigen challenge.

Detection of coxsackievirus A10 in multiple tissues of a fatal infant sepsis case - Corrected Proof

2012-01-04

Abstract: Non-polio enteroviruses are a common cause of childhood infections varying in symptomatology and severity. While infections with many of the enterovirus serotypes can be severe and even fatal, coxsackievirus A10 (CVA10) has most commonly been associated with more mild disease. Here we present the detection of CVA10 in multiple organ tissues in the investigation of an infant death.

Prevalence of TMC278 (rilpivirine) associated mutations in the Frankfurt Resistance Database - Corrected Proof

Claudia Reinheimer, Hans W. Doerr, Martin Stürmer — 2012-01-04

Abstract: Background: Analysis of the 3D structure of the HIV-1 reverse transcriptase led to the development of TMC278 (rilpivirine), a next-generation nonnucleoside reverse transcriptase inhibitor (NNRTI), which proved to be effective against wild-type HIV-1 strains and NNRTI-resistant mutants emerging after failure of NNRTI-containing therapy regimens. Recently, rilpivirine associated mutations (e.g. at positions 138, 181 or 101) have been described in vitro and in vivo; however, some of these mutations have also been observed in the past.Objective: Objective of our investigation was to determine the prevalence of mutations E138K, Y181I/V, and K101E/P before the approval of rilpivirine.Study design: The Frankfurt Resistance Database consists of 7295 samples which have been sent for resistance testing since 1995.Results: The E138K, Y181I/V, and the K101E mutations were found in 0.4%, 0.9%, and 2.4% of the patients, respectively.Conclusions: Based on these findings we do not expect a broad cross-resistance to rilpivirine due to previous treatment failures of NNRTI-containing regimens.

Epstein-Barr Virus load and immune activation in Human Immunodeficiency Virus type 1-infected patients - Corrected Proof

2012-01-04

Abstract: Background: Patients infected with HIV-1 are at high risk of developing Epstein-Barr Virus (EBV)-related diseases. Chronic immune activation is a hallmark of HIV-1 pathogenesis and may play a role in B-cell stimulation and expansion of EBV-infected cells.Objectives: The aim of the study was to define the relationship between parameters of immune activation and EBV load in HIV-1-infected subjects.Study design: A total of 156 HIV-1-infected patients were studied. EBV types 1 and 2 were quantified on peripheral blood mononuclear cells by multiplex real-time PCR. Plasma levels of cytokines and lipopolysaccharide (LPS) were determined by immunoenzymatic assays. B-cell activation was analyzed by flow cytometry.Results: EBV-DNA was detected in 114 patients, and in all but 3 was EBV type 1. The median [interquartile] EBV-DNA load was 43[1–151] copies/105 PBMC. EBV-DNA load was higher in patients with detectable HIV-1 plasma viremia, despite good immunological status (CD4>500 cells/μl), than in patients with undetectable HIV-1 plasma viremia regardless of immunological status (46[5–136] copies/105 cells vs 17[1–56] copies/105 cells, p=0.008). Patients with high EBV-DNA load (>median value) had higher levels of LPS and proinflammatory cytokines (IL-6, IL-10 and TNF-α) than patients with low EBV load. Furthermore, percentages of activated B-cells correlated with EBV-DNA load (rs=0.754; p<0.001).Conclusions: Overall, these findings indicate a strong association between HIV-1 viremia, markers of immune activation and EBV load and suggest that persistence of HIV-1 viremia and immune activation, regardless of peripheral CD4 cell depletion/repopulation, may favor expansion of EBV-infected cells and onset of EBV-related malignancies.

Trichodysplasia spinulosa is characterized by active polyomavirus infection - Corrected Proof

2011-12-26

Abstract: Background: Recently a new polyomavirus was identified in a patient with trichodysplasia spinulosa (TS), a rare follicular skin disease of immunocompromised patients characterized by facial spines and overgrowth of inner root sheath cells. Seroepidemiological studies indicate that TSPyV is ubiquitous and latently infects 70% of the healthy individuals.Objective: To corroborate the relationship between active TSPyV infection and TS disease by analyzing the presence, load, and precise localization of TSPyV infection in TS patients and in controls.Study design: TS lesional and non-lesional skin samples were retrieved from TS patients through a PubMed search. Samples were analyzed for the presence and load of TSPyV DNA with quantitative PCR, and for expression and localization of viral protein with immunofluorescence. Findings obtained in TS patients (n=11) were compared to those obtained in healthy controls (n=249).Results: TSPyV DNA detection was significantly associated with disease (P<0.001), with 100% positivity of the lesional and 2% of the control samples. Quantification revealed high TSPyV DNA loads in the lesional samples (∼106copies/cell), and low viral loads in the occasionally TSPyV-positive non-lesional and control samples (<102copies/cell). TSPyV VP1 protein expression was detected only in lesional TS samples, restricted to the nuclei of inner root sheath cells over-expressing trichohyalin.Conclusions: The high prevalence and load of TSPyV DNA only in TS lesions, and the abundant expression of TSPyV protein in the affected hair follicle cells demonstrate a tight relation between TSPyV infection and TS disease, and indicate involvement of active TSPyV infection in TS pathogenesis.

Epidemiological and genetic analyses of a diffuse outbreak of hepatitis A in Japan, 2010 - Corrected Proof

2011-12-26

Abstract: Background: Hepatitis A virus (HAV) is still one of the most common causative agents of acute hepatitis in Japan. Although a relatively small number of annual acute hepatitis A cases (approximately 100–150, 0.78–1.17 per million) were recently reported, a larger number of cases (346, 2.71 per million) were reported in 2010.Objectives: To investigate the causes of the 2010 HAV resurgence in Japan by using molecular epidemiological and genetic analyses.Study design: HAV specimens were obtained from 61 cases from 22 different prefectures. These viral specimens were genotyped by PCR amplification and sequencing of the VP1/2A region of HAV genome.Results: Phylogenetic analysis revealed that 61 HAV strains could be divided into three genotypes: IA (44 cases), IB (1 case) and IIIA (16 cases). The IA genotype consisted of two genomic sub-lineages. The sequences of one of the two IA sub-lineages (corresponding to 31 cases) were very similar, 26 of these 31 isolates had 100% identity. The other IA sub-lineage corresponded to strains endemic to Japan. The sequences of Japanese IIIA strains were similar to those of strains that caused a large epidemic in the Republic of Korea from 2007 to 2009.Conclusions: The resurgence of HAV in 2010 can be attributed to importation of two newly emerged HAV genotypes.

Comparison of six different specimen types for Epstein-Barr viral load quantification in peripheral blood of pediatric patients after heart transplantation or after allogeneic hematopoietic stem cell transplantation - Corrected Proof

2011-12-19

Abstract: Background: Epstein-Barr Virus (EBV) a gamma-herpes virus is associated with a spectrum of lymphoid and epithelial malignancies including posttransplant lymphoproliferative disorders (PTLD). EBV-load measurement has been shown to be important for the monitoring of these patients. However, in contrast to the viral quantification of human immunodeficiency virus or human hepatitis C virus, the EBV-load measurement has not been completely standardized as yet.Objectives: In this study, we compared the EBV DNA levels in whole blood (WB), plasma, peripheral mononuclear cells (PBMC) and B-cells (BC) in children and adolescents after heart transplantations (HTx) and allogeneic hematopoietic stem cell transplantations (HSCT).Study design: In a period of 2 years (from May 2007 to May 2009) we collected 547 samples of 96 cardiac transplant recipients and 248 samples of 37 patients who underwent HSCT. For EBV DNA quantification we used a duplex real-time PCR (ABI Prism 7500, Applied Biosystems). Additionally, EBV-load of PBMC and BC were normalized with respect to endogenous cell DNA.Results: In both patient populations we found no significant difference of test sensitivity for the EBV detection. In PBMC as well as BC, there was a high correlation between the analysis of cells with and without normalization in both populations. Spearman's correlation coefficient ρ between PBMC without and PBMC with normalization was ρ=0.98 (P<0.0001) in patients after HTx and ρ=0.99 (P<0.0001) in patients after HSCT. Correlation between BC with and without normalization was ρ=0.98 (P<0.0001) in patients after HTx and ρ=0.995 (P<0.0001) in patients after HSCT. When comparing the different blood compartments for EBV quantification in both populations, the strongest correlations were found between the EBV DNA levels in WB and PBMC (HTx: ρ=0.93, P<0.0001; HSCT: ρ=0.81, P<0.0001) followed by PBMC and BC (HTx: ρ=0.87, P<0.0001; HSCT: ρ=0.81, P<0.0001) as well as WB and BC (HTx: ρ=0.86, P<0.0001; HSCT: ρ=0.75, P<0.0001). In contrast, the correlation coefficients between plasma and the other blood compartments (WB as well as PBMC or BC) were lower.Six patients developed seven episodes of PTLD (five patients after HTx and one after renal transplantation). Analyzing the different blood compartments, we found that a threshold of WB ≥20,000EBV-copies/ml and plasma ≥1000EBV-copies/ml had the highest sensitivities and specificities (WB: sensitivity 100%, specificity 87% and plasma: sensitivity 88%, specificity 98%).Conclusion: Normalization towards an endogenous control does not seem to be necessary for EBV quantification in peripheral blood. The analysis of whole blood correlates well with B-cells and PBMC. Routine screening of EBV DNA in whole blood appeared to be a useful tool supplemented by EBV-load measurement in plasma to discriminate chronic high EBV-load carrier without risk for PTLD from those who are at risk for PTLD. Values in whole blood higher than 20,000EBV-copies/ml WB and plasma values higher than 1000EBV-copies/ml plasma indicated PTLD in our series.

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.