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  • Demographic, clinical, and virological characteristics of patients with a laboratory-confirmed diagnosis of influenza during during three consecutive seasons, 2015/2016 to 2017/18, in the Republic of Iran
    J. Clin. Virol. (IF 3.020) Pub Date : 2020-01-16
    Alireza Nateghian; Mohammad Mehdi Gouya; Mahmood Nabavi; Hanieh Soltani; Seyed Vahid Mousavi; Elmira Agah; Hosein Erfani; Peiman Parchami; Mohammadnasr Dadras; Joan L. Robinson

    Background There are minimal data on the differences in demographics, clinical presentations and outcomes for patients with different types and sub-types of influenza in the Middle East. Objectives To use population-based data from Iran to investigate factors associated with unfavorable disease outcome. Study Design Clinical data were compiled from the Iranian Ministry of Health for patients of all ages who fulfilled the severe acute respiratory infections (SARI) definition according to World Health Organization criteriatested for any reason and found to have and had laboratory proven influenza September 21, 2015 through March 20, 2018. Pulmonary, cardiac, renal, hematologic and neurologic complications were recorded. Results were compared by type, age, gender and health status. Multivariate analysis was used to analyze risk factors for complications and death. Results Of 11080 enrolled patients, 10046 (90.7%) were inpatients, 2254 (20.4%) were children, 8403 (75.8%) had influenza A, 2599 (23.5%) had influenza B, and 78 (0.7%) had unidentified types. Fever was less common in older patients (OR 0.99; 95% CI 0.98-0.99, p<0.001 and in those with comorbidity (OR 0.87; 95% CI 0.77-0.97, p=0.013). Although the rate of complications was lower with A(H1N1) pdm09 influenza than with A(H3N2) infection (12.8% versus 15.6%, p=0.001), the mortality rate was higher (7.0% versus 3.0%, p<0.001). Complications occurred more often during late versus early influenza season (OR 1.22; 95% CI 1.08-1.37, p=0.002). Patients with type B influenza (OR 0.85; 95% CI 0.74-0.98, p=0.025), or who presented with sore throat (OR 0.74; 95% CI 0.65-0.84, p<0.001) were less likely to develop complications. The risk of developing complications was increased in patients who had chronic heart disease (OR 1.51; 95% CI 1.29-1.76, p<0.001), chronic pulmonary disease (OR 1.62; 95% CI 1.37-1.91, p<0.001), diabetes (OR 1.24; 95% CI 1.03-1.50, p=022), or epilepsy (OR 1.55; 95% CI 1.17-2.05). Older age and male gender increased the risk of death but not of complications. Conclusions The clinical features, complications and outcomes of influenza vary by age and by viral type and sub-type. Comorbidites appear to be more important than age in predicting complications.

  • Performance evaluation of four point-of-care HIV tests using unprocessed specimens
    J. Clin. Virol. (IF 3.020) Pub Date : 2020-01-16
    Pollyanna R. Chavez; Heather M. Bradley; Laura G. Wesolowski; Lauren R. Violette; David A. Katz; Lisa A. Niemann; Vanessa M. McMahan; Sarah McDougal; Andy M. Cornelius-Hudson; Steven F. Ethridge; Joanne D. Stekler; Kevin P. Delaney

    Background The performance of recently approved point-of-care (POC) HIV tests should be assessed using unprocessed specimens. Objective: To evaluate the sensitivity and specificity of four POC HIV tests using whole blood (WB) and two using oral fluid (OF) among persons recruited from health clinics in Seattle, Washington, during September 2015-September 2017. Studydesign Participants were tested with the POC tests, additional plasma and serum were collected for laboratory testing, and participant- reported use of antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) was recorded. Participants testing negative on all tests could reenroll every 90 days. Specimens from persons previously diagnosed with HIV infection as well as from those who were newly diagnosed during the study were included in the sensitivity estimate. Sensitivity and specificity were calculated based on HIV status determined by laboratory testing. Results Of 1,256 visits, 179 were from persons with HIV infection; 120 of these were taking ART. Among 1,077 visits from participants not diagnosed with HIV, PrEP use was reported at 155 (14.4%) visits. Sensitivity was similar among POC WB tests (95.53%-97.21%; p>0.05). Among participants on ART, sensitivity was lower for the same test performed on OF compared to WB (p<0.003). Specificity was high for all tests (99.44%- 100.00%); we did not detect specificity differences with PrEP use. Conclusions These POC tests displayed relatively high sensitivity and specificity using unprocessed specimens, suggesting their effectiveness in identifying HIV infections whenever laboratory-based testing is not feasible. Nonetheless, clients with recent risk should retest to rule out the possibility of a false-negative result.

  • Enterovirus D68 outbreak detection through a syndromic disease epidemiology network
    J. Clin. Virol. (IF 3.020) Pub Date : 2020-01-16
    Lindsay Meyers; Jennifer Dien Bard; Ben Galvin; Jeff Nawrocki; Hubert G.M. Niesters; Kathleen A. Stellrecht; Kirsten St. George; Judy A. Daly; Anne J. Blaschke; Christine Robinson; Huanyu Wang; Camille V. Cook; Ferdaus Hassan; Sam R. Dominguez; Kristin Pretty; Samia Naccache; Katherine E. Olin; Benjamin M. Althouse; Rangaraj Selvarangan
  • Non-structural protein 1 (NS1) of Dengue virus detection correlates with severity in primary but not in secondary Dengue infection
    J. Clin. Virol. (IF 3.020) Pub Date : 2020-01-11
    Celia Martínez-Cuellar; Dolores Lovera; Fernando Galeano; Luis Gatti; Antonio Arbo

    Background Non-structural protein 1 (NS1) of dengue virus circulates in the serum of patients during the acute phase of the disease. Objectives To determine whether NS1 screening can serve in diagnosing primary and secondary infection and to evaluate its utility as a marker for predicting the severity of dengue in children. Study design Patients ≤15 years of age hospitalized for dengue between 2012-2018, with NS1 determination (Panbio, Australia) were included. Clinical y laboratorial characteristics were collected in a standardized data table for analysis of correlation between serotypes, primary or secondary condition of infection, severity, and presence of NS1. Results Of 709 children hospitalized for dengue with NS1 determination, 479 (67.5%) had the positive test. Of the 378 primary cases, 320 (85%) were NS1 (+). while among the 242 secondary cases only 103 (42.5%) were NS1 (+) (p < 0001). Of the 479 patients with NS1 (+), 344 (72%) were warnig-signed cases (WSC) and 94 (19%) were severe cases (SC), being these figures 62% and 34%, in the NS1 negative patients respectively (p < 0.001). There was no difference in the frequency of WSC or SC between patients with NS1 positive or negative test in secondary dengue; however, in primary dengue, the figures were 68% vs 32% (p < 0.001), and 87% vs 12% (p < 0.001), respectively. Conclusions The presence of NS1 positive test is associated with the condition of infection (primary or secondary) and exhibited an increased risk of developing forms with warning signs or severe dengue in primary cases, but not in secondary cases.

  • Human parainfluenza virus circulation, United States, 2011-2019
    J. Clin. Virol. (IF 3.020) Pub Date : 2020-01-09
    Nicholas P. DeGroote; Amber K. Haynes; Calli Taylor; Marie E. Killerby; Rebecca M. Dahl; Desiree Mustaquim; Susan I. Gerber; John T. Watson

    Background Human parainfluenza viruses (HPIVs) cause upper and lower respiratory tract illnesses, most frequently among infants and young children, but also in the elderly. While seasonal patterns of HPIV types 1-3 have been described, less is known about national patterns of HPIV-4 circulation. Objectives To describe patterns of HPIVs circulation in the United States (US). Study design We used data from the National Respiratory and Enteric Virus Surveillance System (NREVSS), a voluntary passive laboratory-based surveillance system, to characterize the epidemiology and circulation patterns of HPIVs in the US during 2011-2019. We summarized the number of weekly aggregated HPIV detections nationally and by US census region, and used a subset of data submitted to NREVSS from public health laboratories and several clinical laboratories during 2015-2019 to analyze differences in patient demographics. Results During July 2011 - June 2019, 2,700,135 HPIV tests were reported; 122,852 (5%) were positive for any HPIV including 22,446 for HPIV-1 (18%), 17,474 for HPIV-2 (14%), 67,649 for HPIV-3 (55%), and 15,283 for HPIV-4 (13%). HPIV testing increased substantially each year. The majority of detections occurred in children aged ≤ 2 years (36%) with fluctuations in the distribution of age by type. Conclusions HPIVs were detected year-round during 2011-2019, with type-specific year-to-year variations in circulation patterns. Among HPIV detections where age was known, the majority were aged ≤ 2 years. HPIV-4 exhibited an annual fall-winter seasonality, both nationally and regionally. Continued surveillance is needed to better understand national patterns of HPIV circulation.

  • Neutrophil-endothelial interactions in respiratory syncytial virus bronchiolitis: an understudied aspect with a potential for prediction of severity of disease
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-12-31
    Amadu Juliana; Rens Zonneveld; Frans B. Plötz; Matijs van Meurs; Jan Wilschut

    Respiratory syncytial virus (RSV) lower respiratory tract infection (LRTI) causes significant morbidity and mortality among young infants worldwide. It is currently widely accepted that neutrophil influx into the airways is a hallmark of the pathophysiology. However, the exact mechanism of neutrophil migration from the vasculature into the alveolar space in RSV LRTI has received little attention. Data shows that endothelial cells become activated upon RSV infection, driving a ‘pro-adhesive state’ for circulating neutrophils with upregulation of endothelial intercellular adhesion molecule-1 (ICAM-1). During RSV LRTI different subsets of immature and mature neutrophils are present in the bloodstream, that upregulate integrins lymphocyte-function associated antigen (LFA)-1 and macrophage (Mac)-1, serving as ICAM-1 ligands. An alveolar gradient of interleukin-8 may serve as a potent chemoattractant for circulating neutrophils. Neutrophils from lung aspirates of RSV-infected infants show further signs of inflammatory and migratory activation, while soluble endothelial cell adhesion molecules (sCAMs), such as sICAM-1, have become measurable in the systemic circulation. Whether these mechanisms are solely responsible for neutrophil migration into the alveolar space remains under debate. However, data indicate that the currently postulated neutrophil influx into the lungs should rather be regarded as a neutrophil efflux from the vasculature, involving substantial neutrophil-endothelial interactions. Molecular patterns of these interactions may be clinically useful to predict outcomes of RSV LRTI and deserve further study.

  • Comparison of virological and serological methods for laboratory confirmation of rubella
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-12-31
    Kiyoko Uchino; Tatsuya Miyoshi; Yoshio Mori; Katsuhiro Komase; Fumika Okayama; Yuri Shibata; Hisayoshi Yoshida; Tomizo Numata; Makoto Takeda; Tomoyuki Tanaka

    Background Work toward rubella elimination has accelerated globally. A reliable laboratory confirmation of rubella-suspected cases is required for effective surveillance in the rubella-elimination phase. The use of adequate specimens is a key to improving the quality of this surveillance. Study design We conducted rubella virus (RUBV) isolation and RUBV genome or anti-RUBV IgM detection on 1,023 specimens from 372 rubella- or measles-suspected cases collected through the national surveillance program in Sakai city of Osaka prefecture, Japan between 2011 and 2013. The resulting data were analyzed by specimen type, collection date, and immunological status. Results Among the three specimen types (throat swab, serum or plasma, and urine) collected through 10 days post-rash onset, the highest success rates for RUBV genome detection and RUBV isolation were obtained using throat swabs. In agreement with previous work, RUBV-specific IgM were undetectable in 50% of the rubella-confirmed cases until 3 days after rash onset. The success rates of RUBV genome detection and RUBV isolation declined in association with the appearance of RUBV-specific antibodies in blood, especially in serum, plasma, or urine samples. Conclusion Throat swabs are the most optimal specimen types for both RUBV genome detection and RUBV isolation; serum/plasma samples may be suboptimal, especially for RUBV isolation. The findings from this study will provide useful information for improving laboratory surveillance for rubella in the elimination phase.

  • How relevant is the HIV low level viremia and how is its management changing in the era of modern ART? A large cohort analysis
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-12-26
    Lucia Taramasso; Laura Magnasco; Bianca Bruzzone; Patrizia Caligiuri; Giorgio Bozzi; Sara Mora; Elisa Balletto; Paola Tatarelli; Mauro Giacomini; Antonio Di Biagio

    Background It is still unclear what might be the best management of people living with HIV (PLWHIV) with low level viremia (LLV) despite being on antiretroviral treatment (ART). Objectives Aim of our study is to describe the clinical management of PLWHIV with LLV followed in a large cohort. Study design Retrospective cohort study. Results We included 1607 adult patients over a three-year period (2015-2017). Follow up continued until June, 30th 2019 or last available visit. We observed a low incidence of LLV (0.9% in 2015, 0.7% in 2016 and 0.4% in 2017), with a total of 21 patients with persistent LLV (pLLV), i.e. two consecutive HIV-RNA determinations of 50-500 copies/ml after at least 4 months of viral suppression. Among them, 12 had low compliance to treatment. Genotype resistance test (GRT) was performed in 14 patients and demonstrated at least one resistance mutation in 85.7%. We described three categories of patients with pLLV: i) those whose ART regimen was not adequate based on GRT; ii) those with presumed suboptimal drug exposure, consequence of low adherence and/or drug-drug interactions and iii) those in which pLLV remained unexplained. For the first two categories, optimization or intensification of ART regimen led to viral suppression in >80% of patients. We observed only 2 (9.5%) virological failures and 1 (4.8%) persistence of LLV in patients who did not switch ART. Conclusions In our cohort, the rate of LLV showed a decline in most recent years. Adherence and previous GRT should be carefully considered with the aim of further reducing the phenomenon.

  • Molecular and clinical characterization of human adenovirus associated with acute respiratory tract infection in hospitalized children
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-12-25
    Meng-chuan Zhao; Ying-hui Guo; Fang-zhou Qiu; Le Wang; Shuo Yang; Zhi-shan Feng; Gui-xia Li

    Background Human adenovirus (HAdV) is a common pathogen in children that can cause acute respiratory tract infection (ARTI), but the molecular epidemiological and clinical information relating to HAdV among hospitalized children with ARTI are few reported in China. Objectives To evaluate the epidemiological, clinical, and molecular characteristics of HAdV infections among hospitalized children with ARTI in Hebei, Northern China from June 2017 to May 2018. Study design A 12-month longitudinal, retrospective study on HAdV, typed by nested polymerase chain reaction targeting the hexon gene’s hypervariable region (typing was merely performed by sequencing of the hexon neutralization epitope and thus genotypes could not be identified unequivocally), associated with ARTI was performed. The epidemiological and clinical data of different types of HAdV were analyzed using statistical product and service solutions (SPSS) 21.0 software. Results HAdV was detected in 330 (3.71%) of the 8906 specimens, with most (88.48%, 292/330) HAdV-positives cases detected among children < 3 years old. HAdV were detected throughout the year with a higher prevalence in spring. 11 types were identified, with HAdV-2 (33.33%, 110/330) as the predominant type, followed by HAdV-3 (21.21%, 70/330) and HAdV-7 (13.94%, 46/330). Of the 330 HAdV-positive specimens, 247 (74.85%) were co-detected with other respiratory pathogens, most commonly rhinovirus (HRV) (58.7%, 145/247). Additionally, patients with HAdV-7 positive had longer duration of fever than HAdV-2 or -3 positive patients. Conclusions During the study period, HAdV-2, HAdV-3 and HAdV-7 were the predominant types identified from children with ARTI in Hebei Province. Pediatric patients with HAdV-7 positive may not present more severe clinical outcome except a longer duration of fever.

    J. Clin. Virol. (IF 3.020) Pub Date : 2019-12-23
    Bruna Morassi Sasso; Michelle Etienne Baptistella Florence; Renata Ferreira Magalhaes; Paulo Eduardo Neves Ferreira Velho; Elemir Macedo de Souza; Maria Leticia Cintra; Rafael Fantelli Stelini

    The goal was to characterize the clinical-epidemiological profile of patients with mucocutaneous tumoural herpes simplex virus (MCT HSV) lesions across the world. Two researchers extracted and independently reviewed data from the literature search engine PubMed/MEDLINE through October 2018. From 110 reported patients, the following data were available: the patients' ages ranged from 7 to 76 years; the majority was male (62.73% - 69/110) and immunosuppression was found in 97.25% (106/109, missing 1) cases, of whom 88 were HIV- related. Lesions size varied from 0.2 to 13 cm, settling in the anogenital region in 76.36% (84/110) patients; 84.13% (53/63, missing 47) complained of pain and multiple recurrences were found in 44.94% (40/89, missing 21) cases. On clinical basis, the initial hypothesis was neoplasia in 36/53 patients. Histopathological diagnosis was achieved in 90% (90/100, missing 10) cases and was sample size-dependent. Type 2 HSV was detected in 86.07% (68/79, missing 31) lesions. MCT HSV lesions recurrence after treatment was reported in 33.96% (18/53, missing 57) patients. Pathophysiology is poorly understood. Physicians should be aware of MCT HSV lesions in immunosuppressed patients to avoid inappropriate therapeutic strategies.

  • Antiviral activity of ribavirin and favipiravir against human astroviruses
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-12-17
    Andrew B Janowski; Holly Dudley; David Wang

    Background Recent recognition of invasive astrovirus infections, including encephalitis and viremia in humans, have highlighted the need for effective anti-astrovirus therapeutics. However, there is a paucity of data regarding the in vitro activity of broad-spectrum RNA antivirals against astroviruses, including ribavirin and favipiravir. Objectives We quantified the EC50 values for ribavirin and favipiravir against two human astrovirus strains, astrovirus VA1 (VA1) and human astrovirus 4 (HAstV4). Study Design Caco-2 cells were infected with VA1 or HAstV4 in the presence of ribavirin or favipiravir (dose range 0.1-1000 μM), and the cells were maintained in media containing the drugs for 72 hours. Viral RNA was extracted and quantified by qRT-PCR. As a surrogate for cytotoxicity, cellular adenosine triphosphate (ATP) from each drug treatment was also measured. Results VA1 replication was inhibited 10-100-fold by both ribavirin (EC50 = 154μM) and favipiravir (EC50 = 246 μM). In contrast, ribavirin inhibited HAstV4 replication (EC50 = 268 μM) but favipiravir only reduced replication by 44% at the highest dose. Mild reductions in ATP (17-31%) was only observed at the highest concentration of ribavirin (1000 μM) and no significant decrease in ATP was detected for any concentration of favipiravir. Conclusions Ribavirin inhibited both human astrovirus species and favipiravir was only active against VA1. In the future, the in vivo efficacy of these drugs could be tested with development of an animal model of human astrovirus infection

  • Performance evaluation of the Panther Fusion® respiratory tract panel
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-12-10
    Jolanda J.C. Voermans, Daphne G.J.C. Mulders, Suzan D. Pas, Marion P.G. Koopmans, A.A. van der Eijk, R. Molenkamp

    Background Respiratory tract infections are among the most common infections during winter season. Rapid diagnostics is required for clinical decision making regarding isolation of patients and appropriate therapy. Objectives The aim of this study was to evaluate the analytical and clinical performance characteristics of the Panther Fusion® respiratory panel using published laboratory-developed real-time PCR assays (LDT). Study design Analytical sensitivity of Panther Fusion® Flu A/B/RSV was assessed by testing dilutions of cell culture isolates. Clinical performance assessment included the complete Panther Fusion® respiratory panel (Flu-A/B/RSV, PIV1-4 and AdV/hMPV/RV) and consisted of a retrospective and a prospective study-arm. The retrospective evaluation included 201, stored (−80 °C) samples collected between February 2006 and January 2017. Prospective evaluation was performed on 1047 unselected pretreated respiratory tract samples from patients presented to our hospital between November 2017 and May 2018. Results Analytical sensitivity was generally slightly lower for the Panther Fusion® assays. Clinical specificity and sensitivity was between 96%-100% and 71.9% -100%, respectively. Discrepant results were found in 146 samples of which 88 samples tested LDT positive / Panther Fusion® negative and 58 samples were LDT negative / Panther Fusion® positive. A total of ten discrepant samples with Ct-values <30 were sequenced to confirm the presence of 7 RV-C not-detected by LDT and 1 RV-A and 2 ADV-2 not detected by Panther Fusion®. Conclusions The Panther Fusion® provides a random-access system with continuous loading and much shorter sample-to-answer times compared to LDT, albeit with a slightly less clinical sensitivity compared to the LDT.

  • Blood Viral Load in the Diagnostic Workup of Congenital Cytomegalovirus Infection
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-29
    Mina Smiljkovic, Jean-Baptiste Le Meur, Brigitte Malette, Isabelle Boucoiran, Anne-Frédérique Minsart, Valérie Lamarre, Bruce Tapiero, Christian Renaud, Fatima Kakkar

    Background There is limited data on the role of cytomegalovirus (CMV) blood quantitative polymerase chain reaction (qPCR) in the diagnostic workup of congenital CMV (cCMV) infection. Objectives The objective of this study was to determine if CMV blood qPCR at the time diagnosis could differentiate between symptomatic and asymptomatic infants according to the recent consensus classification. Study design Retrospective study of children diagnosed with cCMV infection at CHU Sainte-Justine, Montreal, Canada, between 2008 and 2016. Cases for whom qPCR was done at baseline (<4 weeks of age) alongside a complete diagnostic workup were included. The association between CMV blood viral load (VL) and clinical severity group was determined. The probability of having moderate to severe symptoms was assessed using univariate logistic regression analysis. Results Forty-seven patients were included in the analysis. Median VL was significantly higher among infants with moderate to severely symptomatic disease vs. those asymptomatic or asymptomatic with isolated sensorineural hearing loss (SNHL) (13 736 vs. 1876 copies/ml, p = 0.004), infants with moderate to severe disease or asymptomatic with isolated SNHL vs. asymptomatic (17 736 vs. 1496 copies/ml, p < 0.001), and in infants with baseline neurological involvement vs. those without (17 317 vs. 2641 copies/ml, p = 0.03). Using logistic regression, an infant would have a > 75% probability of being moderate to severely symptomatic above 18 770 copies/ml, with a threshold of 100 000 copies/ml approaching a 100% probability. Conclusions Our baseline assessment of CMV blood VL suggests that that the level of CMV viremia correlates with symptom severity.

  • The laboratory investigation of a measles outbreak in the eve of its elimination in Sri Lanka
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-26
    Buddhini Samaraweera, Adhyana Mahanama, Almarz Z Ahamad, Gayan I Wimalaratne, Janaki Abeynayake

    Background Measles is highly contagious and cause significant morbidity and mortality. Sri-Lanka has the goal to eliminate endogenous measles by 2020 in par with WHO. Objective To describe laboratory confirmation and genotype distribution of measles cases during the outbreak occurred from mid-March to May 2019, Sri-Lanka Study design This retrospective study was conducted at National Measles Reference Laboratory (NMRL), Sri-Lanka. All samples received were tested according to the testing flow chart at NMRL with WHO recommended kits. Blood samples were tested for anti-measles IgM and IgG with IgG avidity for IgG positives. Samples within 5days post-onset rash were tested with measles real-time RT-PCR. Products of genotyping PCR were sent to Regional Reference Laboratory, Thailand for sequencing. Subsequent phylogenetic analysis was done at NMRL. Data were analyzed by descriptive statistics. Results A total of 182 blood and 46 throat/nasopharyngeal swabs were received from 195 suspected cases and 37(19%) were positive for measles by anti-measles IgM, rRT-PCR or both. Majority was females, with mean age of 20 years. Cases represented three main geographical areas; Western-35%, Central-32% and Southern-27%. High avidity IgG was detected in 27/37(73%). Sequencing data of six cases (4 from Western and 2 from Central province) revealed genotype D8. Conclusion Nineteen percent of the suspected patients were measles positive with 73% having re-infections. Majority were 22 years or over. Measles genotype was D8 in two provinces, suggesting the spread of virus within the country. Laboratory confirmation with measles PCR; IgG avidity and sequencing/genetic analysis is critical in the verge of measles elimination.

  • Performance Evaluation of the Vela Dx Sentosa Next-Generation Sequencing System for HIV-1 DNA Genotypic Resistance
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-26
    Stéphanie Raymond, Florence Nicot, Florence Abravanel, Luce Minier, Romain Carcenac, Caroline Lefebvre, Agnès Harter, Guillaume Martin-Blondel, Pierre Delobel, Jacques Izopet

    Background Patients on antiretroviral therapy could benefit from HIV-1 DNA resistance genotyping for exploring virological failure with low viral load or to guide treatment simplification. Few new generation sequencing data are available. Objective To check that the automated deep sequencing Sentosa platform (Vela DX) detected minority resistant variants well enough for HIV DNA genotyping. Study design We evaluated the Sentosa SQ HIV genotyping assay with automated extraction on 40 DNA longitudinal samples from treatment-experienced patients by comparison with Sanger sequencing. HIV drug resistance was interpreted using the ANRS algorithm (v29) at the threshold of 20% and 3%. Results The Sentosa SQ HIV genotyping assay was 100% successful to amplify and sequence PR and RT and 86% to amplify and sequence IN when the HIV DNA load was >2.5 log copies/million cells. The Sentosa and Sanger sequencing were concordant for predicting PR-RT resistance at the threshold of 20% in 14/18 samples successfully sequenced. A higher level of resistance was predicted by Sentosa in three samples and by Sanger in one sample. The prevalence of resistance was 7% to PI, 59% to NRTI, 31% to NNRTI and 20% to integrase inhibitors using the Sentosa SQ genotyping assay at the threshold of 3%. Seven additional mutations <20% were detected using the Sentosa assay. Conclusion Automated DNA extraction and sequencing using the Sentosa SQ HIV genotyping assay accurately predicted HIV DNA drug resistance by comparison with Sanger. Prospective studies are needed to evaluate the clinical interest of HIV DNA genotyping.

  • Next generation sequencing of human enterovirus strains from an outbreak of enterovirus A71 shows applicability to outbreak investigations
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-17
    Sacha Stelzer-Braid, Matthew Wynn, Richard Chatoor, Matthew Scotch, Vidiya Ramachandran, Hooi-Ling Teoh, Michelle A. Farrar, Hugo Sampaio, Peter Ian Andrews, Maria E. Craig, C. Raina MacIntyre, Hemalatha Varadhan, Alison Kesson, Philip N. Britton, James Newcombe, William D. Rawlinson

    Background The most recent documented Australian outbreak of enterovirus A71 (EV-A71) occurred in Sydney from 2012 to 2013. Over a four-month period more than 100 children presented to four paediatric hospitals with encephalitic presentations including fever and myoclonic jerks. The heterogeneous presentations included typical encephalomyelitis, and cardiopulmonary complications. Objectives To characterise the genomes of enterovirus strains circulating during the 2013 Sydney EV-A71 outbreak and determine their phylogeny, phylogeography and association between genome and clinical phenotype. Study design We performed an analysis of enterovirus (EV) positive specimens from children presenting to hospitals in the greater Sydney region of Australia during the 2013 outbreak. We amplified near full-length genomes of EV, and used next generation sequencing technology to sequence the virus. We used phylogenetic/phylogeographic analysis to characterize the outbreak viruses. Results We amplified and sequenced 23/63 (37 %) genomes, and identified the majority (61 %) as EV-A71. The EV-A71 sequences showed high level sequence homology to C4a genogroups of EV-A71 circulating in China and Vietnam during 2012-13. Phylogenetic analysis showed EV-A71 strains associated with more severe symptoms, including encephalitis or cardiopulmonary failure, grouped together more closely than those from patients with hand, foot and mouth disease. Amongst the non-EV-A71 sequences were five other EV subtypes (representing enterovirus subtypes A and B), reflecting the diversity of EV co-circulation within the community. Conclusions This is the first Australian study investigating the near full-length genome of EV strains identified during a known outbreak of EV-A71. EV-A71 sequences were very similar to strains circulating in Asia during the same time period. Whole genome sequencing offers additional information over routine diagnostic testing such as characterisation of emerging recombinant strains and inform vaccine design.

  • Use of whole-genome sequencing in the molecular investigation of care-associated HCoV-OC43 infections in a hematopoietic stem cell transplant unit
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-16
    Delphine Beury, Léa Fléchon, Florence Maurier, Ségolène Caboche, Jean-Stéphane Varré, Hélène Touzet, Karine Faure, Jean Dubuisson, David Hot, Benoit Guery, Anne Goffard

    Background While respiratory viral infections are recognized as a frequent cause of illness in hematopoietic stem cell transplantation (HSCT) recipients, HCoV-OC43 infections have rarely been investigated as healthcare-associated infections in this population. Objectives In this report, HCoV-OC43 isolates collected from HSCT patients were retrospectively characterized to identify potential clusters of infection that may stand for a hospital transmission. Study design Whole-genome and S gene sequences were obtained from nasal swabs using next-generation sequencing and phylogenetic trees were constructed. Similar identity matrix and determination of the most common ancestor were used to compare clusters of patient’s sequences. Amino acids substitutions were analysed. Results Genotypes B, E, F and G were identified. Two clusters of patients were defined from chronological data and phylogenetic trees. Analyses of amino acids substitutions of the S protein sequences identified substitutions specific for genotype F strains circulating among European people. Conclusions HCoV-OC43 may be implicated in healthcare-associated infections.

  • Evaluation of the performance of the cepheid xpert HIV‐1 viral load assay for quantitative and diagnostic uses
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-15
    Laura Wesolowski, William Fowler, Wei Luo, Vickie Sullivan, Silvina Masciotra, Tara Smith, Rebecca Rossetti, Kevin Delaney, Emeka Oraka, Pollyanna Chavez, Steven Ethridge, William M. Switzer, S. Michele Owen

    Background Cepheid's Xpert HIV‐1 Viral Load (Xpert VL), a simplified, automated,single-use quantitative assay used with the GeneXpert System, is not FDAapproved. Objectives Using stored plasma, we conducted a study to assess the ability of Xpert VL to quantify viral load relative to the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 (Cobas VL) and to examine the use of the Xpert VL as a qualitative diagnostic test. Study design Following HIV-1 viral stock dilutions, we conducted a probit analysis to identify the concentration where 95 % of specimens had quantified VLs. We also examined Xpert and Cobas log VL correlation in linearity panels; compared the proportion of 220 seroconverter specimens with virus detected using McNemar's test; and tested specimens from persons with untreated, established HIV-1 infection (n=149) and uninfected persons (n=497). Furthermore, we examined Xpert VL as a qualitative test in seroconverter specimens with early (n=20) and later (n=68) acute infections. Results At 1.80 log10 copies/mL, 95 % of specimens had quantifiable virus using Xpert VL. Xpert and Cobas VLs were highly correlated (R2=0.994). The proportion of seroconverter specimens with virus detected using Cobas and with Xpert VL was not statistically different (p=0.0578). Xpert VL detected 97.9 % of established infections, and specificity was 99.80 % (95 % CI 98.87%–99.99%). Xpert VL detected 90 % and 98.5 % of early and later acute infections, respectively. Conclusions If approved, Xpert VL could allow U.S. laboratories that cannot bring on large, complex testing platforms to conduct HIV monitoring. An approval for diagnostic use may provide timely identification of HIV infections.

  • Next-generation sequencing shows marked rearrangements of BK polyomavirus that favor but are not required for polyomavirus-associated nephropathy
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-13
    Hanna Liimatainen, Lukas Weseslindtner, Robert Strassl, Stephan W. Aberle, Gregor Bond, Eeva Auvinen

    Background BKPyV is associated with polyomavirus-associated nephropathy (PVAN), a major cause of graft rejection in kidney transplant recipients (KTRs). Mutations occur in the transcriptional control region (TCR) of BKPyV, but whether they are required for the development of PVAN is not completely understood. To this end, we characterized BKPyV TCRs from KTRs to assess whether TCR mutations are associated with PVAN. Study design We analyzed urine and plasma samples of fifteen KTRs with biopsy-confirmed PVAN, presumptive PVAN, or probable PVAN in order to explore the contents of the BKPyV virome. BKPyV TCRs were amplified and deep sequenced to characterize the viral strains. Alterations in block structures and transcription factor binding sites were investigated. Results The majority of sequences in both urine and plasma samples represented archetype BKPyV TCR. Minor populations harboring rearranged TCRs were detected in all patient groups. In one biopsy-confirmed PVAN patient rearranged TCRs predominated, and in another patient half of all reads represented rearranged sequences. Conclusions Although archetype BKPyV predominated in most patients, highest proportions and highest numbers of rearranged strains were detected in association with PVAN. TCR mutations seem not necessary for the development of PVAN, but immunosuppression may allow increased viral replication giving rise to TCR variants with enhanced replication efficiency.

  • Prediction of unfavorable outcomes in West Nile virus neuroinvasive infection – result of a multinational ID-IRI study
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-11
    Corneliu Petru Popescu, Simin Aysel Florescu, Rodrigo Hasbun, Arjan Harxhi, Razi Evendar, Hasip Kahraman, Ami Neuberger, Daniel Codreanu, Mihaela Florentina Zaharia, Selma Tosun, Emanoil Ceausu, Simona Maria Ruta, Gorana Dragovac, Natalia Pshenichnaya, Galina Gopatsa, Olga Shmaylenko, Éva Nagy, Jelena Djekic Malbasa, Hakan Erdem

    Background WNV causes 1.4% of all central nervous system infections and is the most common cause of epidemic neuro-invasive disease in humans. Objectives Our main objective was to investigate retrospectively West Nile virus neuroinvasive disease (WNND) cases hospitalized during 2010- 2017 and identified factors that can influence prognosis. Study design We documented the demographic, epidemiologic, clinical and laboratory data of WNND and identified factors that can influence prognosis. The data were recruited through Infectious Diseases International Research Initiative (ID-IRI), which serves as a network for clinical researches. Results We investigated 165 patients with WNND in 10 countries from three continents. 27 patients died and the mortality rate was 16.4%. In an univariate analysis age, congestive heart failure, neoplasm and ischemic heart disease (p < 0.001), neuropsychiatric disorders (p = 0.011), chronic hepatitis (p = 0.024) and hypertension (p = 0.043) were risk factors for death. Fatal evolution was also correlated with ICU addmission, disorientation, speech disorders, change in consciousnes, coma, a low Glasgow coma score, obtundation, confusion (p < 0.001), history of syncope (p = 0.002) and history of unconsciousness (p = 0.037). In a binomial logistic regresssion analysis only age and coma remained independent prediction factors for death. We created an equation that was calculated according to age, co-morbidities and clinical manifestations that may be used to establish the prognosis of WNND patients. Conclusions WNND remain an important factor for morbidity and mortality worldwide, evolution to death or survival with sequelae are not rare. Our study creates an equation that may be used in the future to establish the prognosis of WNND patients.

  • Panther Fusion® Respiratory Virus Assays for the Detection of Influenza and other Respiratory Viruses
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-09
    Kathleen A. Stellrecht, Jesse L. Cimino, Lisa I. Wilson, Vincente P. Maceira, Shafiq A. Butt

    Background Nucleic acid amplification tests (NAATs), such as PCR, are preferred for respiratory virus testing, due to superior diagnostic accuracy and faster turnaround time. Panther Fusion® Respiratory Assays (Fusion), which includes FluA/B/RSV (FFABR), Paraflu and AdV/hMPV/RV, offers a modular approach to syndromic testing on a fully automated platform while improving gene targets and expanding the test menu. Objectives and study design We evaluated Fusion using 275 consecutive nasopharyngeal specimens previously used in an analysis of five PCRs, as well as 225 archived specimens. Results Of the combined 500 specimens, 134 were positive for influenza A (FluA), 54 for FluB, 65 for RSV, 64 for parainfluenza (PIV), 24 for adenovirus (AdV), 21 for humanmetapneumovirus (hMPV), and 40 for rhinovirus (RV) with Fusion. Of the positive samples Fusion correlated with historical results for all but one, despite multiple freeze-thaws cycles of this collection. Fusion was positive for an additional 33 samples, including 11 FluAs, 7 RSVs, 3 PIV3s, 3 AdV, 6 hMPV and 3 RVs. These samples were retested with corresponding Prodesse (Pro) assays using quadruple sample volume. This resolver test confirmed Fusion results for an additional 4 FluAs, 4 RSVs, 1 PIV3 and 3 AdVs. The sensitivity and specificity ranges of Fusion were 99-100% and 98-100%. Limit of detection (LOD) analyses were performed on a variety of Flu isolates. The LODs ranged from 2.69-2.99 log copies/ml and demonstrated superior LOD as compared to previously published data for some assays or to concurrent analyses with two new commercial tests.

  • The performance of a new point-of-care HIV virus load technology to identify patients failing antiretroviral treatment
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-09
    Diana Mariani, Marcelo C.V.M. de Azevedo, Isabelle Vasconcellos, Luiz Ribeiro, Cassia Alves, Orlando C. Ferreira, Amilcar Tanuri

    Background A new point-of-care (POC) HIV virus load technology has been recently developed and designed to be utilized in decentralized settings. Alere Technologies GmbH*, Germany, developed the mPIMA HIV-1/2 V L plasma test which uses real time PCR technology with 50 μl and a turnaround time of one hour. Objective Analyze the performance of mPIMA to detect and quantify HIV-1 and HIV-2 and compare with Abbott M2000 assay fooling patients HIV-1 failing ARV therapy. Study design In this study we evaluate the mPIMA HIV-1/2 V L plasma test using 413 specimens from 270 patients failing ARV therapy, and compared its performance with Abbott RealTime HIV-1 Viral Load assay on the m2000 system. In addition, were determined VL in plasma specimens obtained from HIV-2 infected patients. Results The results strongly indicate that mPIMA HIV-1/2 V L plasma test can determine HIV-1 with concordance of 88.9% (95% CI 85.4-91.7) the reference test when 1,000 HIV-1 VL threshold was used as WHO cutoff to identify therapy failure. The overall correlation between HIV-1 VL was 0,928 (Pearson correlation coefficient of Linear regression) and the Bland-Altman showed a mean difference of -0.20 Log cp/mL between the two technology. mPIMA HIV-1/2 V L plasma test was also able to measure HIV-2 viral load in 16 specimens from Guinea-Bissau HIV-1/HIV-2 positive samples. Conclusions These data support the use of mPIMA HIV-1/2 V L plasma test to follow up patients and select patients failing ART, guiding immediate clinical decisions such as adherence counseling or ART regimen switch during the patient consultation.

    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-09
    Paolo A. Grossi, Fausto Baldanti, Massimo Andreoni, Carlo Federico Perno

    Transplant represents an effective strategy in the management of chronic organ dysfunction. Nonetheless, life threatening risks remain, especially in the post-transplant; among them, human cytomegalovirus (CMV) is a major concern, currently causing active infections in at least one-third of transplant recipients. Microbiologist and transplant scientific societies redefined guidance on CMV disease prevention and the best use for universal prophylaxis and pre-emptive virological monitoring. Developments in molecular diagnostic supported the spread of the pre-emptive strategy, and quantitative Real Time-PCR assays has unravelled the potential of viral load measurement as a predictor of the infection development in CMV post-transplant management. However, despite the WHO 1st CMV International standard, the standardisation of diagnostic and clinical practice has been limited by the absence of algorithms for calculating conversion factor to International Units and the lack of shared monitoring procedure, both at national and international level. At a regional level, the Italian scientific societies, AMCLI (Italian Clinical Microbiologist Association), SITO (Organ Transplant Italian Society), GITMO (Italian Group for Bone Marrow Transplant), recently tried to define a consensus for post-transplant monitoring. The concerted practice encompasses molecular quantitative PCR assays technical aspects and endorses the relevance of immunologic monitoring for improvement in patient risk stratification and prognosis. Here, we provide an overview of the state of the art of CMV management strategies, with a specific focus on the clinical practices and on the scientific societies' initiatives that aim to implement international standardization guidelines at a national level.

  • Association between Hepatitis C and B viruses and Head and Neck Squamous Cell Carcinoma
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-04
    Sara Donà, Daniele Borsetto, Jonathan Fussey, Valeria Biscaro, Elisa Vian, Giacomo Spinato, Anna Menegaldo, Maria Cristina Da Mosto, Roberto Rigoli, Jerry Polesel, Paolo Boscolo-Rizzo

    Background Hepatitis B and C viruses are known to be carcinogenic and have been associated with the development of non-Hodgkin’s lymphoma as well as hepatocellular carcinoma. The incidence of head and neck cancer is increasing worldwide, and early diagnosis is vital in order to achieve good oncological outcomes. Objectives To investigate the association between chronic hepatitis B and C infection, and head and neck squamous cell carcinoma (HNSCC). Study design We performed a retrospective case control study with 774 head and neck squamous cell carcinoma (HNSCC) patients undergoing treatment, and 1518 cancer-free controls undergoing hernia surgery. Hepatitis B and C serologies were tested prior to treatment, and cases and controls were age- and sex-matched before analysing rates of infection. Results HNSCC patients were more likely than controls to have evidence of chronic hepatitis B (OR = 2.76; CI 95%, 1.64-4.64) and hepatitis C (OR = 2.59; 95% CI, 1.46-4.60) infection. No substantial association was found between hepatitis B and C infection and other known risk factors for head and neck cancer. Conclusions These findings suggest a positive association between both hepatitis B and hepatitis C chronic infection, and HNSCC. More work is needed to establish a causal role, however an awareness of the possibility of increased risk of HNSCC may lead to earlier diagnosis and better outcomes in patients with hepatitis B and C.

  • Performance of CDC Trioplex qPCR during a Dengue Outbreak in Brazil
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-04
    Tatiana Elias Colombo, Alice Freitas Versiani, Karina Rocha Dutra, Julia Guimarães Dias Rubiato, Tayna Manfrin Galvão, Andréia Francesli Negri Reis, Maurício Lacerda Nogueira

    Background In recent years real‑time reverse transcription polymerase chain reaction (real-time RT-PCR) has become a leading technique for nucleic acid detection and quantification of flaviviruses, including Dengue virus (DENV). Trioplex real-time RT-PCR has the advantages of providing the concurrent detection of Zika virus (ZIKV), DENV, and Chikungunya virus (CHIKV) RNA in human serum. Objective This study sought to compare the sensitivity and specificity of the Trioplex real-time RT-PCR assay to those provided by CDC DENV TaqMan® RT-qPCR assay and conventional PCR when used for DENV detection in the context of a dengue epidemic. Study Design We analyzed 1656 serum samples from symptomatic patients with acute febrile disease for 5 days less between December 2018 and May 2019. The samples were tested using the various PCR-based assays. Results Of the 1656 serum samples analyzed, 713 (43%) were laboratory-confirmed as arboviruses: 99.86% (712/713) were confirmed as DENV and 0.14% (1/713) were confirmed as ZIKV. Next, 590 samples were selected, and of these, 331 samples (56.1%) were determined to be positive (Ct < 38) and 259 samples (43.9%) were determined to be negative (Ct > 38) using the Trioplex real-time RT-PCR assay. The multiplex method found that the test exhibits 95% sensitivity and 100% specificity. Conclusion This evaluation demonstrates the capacity of the Trioplex real-time RT-PCR assay to detect DENV at a high sensitivity and specificity in a geographic area with a current dengue outbreak and a lower co-circulation of other arboviruses – such as ZIKV and CHIKV, and the results prove it´s applicability as clinical screening test that can serve as a confirmatory test.

  • Detection and Quantification of Human Immunodeficiency Virus and Hepatitis C Virus in Cadaveric Tissue Donors Using Different Molecular Tests
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-11-04
    Victoria Stadler Tasca Ribeiro, Sonia Mara Raboni, Paula Hansen Suss, Juliette Cieslinski, Letícia Kraft, Jucélia Stadinicki dos Santos, Luciane Pereira, Felipe Francisco Tuon

    BACKGROUND Tissue banks evaluate potential donors with several serological screening tests for infectious diseases. These tests can vary according to the tissue and the patient’s origin, including risk for endemic infectious diseases [[1], [2], [3], [4], [5]]. Despite the rigorous microbiological and viral screening of tissue donors, transmission of infectious diseases has been reported [[6], [7], [8], [9], [10]], such as cytomegalovirus, rabies, prion disease, hepatitis B virus, hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Several analytical methods can be performed in tissue donors samples for HIV and HCV diagnosis, including testing for antibodies, antigens and nucleic acids (NAT) [4,[11], [12], [13], [14]]. All these tests are specific and sensitive; however, NAT and other molecular-based methods can detect the viruses sooner than antibody and antigen tests, increasing the safety of the donated tissues [4,5,[15], [16], [17], [18]].Cadaveric serum and plasma specimens are usually collected after circulation and are often of poor quality. This is due to effects of hemolysis, hemodilution, autolysis, bacterial growth, and other factors, which can interfere in immunological tests (antigen and antibody) [18]. OBJECTIVES Considering the shortage of available tests for cadaveric specimens that are developed, validated for use and standardized in Brazil, the aim of this study was to evaluate the specificity, sensitivity, and accuracy of different commercial molecular tests for the detection and quantification of HIV and HCV in peripheral blood samples from cadaveric donors through spiked samples.

  • A decade of Enterovirus genetic diversity in Belgium
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-10-25
    Elke Wollants, Leen Beller, Kurt Beuselinck, Mandy Bloemen, Katrien Lagrou, Marijke Reynders, Marc Van Ranst

    Background Enteroviruses are responsible for a wide range of clinical symptoms.Enterovirus D68 was already known to cause mild to severe respiratory infections, but in the last few years, it has also been associated with neurological symptoms and acute flaccid paralysis. Objectives In this epidemiological surveillance in Belgium, 1521 enteroviruspositive samples were genotyped. Study design Enterovirus-positive patient samples were collected from the University Hospitals Leuven and other hospitals and medical practices in Belgium from 2007 to 2018. Molecular typing was done by RT-PCR using different primers sets. EV-A and EV-B were typed by sequencing part of VP1. For EVC and EV-D, the VP4/VP2 region was used together with the non-coding region. Results In this epidemiological survey with samples collected over 12 years, 35 different EV types were detected in 1521 patient samples. Enterovirus species B was by far the most dominant species in our samples (93%).Echovirus 30 was most frequently found (24%), followed by echovirus 6 (8%) and echovirus 9 (7%). In 2018, there was an outbreak for the first time of enterovirus D68 with severe respiratory infections but no acute flaccid paralysis. Phylogenetic analyses showed that the collected outbreak strains coexist in different clades. Conclusions For more than a decade, the circulating enterovirus strains were investigated in Belgium. During this time span, echovirus 30 was the most frequently detected and peaked every 3 years. Enterovirus D68 began an upsurge in 2018, but thus far without being clinically associated with acute flaccid paralysis.

  • Performance and Usability of mPIMATM HIV 1/2 Viral Load test in point of care settings in Kenya
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-10-23
    Priska Bwana, Joshua Ageng’o, Jeff Danda, Joseph Mbugua, Allan Handa, Matilu Mwau

    Background HIV viral load testing is the standard of care for monitoring antiretroviral therapy. In resource-limited settings such as Kenya, access to HIV viral load monitoring is suboptimal due to reliance on centralized laboratory based in vitro diagnostics. Point of care technologies have the potential to improve access and reduce test to result turnaround time. Objective To determine the performance and usability of the mPIMATM HIV-1/2 Viral Load (VL) test in point of care settings in Kenya. Method This was a cross-sectional study conducted among 568 HIV positive adults recruited from selected health facilities in Western Kenya between June and November 2018. Five hundred and sixty-six plasma samples (566) were tested successfully on AbbottTM RealTime HIV-1 quantitative test (reference assay) and mPIMATM HIV-1/2 Viral Load test to determine diagnostic accuracy. Usability data was collected through simple structured questionnaires. Statistical analysis was done using Stata/MP Version 14 for Mac OSX. Concordance and misclassification values were calculated at the clinical cut-off of 1000 copies/ml. Results The positive, negative and overall agreement of the mPIMATM HIV-1/2 V L test were 95.45% (95% CI 89.49-98.11%), 95.96% (95% CI 93.66-97.44%) and 95.86% respectively. All users (7/7, 100%) reported that the machine was easy to use and that the results interpretation and workflow were simple. The test to result turnaround time was 69 minutes. All clinicians (4/4, 100%) felt that a Point of care test would fit easily within their workflow and would facilitate decision-making. There were 44 (7.77%) errors in 566 tests; 38 (6.71%) were user related and four (4, 0.71%) were software related. Conclusion The mPIMATM HIV-1/2 V L test can be used interchangeably with reference assays for HIV viral load monitoring. At the point of care, mPIMATM‘s simple workflow, ease of use and short test to result turnaround time have the potential to improve access to HIV viral load monitoring.

  • Clinical performance of the HPV-Risk assay on cervical samples in SurePath medium using the VALGENT-4 panel
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-10-12
    D.A.M. Heideman, L. Xu, A.T. Hesselink, S. Doorn, D.M. Ejegod, H. Pedersen, W.G.V. Quint, J. Bonde, M. Arbyn

    Background The VALidation of HPV GENoyping Tests (VALGENT)framework is designed for comparison and clinical validation of HPV assays. Objectives To evaluate the accuracy of the HPV-Risk assay within VALGENT-4, relative to clinically validated comparator HPV tests. Study design The VALGENT-4 panel comprises consecutive SurePath cervical samples from routine screening (n⿿=⿿998), of which 51 had abnormal cytology and 13 women had cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+), enriched with SurePath cervical samples from 297 women with abnormal cytology and 109 CIN2⿿+⿿. HPV-Risk assay was performed on DNA extracted panel samples (n⿿=⿿1,295), blinded to clinical data, cytology results, and results from other HPV assays evaluated in VALGENT-4. All assay results were reported to the central VALGENT coordination institute for data and statistical analysis. HPV prevalence was analysed and accuracy for detection of CIN grade 3 or worse (CIN3+) and CIN2+ were assessed relative to GP5+/6+-PCR-EIA and GP5+/6+-PCR-EIA-LMNX. Results The sensitivity of the HPV-Risk assay for detection of CIN3+ and CIN2+ was similar to that of GP5+/6+-PCR-EIA (relative sensitivity for CIN3⿿+⿿1.01; 95%CI:0.97-1.06; pMcN⿿=⿿1.000, and for CIN2⿿+⿿1.01; 95%CI:0.96-1.06; pMcN⿿=⿿1.000) at significantly higher specificity (relative specificity 1.04; 95%CI:1.02-1.06; pMcN<0.001). The accuracy of the HPV-Risk assay for CIN3+ and CIN2+ was non-inferior compared to GP5+/6+-PCR- EIA and GP5+/6+-PCR-EIA-LMNX, with all p-values ⿤0.002. HPV16/18 genotype agreement between HPV-Risk assay and GP5+/6+-PCR-LMNX was high. Conclusions: The HPV-Risk assay demonstrated non-inferiority to clinically validated comparator assays on cervical samples in SurePath medium using the VALGENT-4 panel, and is therefore suitable for cervical cancer screening.

  • Prevalence of baseline HCV NS5A resistance associated substitutions in genotype 1a, 1b and 3 infection in Australia
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-10-03
    T. Papaluca, J. O’Keefe, S. Bowden, J.S. Doyle, M. Stoove, M. Hellard, A.J. Thompson

    Background Direct-acting antivirals (DAA) have revolutionised hepatitis C virus (HCV) treatment, and most regimens include an NS5A inhibitor. Certain amino-acid substitutions confer resistance to NS5A inhibitors, termed resistance-associated substitutions (RAS). If present at baseline, they can reduce virological response rates. Population-based sequencing (PBS) is generally used for baseline sequencing, however next generation sequencing (NGS) reduces the threshold for detection of sequences encoding RAS from 20% to 5%. We determined the prevalence of NS5A RAS at baseline amongst Australian chronically infected with genotype (GT)1a, GT1b and GT3 HCV, using both PBS and NGS. Methods Samples from DAA-naïve individuals were received at the Victorian Infectious Disease Reference Laboratory between June 2016 and December 2018. All samples were analysed for NS5A RAS using PBS. A subset of GT1 HCV samples were processed using NGS technology (Vela Diagnostics, Singapore) to determine the improvement in sensitivity. Results In total, 672 samples were analysed using PBS. The baseline prevalence of NS5A RAS was 7.6% for GT1a (n = 25/329), 15.7% for GT1b (n = 8/51) and 15.1% for GT3 (n = 44/292). NGS only marginally increased sensitivity for NS5A RAS at baseline in GT1a (16% vs 17%) and GT1b (29% vs 36%). Conclusion The prevalence of NS5A RAS in GT1a HCV in Australia was low compared with international data, and was similar to other reported international prevalence for GT1b and GT3 infection. NGS at baseline only marginally increased sensitivity for the detection of NS5A RAS in patients with GT1 HCV and cannot be recommended for routine use at baseline in clinical practice.

  • Using the VALGENT-3 framework to assess the clinical and analytical performance of the RIATOL qPCR HPV genotyping assay
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-09-20
    I. Benoy, L. Xu, D. Vanden Broeck, M. Poljak, A. Ostrbenk, M. Arbyn, J. Bogers

    Background and objective The VALGENT framework is developed to assess the clinical performance of HPV tests that offer genotyping capability. Samples from the VALGENT-3 panel are used to identify an optimal viral concentration threshold for the RIATOL qPCR HPV genotyping assay (RIATOL qPCR) to assure non-inferior accuracy to detect high-grade cervical intraepithelial neoplasia (CIN), compared to Qiagen Hybrid Capture 2 (HC2), a standard comparator test validated for cervical cancer screening. Study design The VALGENT-3 panel comprised 1,300 samples from women participating in the Slovenian cervical cancer screening programme, enriched with 300 samples from women with abnormal cytology. In follow- up, 126 women were diagnosed with CIN2+ (defined as diseased) and 1,167 women had two consecutive negative Pap smears (defined as non-diseased). All 1,600 samples were analyzed with the RIATOL qPCR. Viral concentration was expressed as viral log10 of the number of copies/ml. A zone of viral concentration cut-offs was defined by relative ROC analysis where the sensitivity and specificity were not inferior to HC2. Results The RIATOL qPCR had a sensitivity and specificity for CIN2+ of 97.6% (CI: 93.2-99.5%) and 85.1% (CI: 82.9-87.1%), respectively, when the analytical cut off was used. At a cut off of 6.5, RIATOL qPCR had a sensitivity of 96.0% (CI: 91.0-98.7%) and a specificity of 89.5% (87.6- 91.2%). At optimized cut off, accuracy of the qPCR was non-inferior to the HC2 with a relative sensitivity of 1.00 [CI: 0.95-1.05 (p = 0.006)] and relative specificity of 1.00 [CI: 0.98-1.01 (p = 0.0069)]. Conclusions The RIATOL qPCR has a high sensitivity and specificity for the detection of CIN2 + . By using a fixed cut-off based on viral concentration, the test is non-inferior to HC2. HPV tests that provide viral concentration measurements or other quantifiable signals allow flexibility to optimize accuracy required for cervical cancer screening.

  • National and regional modeling of distinct RSV seasonality thresholds for antigen and PCR testing in the United States
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-09-20
    Christopher S. Ambrose, Lisa L. Steed, Mike Brandon, Kara Frye, Ifedapo R. Olajide, Gina Thomson

    Background PCR tests now outnumber antigen tests for the diagnosis of respiratory syncytial virus (RSV) infection in the US. Recent analyses have shown that the traditional 10% positivity threshold to define an RSV season by rapid antigen testing was inappropriate for real-time PCR testing, for which 3% positivity appeared more appropriate. Objective To respectively model antigen (10%) and PCR (3%) positivity thresholds at national and regional levels using a large dataset of RSV testing results from US hospital-affiliated laboratories. Study Design From 2011–2016, 599 laboratories participated in a national RSV surveillance program (RSVAlert®). For laboratories with ≥10 tests for ≥30 weeks of a season, national and regional test numbers and positivity were summarized by test type overall, by season, and weekly within each season. Test type positivity thresholds were used to calculate season onset and offset. Results A seasonal average of 543,387 RSV tests was reported. PCR testing increased from 26% in 2011–2012 to 72% in 2015–2016. Overall, national positivity was 15.6% for antigen and 8.3% for PCR testing. National RSV season onsets and offsets were comparable using the 10% antigen and 3% PCR thresholds, but PCR-defined seasons generally started and ended later than antigen-defined seasons. Regionally, there were fewer outlier estimates of RSV season length when the predominant regional test type was used to define the season. Conclusion RSV positivity rates differed by test type, likely due to differential clinical use of the tests. These findings support the use of distinct positivity thresholds by test type.

  • Standardising surveillance of hepatitis E virus infection in the EU/EEA: a review of national practices and suggestions for the way forward
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-09-19
    Cornelia Adlhoch, Zdenka Manďáková, Steen Ethelberg, Jevgenia Epštein, Ruska Rimhanen-Finne, Julie Figoni, Sally A. Baylis, Mirko Faber, Kassiani Mellou, Niamh Murphy, Joanne O’Gorman, Maria Elena Tosti, Anna Rita Ciccaglione, Agnetha Hofhuis, Hans Zaaijer, Heidi Lange, Rita de Sousa, Ana Avellón, Samreen Ijaz

    Background Hepatitis E virus (HEV) infection is not notifiable at EU/EEA level, therefore surveillance relies on national policies only. Between 2005 and 2015, more than 20,000 cases were reported in EU/EEA countries. HEV testing is established in 26 countries and 19 countries sequence HEV viruses. Objective and study design WHO's European Action plan for viral hepatitis recommends harmonised surveillance objectives and case definitions. ECDC's HEV expert group developed minimal and optimal criteria for national hepatitis E surveillance to support EU/EEA countries in enhancing their capacity and to harmonise methods. Results The experts agreed that the primary objectives of national surveillance for HEV infections should focus on the basic epidemiology of the disease: to monitor the incidence of acute cases and chronic infections. The secondary objectives should be to describe viral phylotypes or subtypes and to identify potential clusters/outbreaks and possible routes of transmission. Seventeen of 20 countries with existing surveillance systems collect the minimal data set required to describe the epidemiology of acute cases. Eleven countries test for chronic infections. Twelve countries collect data to identify potential clusters/outbreaks and information on possible routes of transmission. Discussion Overall, the majority of EU/EEA countries collect the suggested data and meet the outlined requirements to confirm an acute case.

  • One-step pentaplex real-time polymerase chain reaction assay for detection of zika, dengue, chikungunya, West nile viruses and a human housekeeping gene
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-09-16
    Nischay Mishra, James Ng, Jennifer L. Rakeman, Michael J. Perry, Dominick A. Centurioni, Amy B. Dean, Adam Price, Riddhi Thakkar, Andreina Garcia Angus, Phillip Williamson, Eric Delwart, Christine Carrington, Nikita Sahadeo, Che Xiaoyu, Thomas Briese, Rafal Tokarz, W. Ian Lipkin

    Background Recent emergence of Zika virus (ZIKV), and the global spread of dengue (DENV), chikungunya (CHIKV) and West Nile viruses (WNV) raised urgent need of accurate and affordable molecular diagnosis of these clinically indistinguishable arboviral infections. Objectives We established a pentaplex real-time reverse transcription PCR (rRT-PCR) assay (CII-ArboViroPlex rRT-PCR) for specific and sensitive detection of the African and American genotypes of ZIKV, all four serotypes of DENV, CHIKV, WNV and a housekeeping gene as internal control in single reaction. Study design Specific primers and probe sets were designed for ZIKV, DENV, CHIKV, WNV and RNase P (housekeeping gene) and tested for in-vitro transcribed RNA standards, virus cultures, clinical samples positive for ZIKV, DENV, CHIKV and WNV and limit of detection (LOD) were determined for each. Results Using ten-fold serially diluted in-vitro transcribed RNA, CII- ArboViroPlex rRT-PCR assay has LOD of 100 RNA copies/reaction (Rn) for ZIKV in serum or urine, 100 RNA copies/Rn for DENV in serum, and 10 RNA copies/Rn for CHIKV and WNV in serum. LODs from sera spiked with quantitated viral stocks were 2.6 × 102 GEQ/Rn for ZIKV, 2.2 × 101 GEQ/Rn for DENV-1, 9.4 × 100 GEQ/Rn for DENV-2, 2.3 × 102 GEQ/Rn for DENV-3, 1.4 × 103 GEQ/Rn for DENV-4, 2.7 × 102 GEQ/Rn for CHIKV, and 1.05 × 101 GEQ/Rn for WNV. Conclusions The CII-ArboViroPlex rRT-PCR assay is a quantitative one-step pentaplex rRT-PCR assay for the molecular detection and differential diagnosis of ZIKV, DENV, CHIKV, WNV and a human housekeeping gene control in a single- PCR reaction.

  • Hepatitis E virus infections in Europe
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-09-08
    Jacques Izopet, Pauline Tremeaux, Olivier Marion, Marion Migueres, Nicolas Capelli, Sabine Chapuy-Regaud, Jean-Michel Mansuy, Florence Abravanel, Nassim Kamar, Sébastien Lhomme

    Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide. The systematic use of improved tools for diagnosing and genotyping has completely changed our understanding of the epidemiology and clinical consequences of HEV infection. Most cases of HEV in Europe arise from infected animals such as pigs, wild boar, deer and rabbits. Zoonotic HEV genotypes (HEV genotypes 3-8) are mainly food-borne or transmitted by direct contact, but recent data suggest that infection can also be water-borne or even iatrogenic throught contamined blood products. HEV-3 is the most prevalent genotype in Europe but the geographic distributions of the 3 major clades and subgenotypes (HEV-3abjkchi, HEV-3efg, and HEV-3ra) differ. Most HEV-3 infections are asymptomatic but they can result in severe acute hepatitis in patients with chronic liver disease, chronic hepatitis in immunocompromised patients, and to extra-hepatic manifestations. Despite more frequent reports of symptomatic hepatitis E cases across Europe, systems for monitoring HEV infections vary greatly. Severe HEV-associated illnesses, hospitalizations and deaths are probably underestimated. The seroprevalence and incidence of locally acquired hepatitis E varies between and within European countries and over time. The precise origin of these variations is uncertain but may be linked to environmental factors or the degree to which HEV contaminates the human food chain. Collaborative initiatives such as the establishment of the One Health platform for HEV sequences (HEVnet database) will be very useful for a better understanding of the epidemiology of HEV in Europe and the development of effective prevention strategies.

  • Host genetic background affects the course of infection and treatment response in patients with chronic hepatitis B
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-09-03
    Magda Rybicka, Anna Woziwodzka, Tomasz Romanowski, Alicja Sznarkowska, Piotr Stalke, Marcin Dręczewski, Krzysztof Piotr Bielawski

    Background Hepatitis B<-- --> virus (HBV) utilizes proteins encoded by the host to infect hepatocytes and replicate. Recently, several novel host factors have been identified and described as important to the HBV lifecycle. The influence of host genetic background on chronic hepatitis B (CHB) pathogenesis is still poorly understood. Objectives Here, we aimed to investigate the association ofNTCP, FXRα, HNF1α, HNF4α, and TDP2 genetic polymorphisms with the natural course of CHB and antiviral treatment response. Study design We genotyped 18 single-nucleotide polymorphisms using MALDI-TOF mass spectrometry in 136 patients with CHB and 100 healthy individuals. We investigated associations of the selected polymorphisms with biochemical, serological and hepatic markers of disease progression and treatment response. Results No significant differences in genotypic or allelic distribution between CHB and control groups were observed. Within TDP2, rs3087943 variations were associated with treatment response, and rs1047782 modified the risk of advanced liver inflammation. Rs7154439 within NTCP was associated with HBeAg seroconversion after 48 weeks of nucleos(t)ide analogue treatment. HNF1α genotypes were associated with treatment response, liver damage and baseline HBeAg presence. HNF4α rs1800961 predicted PEG-IFNα treatment-induced HBsAg clearance in long-term follow up. Conclusions This study indicates host genetic background relevance in the course of CHB and confirms the role of recently described genes for HBV infection. The obtained results might serve as a starting point for validation studies on the clinical application of selected genetic variants to predict individual risks of CHB-induced liver failure and treatment response.

  • Multiplex analysis of Human Polyomavirus diversity in kidney transplant recipients with BK virus replication
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-27
    Yilin Wang, Robert Strassl, Ilkka Helanterä, Stephan W. Aberle, Gregor Bond, Klaus Hedman, Lukas Weseslindtner

    Background While the pathogenicity of the two initially identified Human Polyomaviruses (HPyVs), BK Virus (BKPyV) and JC Virus (JCPyV) has been intensely studied, there is only limited data, on whether the occurrences of the recently discovered HPyVs correlates with high level BKPyV replication and progression towards Polyomavirus associated nephropathy (PVAN). Methods Therefore, we performed a comprehensive longitudinal genoprevalence analysis of 13 HPyVs using a novel multiplex assay including 400 serum and 388 urine samples obtained from 99 kidney transplant recipients (KTRs), grouped by quantitative BKPyV DNA loads and evidence of manifest BKPyV associated disease (histologically verified PVAN, high urinary decoy cell levels and concurrent decrease of renal function). Results In total, 3 different non-BKPyV/JCPyV HPyVs, Human Polyomavirus 9, Merkel Cell Polyomavirus (MCPyV) and Trichodysplasia Spinulosa associated Polyomavirus were detected in 11 blood and 21 urine samples from 21 patients. Although DNAemia of these viruses occurred more frequently during high level BKPyV DNAemia and PVAN, the increase of the detection frequency due to progression of BKPyV replication did not reach statistical significance for blood samples. The positive detection rate of MCPyV in urine, however, was significantly higher during BKPyV DNAemia in 19 KTRs of our cohort who suffered from histologically verified PVAN (p = 0.005). In one individual with PVAN, continuous long-term shedding of MCPyV in urine was observed. Conclusion In our cohort the recently discovered HPyVs HPyV9, TSPyV and MCPyV emerged in blood from KTRs with variable kinetics, while detection of MCPyV DNAuria occurred more frequently during BKPyV DNAemia in patients with PVAN.

  • Influenza virus specific CD4+ and CD8+ T cell mediated immunity induced by infection and vaccination
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-24
    Janina Jansen, Thomas Gerlach, Husni Elbahesh, Guus F. Rimmelzwaan, Giulietta Saletti

    Influenza A and B virus infections are a major cause of respiratory disease in humans and are responsible for substantial morbidity and mortality worldwide. Vaccination against influenza mainly aims at the induction of virus neutralizing serum antibodies, which are an important correlate of protection provided that the antibodies match the strains causing the outbreaks antigenically. In addition, virus-specific T cells are known to contribute to protective immunity to influenza virus infections by limiting duration and severity of the disease. As the majority of virus-specific T cells recognize epitopes located in relatively conserved proteins, like the Nucleoprotein and Matrix 1 protein, they display a high degree of cross-reactivity with a wide range of influenza viruses, including newly emerging viruses of alternative subtypes. Advancing our understanding of influenza virus-specific T cell responses and their role in protective immunity against influenza will aid the rational design of novel vaccines that could induce robust, broad and long-lasting immune responses. Here, we discuss the contribution of influenza virus-specific CD4+ and CD8 + T cells to protective immunity against influenza infection and the requirements and strategies for their induction by natural infection or vaccination, especially in children.

  • Treatment as Prevention enrolling at least 75 percent of individuals on ART will be needed to significantly reduce HIV prevalence in a HIV cohort
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-24
    Hélène Jeulin, Eliette Jeanmaire, John M. Murray, Brice Malve, Marie André, Hugues Melliez, Jean-Philippe Lanoix, Laurent Hustache-Mathieu, Marialuisa Partisani, François Goehringer, Thierry May, Evelyne Schvoerer

    Background “Treatment as Prevention” (TasP) aims to reduce new HIV infections through higher enrolment on suppressive antiretroviral therapy (ART). Objectives We studied the current epidemic and possible impact of TasP in a French HIV cohort including MSM and migrant subjects. Study design Socio-demographic, clinical and laboratory variables were collected during the follow-up of 6995 HIV-infected patients. The numbers of individuals living with HIV in each year were estimated from diagnoses up to that year minus recorded deaths. Patients were classified according to gender, transmission mode, country of birth and treatment status. Results The cohort includes 6995 individuals diagnosed from 1985 to 2015, of whom 72% were men. Unprotected sexual intercourse was the main mode of transmission. Women were more likely to be migrants (45% versus 13%), whereas men were more likely to have been born in France (52% versus 27%). Diagnoses were more correlated with untreated than treated prevalence in each group. MSM diagnoses was strongly correlated to untreated prevalence whatever the country of birth (p < 0.0001). However, heterosexual diagnoses were better correlated with prevalence within individual country groups (b = 0.29 female diagnoses/year per untreated male born in France, compared to b = 0.73 for foreigners). Using these transmission rates, mathematical modelling estimated that enrolling 75% of untreated individuals per year would decrease diagnoses ten-fold by 2021. Conclusions Enrolling at least 75% of individuals on ART is necessary to substantially impact numbers of new HIV infections in this cohort. Treatment as prevention will actually be effective to reduce HIV prevalence.

  • Blood parameters in fetuses infected with cytomegalovirus according to the severity of brain damage and trimester of pregnancy at cordocentesis
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-20
    Ameth Hawkins-Villarreal, Ana L. Moreno-Espinosa, Elisenda Eixarch, M. Angeles Marcos, Raigam J. Martinez-Portilla, Laura Salazar, Laura Garcia-Otero, Marta Lopez, Antoni Borrell, Francesc Figueras, Anna Goncé

    Background and objective Cytomegalovirus (CMV) remains a major cause of congenital infection and disease. During pregnancy, symptomatic cases can be detected through ultrasound (US) features, nevertheless, prognostic assessment is difficult. The aim of this study was to assess the predictive value of specific blood parameters in CMV infected fetuses. Study design Twenty-eight CMV-infected fetuses in which a cordocentesis had been performed were included. Fetuses were considered severely or mildly affected according to prenatal US/MRI brain damage. Fetal blood parameters were assessed for the prediction of severe brain abnormalities, and compared according to the trimester of pregnancy. Logistic regression and receiver operating curve analysis were performed. Results Thrombocytopenia (≤100,000/mm3; p:0.03) and high levels of gamma-glutamyl transpeptidase (GGT) (≥151 IU/L; p:0.02) signaled severity. For the prediction of brain damage, GGT levels ≥ 183 UI/l achieved 71% sensitivity, 83% specificity (AUC: 0.78), and OR of 2.05 (95% CI: 1.22-3.43) per 100 IU/l increase, adjusted for gestational age. However, thrombocytopenia (91% vs 50%; p: 0.04), β2 microglobulin >10.4 mg/L (60% vs 0% p: 0.03), CMV-DNA >50,000 copies/ml (80% vs 25%; p: 0.02), and positive IgM (70% vs 17%; p: 0.04) were observed significantly more often in severely damaged fetuses sampled ≤28 weeks than thereafter. Conclusion In CMV infected fetuses, thrombocytopenia and high levels of GGT are associated with severe US/MRI brain abnormalities. Nevertheless, among severely affected fetuses, blood parameters, with exception of GGT, change according to gestational age. Fetal blood could be less predictive of brain damage in the third trimester.

  • Cytotoxic CD4+ T-cells during HIV infection: targets or weapons?
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-16
    Alexandra Sanchez-Martinez, Federico Perdomo-Celis, Liliana Acevedo-Saenz, Maria T. Rugeles, Paula A. Velilla

    Classically, CD4+ T-cells have been referred as cytokine-producing cells and important players in immune responses by providing soluble factors that potentiate several effector immune functions. However, it is now evident that CD4+ T-cells can also elaborate cytotoxic responses, inducing apoptosis of target cells. Cytotoxic CD4+ T cells (CD4+ CTLs), exhibit cytolytic functions that resemble those of CD8+ T-cells; in fact, there is evidence suggesting that they may have a role in the control of viral infections. In this article, we discuss the role of CD4+ CTLs during HIV infection, where CD4+ CTLs have been associated with viral control and slow disease progression. In addition, we address the implication of CD4+ CTLs in the context of antiretroviral therapy and the partial reconstitution of CD8+ T-cells effector function.

  • Automation and standardisation of clinical molecular testing using pcr.ai – a comparative performance study
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-14
    AR MacLean, R Gunson

    Background We undertook a prospective clinical study to evaluate PCR.Ai’s (www.pcr.ai) accuracy and impact when automating the manual data-analysis and quality control steps associated with routine clinical pathogen testing using real-time PCR (qPCR). Objectives We evaluated the impact of PCR.Ai when used as the final interpretation/verification step for routine in-house qPCR tests for respiratory pathogens and for norovirus for a total of 22,200 interpretations. Study Design We compared PCR.Ai to our existing manual interpretation, to determine accuracy and hands-on time savings. PCR.Ai was accurate. Results and Conclusions There was 100% concurrence between validated respiratory virus and norovirus detection by our manual routine analysis method and PCR.Ai. Furthermore, there were significant routine savings with PCR.Ai of 45 minutes/respiratory run and 32 minutes/norovirus run. Our conclusion is that PCR.Ai is a highly accurate time-saving tool that reduces complexity of qPCR analysis and hence the need for specialists and hands-on time. It demonstrated capabilities to enable us to get results out more quickly with lower costs and less risk of errors.

  • Detection of BKV Encoded Mature MicroRNAs in Kidney Transplant Patients: Clinical and Biologic Insights
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-12
    Yuchen Huang, Gang Zeng, Parmjeet S. Randhawa

    Background Polyomavirus BK (BKV) encodes two mature miRNAs that regulate the viral life cycle. Objectives This study investigated the autoregulatory and immunomodulatory effects of these miRNAs that have been defined in culture systems, but subject to only limited exploration in clinical samples. Methods BKV-miR-B1-5p, BKV-miR-BJ1-3p, BKV DNA and BKV VP-1 mRNA levels were measured in 32 paired obtained plasma & urine samples from kidney transplant patients with (a) early stage infection manifesting as viruria, and (b) later stage infections complicated by viremia. Results All patients showed abundant urine miRNAs (7.84E + 02-1.91E + 06 copies/ml, but plasma miRNA was below the limit of detection. There was no statistically significant difference in urinary miRNA levels between viruric and viremic patients. Median 5p miRNA load was 4-6 logs lower than the BKV genomic load. Higher miRNA levels in the urine were associated not with lower but higher urinary viral loads. BKV preferentially used the 3p miRNA for its interactions with host cell mRNAs. The mean ratio of 5p/3p in patients with viruria was 0.09, and 0.03 in patients with viremia. Conclusions The data suggest that immune evasion functions of BKV miRNAs over-ride the negative autoregulatory feedback effects in kidney transplant patients with active viral replication.

  • HPV16 whole genome minority variants in persistent infections from young Dutch women
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-12
    Sonja Lagström, Pascal van der Weele, Trine Ballestad Rounge, Irene Kraus Christiansen, Audrey J. King, Ole Herman Ambur

    Background Chronic infections by one of the oncogenic human papillomaviruses (HPVs) are responsible for near 5% of the global cancer burden and HPV16 is the type most often found in cancers. HPV genomes display unexpected levels of variation when deep-sequenced. Minor nucleotide variations (MNVs) may reveal HPV genomic instability and HPV-related carcinogenic transformation of host cells. Objectives The objective of this study was to investigate HPV16 genome variation at the minor variant level on persisting HPV16 cervical infections from a population of young Dutch women. Study design 15 HPV16 infections were sequenced using a whole-HPV genome deep sequencing protocol (TaME-seq). One infection was followed over a three-year period, eight were followed over a two-year period, three were followed over a one-year period and three infections had a single sampling point. Results and conclusions Using a 1% variant frequency cutoff, we find on average 48 MNVs per HPV16 genome and 1717 MNVs in total when sequencing coverage was >100 × . We find the transition mutation T > C to be the most common, in contrast to other studies detecting APOBEC-related C > T mutation profiles in pre-cancerous and cancer samples. Our results suggest that the relative mutagenic footprint of HPV16 genomes may differ between the infections in this study and transforming lesions. In addition, we identify a number of MNVs that have previously been associated with higher incidence of high-grade lesions (CIN3+) in a population study. These findings may provide a starting point for future studies exploring causality between emerging HPV minor genomic variants and cancer development.

    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-02
    Sara Cruz, Carla Campos, Mafalda Timóteo, Ana Tavares, Maria São José Nascimento, Rui Medeiros, Hugo Sousa

    Background In developed countries, Hepatitis E virus (HEV) infections, especially by HEV-3, are frequently associated with asymptomatic infection or self-limiting acute hepatitis, although it has been described as a cause of chronic infection, especially in immunocompromised hots. Hematopoietic stem cell transplant (HSCT) recipients have been recognized as an important risk group for HEV infection due to their prolonged immunosuppression state. Objectives We aimed to perform a systematic review of published data to evaluate HEV infection prevalence among HSCT recipients. Study Design Literature search was performed concerning published manuscripts regarding 'hepatitis E virus AND stem cell transplantation' following the Preferred Reporting of Systematic Reviews and MetaAnalyses (PRISMA) guidelines. Statistical analysis was performed using the MetaXL software to estimate the overall prevalence of HEV infection according to the different diagnostic approaches (HEV RNA and anti-HEV IgM and/or IgG detection). Results A total of 7 manuscripts were included for data analysis, with 6 studies performed in Europe and 1 study in China. Regarding HEV RNA detection, the overall HEV infection prevalence was 1.50% (95% CI: 0.70- 2.60). The overall anti-HEV IgM seroprevalence was 2.00% (95% CI: 0.30- 4.50), and anti-HEV IgG was 11.4% (95% CI: 1.80-26.3). Conclusions This systematic review reveals that the overall prevalence of HEV infection in HSCT patients differ according to the diagnostic, thus emphasizing the need of more studies to increase the data regarding prevalence and incidence in HSCT recipients.

  • Self-collected compared with professional-collected swabbing in the diagnosis of influenza in symptomatic individuals: A meta-analysis and assessment of validity
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-02
    Christopher P. Seaman, Luong Thi Tuyet Tran, Benjamin J. Cowling, Sheena G. Sullivan

    Self-collected nasal swabs offer a cheaper alternative to professional-collected swabs for influenza testing. However, the diagnostic accuracy of self-collection has not been quantitatively reviewed. We identified 14 studies that compared diagnostic accuracy of self-collected to professional-collected swabs in influenza symptomatic individuals. Self-collected swabs were found to be highly acceptable, simple and comfortable to use. Data from nine studies were meta-analyzed. Pooled sensitivity was 87% (95% CI: 80%, 92%) and specificity was 99% (95% CI: 98%, 100%), compared to professional-collected swabs in the diagnosis of influenza. Pooled sensitivity and specificity estimates were used to assess the potential bias that would be introduced in studies had self-collected rather than professional-collected samples been used. While self-collected swabbing should not replace the role of clinical testing, our findings support the use of self-collected swabs for influenza research and surveillance. This method will be an important tool for evaluating novel influenza vaccines and vaccination strategies.

  • Reliability of direct varicella zoster virus loop-mediated isothermal amplification method for rapid diagnosis of breakthrough varicella
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-02
    Yuki Higashimoto, Yoshiki Kawamura, Ayumi Kuboshiki, Fumihiko Hattori, Hiroki Miura, Naoko Nishimura, Takao Ozaki, Masaru Ihira, Tetsushi Yoshikawa

    Background Since <-- -->patients with breakthrough varicella (BV) have mild symptoms, clinical diagnosis is difficult. In high vaccine coverage area, as BV occurs sporadically, point of care test is required for controlling varicella outbreak. In this study, the reliability of varicella zoster virus (VZV)-loop mediated isothermal amplification (LAMP) was evaluated for the rapid diagnosis of BV. Study design A total of 328 swab samples collected from patients with suspected varicella were analyzed. For the laboratory diagnosis of varicella, VZV real-time PCR was carried out using DNA extracted from swab samples. Swab samples without DNA extraction were used for VZV-LAMP(direct-LAMP). Results VZV infection was diagnosed by real-time PCR in 285 cases, including 105 natural varicella cases and 180 BV cases. VZV DNA was detected in 250 (87.8%) of the 285 cases by direct-LAMP. The presence and duration of fever, number of skin eruptions, and VZV DNA load were significantly lower in BV than natural varicella. The sensitivity of direct-LAMP for the diagnosis of varicella and BV was 93.3% and 84.4%, respectively. Conclusions Direct LAMP was considered to be useful for rapid diagnosis of BV as it has several advantages such as low cost, ease and rapidity, as compared to real time PCR.

  • Improved Detection of Early Acute, Late Acute, and Occult Hepatitis B Infections by an Increased Sensitivity HBsAg Assay
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-08-02
    Mary C. Kuhns, Vera Holzmayer, Anne L. McNamara, Eva Sickinger, Jan Schultess, Gavin A. Cloherty

    Background Hepatitis B surface antigen (HBsAg) is the primary marker for diagnosis of acute and chronic hepatitis B. Although HBsAg assays have undergone continuous improvement, gaps remain in the detection of early and late acute infection and occult hepatitis B infection (OBI). Objectives The performance of a prototype, improved sensitivity HBsAg assay run on the ARCHITECT and Alinity instruments was evaluated for detection of early and late acute infection and OBI. Study design Seventy seven early acute samples [positive only for hepatitis B viral DNA (HBV DNA)], twelve seroconversion panels spanning late acute infection, and 101 occult samples (HBsAg negative, positive for HBV DNA and anti-HBc) were tested with the prototype assay and ARCHITECT HBsAg Qualitative II. HBsAg gene sequencing was performed to determine genotype and mutations in the immunodominant region. Results Compared with ARCHITECT HBsAg Qualitative II, the prototype assay showed increased detection of NAT yield samples (28/77, 36.4%,), late acute samples (>13 days longer detection of HBsAg for 6/12 panels), and OBI samples (11/101, 10.9%). HBsAg sequence data were obtained for 62 samples. Genotypes represented were A1, A2, B2, B4, C1, C2, C5, D3, E, and H. HBsAg escape mutations were found in 4.8% of NAT yield and 38.9% of OBI samples sequenced. Prototype assay values for 188 samples were equivalent on the ARCHITECT and Alinity instruments. Conclusions The new prototype HBsAg assay will be of diagnostic value in providing improved detection of early acute, late acute, and occult HBV infections.

  • Epidemiology of Aichi virus in fecal samples from outpatients with acute gastroenteritis in Northwestern Spain
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-30
    Enrique Rivadulla, Miguel F. Varela, Jesús L. Romalde

    Background In recent years, Aichi virus (AiV) has been involved in acute viral gastroenteritis outbreaks. However, the common pathogenesis of AiV releases more in subclinical infections underestimating the impact of AiV in human health. Objectives The present study describes the presence and genetic diversity of AiV in patients with gastroenteritis in Northwestern Spain. Study design: A total of 2,667 stool samples, obtained between July 2010 and June 2011, from diarrheic outpatients were studied for detection and molecular characterization of AiV using PCR techniques followed by sequencing and phylogenetic analyses. Results The virus was detected in 124 (5.0%) of the samples among all age groups. Coinfections were also detected, from the 124 positive samples, 72 (58.1%) were positive only for AiV, whereas mixed contaminations with Norovirus genogroup I or genogroup II, Sapovirus, or other enteric pathogens were detected in 52 (41.9%) samples. A total of 70 positive samples could be genotyped, being characterized as genotype A (58.6%) or B (41.4 %). AiV was detected from August to April, being the highest number of AiV positive samples detected during autumn and winter seasons. Conclusions This survey remarks the importance of emerging enteric viruses in patients who require medical assistance, and offers more information about the real importance of AiV as gastroenteritis agent.

  • Clinical Validation of the FluChip-8G Influenza A+B Assay for Influenza Type and Subtype Identification
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-26
    Rebecca H. Blair, Erica D. Dawson, Amber W. Taylor, James E. Johnson, Amelia H. Slinskey, Kelly O’Neil, Andrew W. Smolak, Evan Toth, Kyle Liikanen, Robert S. Stoughton, Catherine B. Smith, Sarah Talbot, Kathy L. Rowlen

    Background The FluChip-8G Influenza A+B Assay is a multiplexed influenza RT-PCR and microarray-based assay with same day turnaround time, developed to subtype seasonal A viruses (H1N1pdm2009 and H3N2), distinguish B viruses as Yamagata or Victoria lineage, and is the only FDA cleared assay capable of positive identification of a wide variety of A subtypes as “non-seasonal” A viruses from human nasal specimens. Objective To evaluate clinical performance of the FluChip-8G Influenza A+B Assay for detection of seasonal influenza viruses in nasal and nasopharyngeal swab specimens, and to evaluate performance for detection of non-seasonal influenza viruses using contrived samples. Study Design For seasonal viruses, a multisite study of the FluChip-8G Influenza A+B Assay using prospectively and retrospectively collected nasal and nasopharyngeal swabs was performed using the FDA-cleared CDC Human Flu Dx Panel as the comparator assay. For non-seasonal viruses, testing was performed at a single site using contrived samples from 100 unique non-seasonal strains representing 41 subtypes. Results Sensitivity (95% CI) and specificity (95% CI) for each target group, respectively, from results of 1689 clinical specimens were: seasonal H1N1pdm2009: 96.4% (87.9-99.0), 99.3% (98.8-99.6), seasonal H3N2: 91.8% (87.7-94.7), 99.7% (99.2-99.9), Influenza B Victoria: 100% (94.0-100.0), 99.9% (99.6-100.0), and Influenza B Yamagata: 95.6% (89.2-98.3), 99.9% (99.6-100.0). The sensitivity and specificity from contrived influenza A non-seasonal viruses was determined to be 99.0% (94.6-99.8) and 100% (96.7-100.0). Conclusion The FluChip-8G Influenza A+B Assay has robust sensitivity and specificity for detecting and identifying all target virus groups, including non-seasonal influenza A, with same day results.

  • Analytical performance evaluation and enhancement of the ADVIA Centaur® HIV Ag/Ab Combo assay
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-25
    Baptiste Demey, Cédric Usureau, Paul Boillod, Sandra Bodeau, Catherine François, Catherine Roussel, Gilles Duverlie, Sandrine Castelain, Etienne Brochot

    Background Fourth-generation immunoassays (such as the ADVIA Centaur® HIV Ag/Ab Combo (CHIV) assay) have improved the early diagnosis of human immunodeficiency virus (HIV), and their sensitivity and specificity usually exceed 99%. In regions with a low prevalence of HIV infection, however, the regular occurrence of false positives interferes with a medical laboratory’s workflow. The additional reagent and staff costs associated with false positives can nevertheless be avoided or reduced by gaining a better knowledge of the CHIV assay’s performance. Objectives/Study design To improve our HIV diagnosis strategy, we retrospectively analyzed all the Centaur® CHIV assays and confirmatory tests performed at Amiens University Medical Center between 2012 and 2018. We used open-source machine learning software to process this large database, develop a predictive model, and identify a new cut-off for Centaur® CHIV index interpretation. Results A total of 56682 HIV serological assay results were analyzed. The results of the CHIV assay were initially reactive or indeterminate for 449 samples. After p24 antigen and/or immunoblotting, there were 171 (38%) false positives and 278 (62%) confirmed true positives. The application of a cut-off of 2.12 led to reclassification of 130 of the 171 false positives as true negatives. Combining our predictive model with medical record analysis reduced the number of false positive CHIV assay results from 171 to 12. Conclusions The efficiency of the Centaur® CHIV assay can be increased by adjusting its cut-off for positivity. This adjustment may reduce the number of unnecessary confirmatory tests and accelerate the delivery of HIV test results.

  • Burden of rotavirus infection in hospitalized elderly individuals prior to the introduction of rotavirus vaccination in Sweden
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-18
    Thomas Beck-Friis, Maria Andersson, Lars Gustavsson, Magnus Lindh, Johan Westin, Lars-Magnus Andersson

    Background Rotavirus gastroenteritis (GE) in the elderly has been much less studied than in children. Objectives The aim of this study was to determine the morbidity and mortality for elderly hospitalized patients with rotavirus GE prior to the introduction of rotavirus vaccination in Sweden, and to investigate the epidemiology of rotavirus genotypes in these patients. Study design All patients 60 years or older who were hospitalized at Sahlgrenska University Hospital, Gothenburg, Sweden, and were rotavirus positive in a clinical diagnostic test from 2009 to 2016, were included. Medical records were reviewed and rotavirus genotyping real-time PCR was performed. Results One hundred and fifty-nine patients were included, corresponding to an annual incidence of hospitalization due to rotavirus GE of 16/100 000 inhabitants aged 60 years or older. G2P[4] was the most common genotype, followed by G1P[8] and G4P[8]. The majority of patients had community-onset of symptoms and no or few pre-existing health disorders. Four patients (2.5%) died within 30 days of sampling. Patients with hospital-onset rotavirus GE had a longer median length of stay following diagnosis compared with patients with community-onset of symptoms (19 vs. 5 days, p = 0.001) and higher 30-day mortality (8.6% (3/35) vs. < 1% (1/124), p = 0.03). Conclusions Hospitalization due to rotavirus GE among the elderly seems to mainly affect otherwise healthy individuals and is associated with low 30-day mortality.

  • Enhancing the concordance of two commercial dengue IgG ELISAs by exchange of the calibrator sample
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-05
    Tom Schüttoff, Awadalkareem Adam, Sven Reiche, Christian Jassoy

    Background : Dengue IgG testing is being recommended before dengue vaccination. Presently, the diagnostic method of choice is the dengue IgG ELISA. Objective : Determine the test performance and concordance of two commercial dengue IgG ELISA kits. Study design : A diagnostic study to examine the sensitivity, specificity, accuracy and concordance of the Panbio Dengue Indirect IgG ELISA kit and the NovaLisa Dengue IgG ELISA kit. Sera (483) were from dengue-endemic regions in Sudan. Test performance characteristics were determined when tests were applied as indicated in the test kits and when the Panbio calibrator sample was used for both tests. Results : The sensitivity of the Panbio and the NovaLisa ELISA were 91.1% and 99.0% and the specificity was 79.4% and 50.9%. The Panbio test was slightly more accurate (87.5% compared with 84.0%). Quantitative measurement readings of the tests correlated. The calibrator samples gave different cutoff values. Replacing the NovaLisa cutoff sample with the Panbio calibrator sample raised the accuracy of the NovaLisa assay to 88% and increased the concordance of the tests from 82.8 to 93%. Conclusions : The study shows that the two dengue IgG ELISAs differed clearly in sensitivity and specificity and gave discordant results for 17.2% of the sera. For the most part the discrepancy depended on the calibrator sample. The findings indicate that an optimized dengue IgG calibrator standard can enhance accuracy and concordance of commercial dengue ELISAs. An optimized standard calibrator would make dengue IgG seroprevalence testing more reliable.

  • Comparative analysis of Four Sample-to-Answer Influenza A/B and RSV Nucleic Acid Amplification Assays Using Adult Respiratory Specimens
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-04
    Dithi Banerjee, Neena Kanwar, Ferdaus Hassan, Kamani Lankachandra, Rangaraj Selvarangan

    Background The use of Sample-to-answer (STA) platforms for the detection of influenza A/B and respiratory syncytial virus (RSV) have greatly improved patient care. These diagnostic assays based on nucleic acid amplification are rapid, accurate and relatively easy to perform. Objectives We compared four such platforms for detecting FluA, FluB, and RSV from adult respiratory specimens: Hologic Panther Fusion® Flu A/B/RSV (Fusion), Cobas® Influenza A/B & RSV (Liat), Luminex Aries® Flu A/B & RSV (Aries), and Diasorin SimplexaTM Flu A/B & RSV (Simplexa). Study Design Nasopharyngeal (NP) swabs (n = 224) from adults were tested on these platforms and results were compared to Center for Disease Control and Prevention recommended real-time RT-PCR assay for influenza A/B and RSV. Subtyping for FluA and FluB was performed for discrepant analysis where applicable. Results Of the 82 FluA, 26 FluB, 15 RSV-positive specimens tested, the positive and negative percentage agreements (PPA and NPA respectively) for FluA detection were 100/100 (Fusion), 95.1/100 (Liat), 92.5/100 (Aries), and 84.1/99.3 (Simplexa); PPA and NPA for FluB detection were 92.3/99.5 (Fusion), 96/99.5 (Liat), 100/99.5 (Aries), and 80.8/100 (Simplexa); and for RSV detection were 100/100 (Fusion), 100/100 (Liat), 88.6/99.5 (Aries), and 73.3/100 (Simplexa). 82 confirmed FluA included 23 pH1N1 and 57 H3N2 strains with 2 strains remaining untyped. Of the 26 confirmed FluB, 25 were of the Yamagata lineage and 1 of unknown lineage. Conclusion Only 2 STA platforms demonstrated >95% PPA for the detection of all three targets while all the 4 platforms demonstrated >95% NPA for FluA, FluB and RSV.

  • Interdependence of diagnostics and epidemiology, a European perspective
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-04
    Mart K. van Genne, Randy Poelman, Hayley Cassidy, Nico E.L. Meessen, Hubert G.M. Niesters

    For some well-known pathogens like influenza or RSV, diagnostic and epidemiological data is available and continuously complement each other. For most other pathogens however, data is not always available or severely delayed. Furthermore, clinical data is needed to assess the burden of disease, which will enhance awareness and help to gain knowledge on emerging pathogens. In this position paper, we discuss the interdependence of diagnostics and epidemiology from a European perspective. In 2004, the European Centre for Disease Prevention and Control (ECDC) was founded to coordinate European wide surveillance and control. At present however, the ECDC still relies on university hospitals, public health institutions and other diagnostic institutions. Close collaboration between all stakeholders across Europe is therefore complex, but necessary to optimize the system for the individual patient. From the diagnostic side, data on detected pathogens should be shared with relevant health institutions in real-time. From the public health side, collected information should be made accessible for diagnostic and clinical institutions in real-time. Subsequently, this information needs to be disseminated across relevant medical disciplines to reach its full potential.

  • Clinical manifestations and outcomes of respiratory syncytial virus infection in adult hospitalized patients
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-07-03
    Benjamas Chuaychoo, Sopita Ngamwongwan, Bualan Kaewnaphan, Niracha Athipanyasilp, Navin Horthongkham, Wannee Kantakamalakul, Nisa Muangman

    Background Respiratory syncytial virus (RSV) is an important virus found in adult hospitalized patients. Objectives To study the clinical outcomes of hospitalized patients aged ≥ 15 years and diagnosed with RSV infection. Study design Both retrospective and prospective cohort studies were conducted at a university hospital between May 2014 and December 2015. Results: RSV was detected in 86 of 1562(5.5%) adult hospitalized patients suspected of respiratory viral infection. Sixty-nine patients were included in the study. RSV was detected by RT-PCR (82.6%), IFA (10.1%), and both RT-PCR and IFA (7.3%). Most patients (87.0%) were aged ≥ 50 years. Cardiovascular diseases, pulmonary diseases, immunocompromised hosts, and diabetes were the major comorbidities. The common manifestations were cough (92.8%), dyspnea (91.3%), sputum production (87.0%), tachypnea (75.4%), wheezing (73.9%), and fever (71.0%). Fifty- five patients (79.7%) were diagnosed with pneumonia. Hypoxemia (SpO2 ≤ 92%) was found in 53.6 % patients. Twenty-five of 69(36.2%) patients developed respiratory failure and required ventilatory support. Cardiovascular complications were found in 24.6% of patients. Congestive heart failure, acute myocardial infarction (MI), new atrial fibrillation, and supraventricular tachycardia were found in 9(13.0%), 7(10.1%), 4(5.8%), and 3(4.3%) of 69 patients, respectively. Overall mortality was 15.9%. Pneumonia (81.8%) and acute MI (18.2%) were the major causes of death. Conclusions Most adult hospitalized patients with RSV infection were of advanced age and had comorbidities. Cardiopulmonary complications were the major causes of death. Management and prevention of RSV infection in these vulnerable groups are necessary.

  • The New Xpert HCV Viral Load Real-time PCR Assay Accurately Quantifies Hepatitis C virus RNA in Serum and Whole-blood Specimens
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-06-22
    Mélanie Wlassow, Lila Poiteau, Françoise Roudot-Thoraval, Isabelle Rosa, Alexandre Soulier, Christophe Hézode, Valérie Ortonne, Jean-Michel Pawlotsky, Stéphane Chevaliez

    Background: Sensitive and accurate hepatitis C virus (HCV) RNA detection and quantification are essential to diagnose and monitor the virological response to antiviral treatment and the emergence of resistance. Objective and study design: The aim of this study was to assess the ability of the new Xpert HCV Viral Load assay to accurately detect and quantify HCV RNA in serum and in whole blood collected on dried blood spot (DBS). Serum and whole blood from a large series of patients chronically infected with different HCV genotypes were tested in parallel for HCV RNA detection and quantification. Results: A significant relationship between HCV RNA levels measured with the Xpert HCV Viral Load assay and the two commercial real-time PCR comparators (Abbott RealTime HCV test and Cobas AmpliPrep/Cobas Taqman HCV 2.0 test) was found in serum as well as in whole blood specimens. Conclusions: The Xpert HCV Viral Load assay accurately quantifies HCV RNA regardless of the HCV genotype and can thus confidently be used to detect active HCV infection in serum and in whole blood specimens.

  • Seasonal influenza-associated intensive care unit admission and death in tropical Singapore, 2011-2015
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-06-18
    Zoe X.Z. Zhang, Win Mar Kyaw, Hanley J. Ho, Min Zhi Tay, Hongjie Huang, Aung Aung Hein, Angela Chow

    Background: Seasonal influenza can cause severe illness leading to intensive care unit (ICU) admission and death. Objective: To define the clinical and epidemiological features of severe seasonal influenza infection and factors associated with mortality. Study design: A retrospective review was conducted on all patients with laboratory-confirmed influenza infection who were either admitted into the ICU or died in the two largest tertiary hospitals in Singapore from 2011-2015. Results: Of 520 patients included in our study, 423 (81.3%) had influenza A infection and the rest with influenza B. Of patients with influenza A infection, 70.0% (296/423) were subtyped, of whom 24.0% (71/296) had A/H1N1pdm2009 and 76.0% (225/296) had A/H3N2. The median age of patients was 72 years (IQR 61-82). Males constituted 53.1% (276/520). Median Charlson comorbidity index score was 1 (IQR 0-3). About 70% had physical or radiological evidence of pneumonia upon admission. In-hospital mortality was 58.1% (302/520). On multiple logistic regression analysis, factors positively associated with mortality were age ≥65 years (adjusted odds ratio, aOR = 3.64, 95%CI 2.21-5.99, p < 0.001), malignancy (aOR = 2.53, 95%CI 1.12-5.73; p = 0.026), and hypoalbuminemia (aOR = 2.16, 95%CI 1.26-3.73; p = 0.005), while antiviral therapy (aOR = 0.33, 95%CI 0.17-0.63; p < 0.001) and ventilation (aOR = 0.23, 95% CI 0.13-0.39; p < 0.001) were negatively associated. Conclusions: Patients with severe seasonal influenza infection were characterized by advanced age, hypoalbuminemia and presence of pneumonia on admission. Age ≥65 years, malignancy, and hypoalbuminemia were associated with increased mortality, and antiviral therapy and ventilation with decreased mortality.

  • Quantification of HIV-DNA and residual viremia in patients starting ART by droplet digital PCR: their dynamic decay and correlations with immunological parameters and virological success
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-06-14
    Claudia Alteri, Rossana Scutari, Christof Stingone, Gaetano Maffongelli, Marta Brugneti, Francesca Falasca, Salvatore Martini, Ada Bertoli, Ombretta Turriziani, Loredana Sarmati, Nicola Coppola, Massimo Andreoni, Maria Mercedes Santoro, Carlo-Federico Perno, Francesca Ceccherini-Silberstein, Valentina Svicher

    Background Accurate quantification of total HIV-DNA and residual-viremia by sensitive assays is extremely useful to optimize monitoring of ART-treated patients. Objectives To evaluate the performances of two ddPCR-based assays for HIV-DNA and residual-viremia quantification, and the correlations of pre-ART HIV-DNA with plasma HIV-RNA, CD4 + T, CD4/CD8 and virological-success (VS) during first-line ART. Study Design Plasma HIV-RNA, total HIV-DNA, CD4 + T, CD4/CD8 were evaluated at baseline of ART, at VS (viral-load <50copies/ml), and at 6 months after VS (6moVS) in 57 newly-diagnosed HIV-1 infected patients, receiving first-line modern ART. HIV-DNA (log10 copies/106CD4 + T) and residual-viremia (copies/ml) were measured with in-house ddPCR assays. Correlations were assessed by Spearman and Jonckheere-Terpstra tests. Results HIV-DNA and residual-viremia assays showed a good linear trend between the expected and obtained values (R2 = 0.9913 and 0.9945); lower limits of detection were 32 copies/106CD4 + T and 2 copies/ml, respectively. At baseline, median (IQR) plasma HIV-RNA and HIV-DNA were 4.88(4.28-5.36)log10 copies/ml and 4.00(3.36-4.51) log10 copies/106CD4 + T cells. Residual-viremia was 8(2-26) and 4(2-12) copies/ml at VS and 6moVS. Pre-ART HIV-DNA positively correlated with plasma HIV-RNA at BL (Rho = 0.708, p < 0.001), and with residual-viremia at VS (Rho:0.383,p = 0.002). Notably, higher HIV-DNA correlated with longer time to achieve VS (median[IQR],weeks: 17.8[12.3-29.0] for HIV-DNA ≥4.5 vs. 7.4[4.1-8.7] for HIV-DNA<4.5, p < 0.001). Furthermore, pre-ART HIV-DNA negatively correlated with CD4 + T and CD4/CD8 at baseline, VS and 6moVS. Conclusions Our results support the adoption of ddPCR-based assays for both HIV-DNA and residual-viremia quantifications and corroborate that pre-ART HIV-DNA is an excellent indicator in predicting viroimmunological response and VS in patients starting ART.

  • HIV-1 Drug Resistance Surveillance among Parturient Women on Anti-retroviral Therapy in the Eastern Cape, South Africa: Implications for Elimination of Mother-To-Child Transmission
    J. Clin. Virol. (IF 3.020) Pub Date : 2019-06-14
    Oladele Vincent Adeniyi, Chikwelu Larry Obi, Daniel Ter Goon, Benson Iweriebor, Anthony Idowu Ajayi, John Lambert, Anthony Okoh

    Background The emergence of HIV drug resistance poses a significant threat to achieving the goal of elimination of mother-to-child transmission. Objectives We assessed the extent and patterns of HIV-1 drug resistance mutations (DRMs) within the context of the public sector prevention of mother-to-child transmission (PMTCT) programme in the Eastern Cape, South Africa. Study design We conducted analysis of the Pol sub-genomic sequence of RNA extracted from plasma samples of women with probable virological failure at delivery between January and May 2018 from two large maternity centres in the Eastern Cape using standard protocols. Partial pol gene covering 1030bp were amplified and sequenced according to previously reported protocol. DRMs were determined by submitting the generated partial pol sequences to the Stanford drug resistance database for query on mutations associated with drug resistance in HIV viruses. We examined the correlates of DRMs using bivariate analysis. Results The age of parturient women ranged from 16 – 43 years. The majority of the parturient women were currently on Efavirenz-based regimen (first line ART) (82.5%) and had been on ART for more than 12 months (65.0%). The prevalence of DRMs was 72.5% (n = 58). The CD4 count demonstrated a negative linear association with the DRMs (p = 0.002). The predominant DRMs were K103 N (n = 43; 74.1%), M184 V (n = 28; 48.3%) and K65R (n = 11; 19%). Among the parturient women on EFV-based regimen treatment; 79.1% already had K103 N while nine patients on protease inhibitor-based regimen still harboured K103 N. The majority of the M184 V mutations were observed in parturient women on first line regimen (n = 23; 82.1%). Conclusions We found a high prevalence of DRMs in women delivering their index babies at high viral loads in the study settings. Drug resistance surveillance using point-of-care reverse transcriptase-PCR strategies for the screening of pregnant women on ART could be a game-changer in the resource-constrained settings.

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