Allison Waters

Norovirus Genotypes Present in Oysters and in Effluent from a Wastewater Treatment Plant during the Seasonal Peak of Infections in Ireland in 2010

ABSTRACT We determined norovirus (NoV) concentrations in effluent from a wastewater treatment plant and in oysters during the peak period of laboratory-confirmed cases of NoV infection in Ireland in 2010 (January to March). Weekly samples of influent, secondary treated effluent, and oysters were analyzed using real-time quantitative reverse transcription-PCR for NoV genogroup I (GI) and genogroup II (GII). The mean concentration of NoV GII (5.87 × 104 genome copies 100 ml−1) in influent wastewater was significantly higher than the mean concentration of NoV GI (1.40 × 104 genome copies 100 ml−1). The highest concentration of NoV GII (2.20 × 105 genome copies 100 ml−1) was detected in influent wastewater during week 6. Over the study period, a total of 931 laboratory-confirmed cases of NoV GII infection were recorded, with the peak (n = 171) occurring in week 7. In comparison, 16 cases of NoV GI-associated illness were reported during the study period. In addition, the NoV capsid N/S domain was molecularly characterized for selected samples. Multiple genotypes of NoV GI (GI.1, GI.4, GI.5, GI.6, and GI.7) and GII (GII.3, GII.4, GII.6, GII.7, GII.12, GII.13, and GII.17), as well as 4 putative recombinant strains, were detected in the environmental samples. The NoV GII.4 variant 2010 was detected in wastewater and oyster samples and was the dominant strain detected in NoV outbreaks at that time. This study demonstrates the diversity of NoV genotypes present in wastewater during a period of high rates of NoV infection in the community and highlights the potential for the environmental spread of multiple NoV genotypes.

Hepatitis C virus in Vietnam: high prevalence of infection in dialysis and multi-transfused patients involving diverse and novel virus variants.

Hepatitis C virus (HCV) is a genetically diverse pathogen infecting approximately 2–3% of the worlds population. Herein, we describe results of a large, multicentre serological and molecular epidemiological study cataloguing the prevalence and genetic diversity of HCV in five regions of Vietnam; Ha Noi, Hai Phong, Da Nang, Khanh Hoa and Can Tho. Individuals (n = 8654) with varying risk factors for infection were analysed for the presence of HCV Ab/Ag and, in a subset of positive specimens, for HCV RNA levels (n = 475) and genotype (n = 282). In lower risk individuals, including voluntary blood donors, military recruits and pregnant women, the prevalence of infection was 0.5% (n = 26/5250). Prevalence rates were significantly higher (p<0.001) in intravenous drug users (IDUs; 55.6%, n = 556/1000), dialysis patients (26.6%, n = 153/575) commercial sex workers (CSWs; 8.7%, n = 87/1000), and recipients of multiple blood transfusions (6.0%, n = 32/529). The prevalence of HCV in dialysis patients varied but remained high in all regions (11–43%) and was associated with the receipt of blood transfusions [OR: 2.08 (1.85–2.34), p = 0.001], time from first transfusion [OR: 1.07 (1.01–1.13), p = 0.023], duration of dialysis [OR: 1.31 (1.19–1.43), p<0.001] and male gender [OR: 1.60 (1.06–2.41), p = 0.026]. Phylogenetic analysis revealed high genetic diversity, particularly amongst dialysis and multi-transfused patients, identifying subtypes 1a (33%), 1b (27%), 2a (0.4%), 3a (0.7%), 3b (1.1%), 6a (18.8%), 6e (6.0%), 6h (4.6%), 6l (6.4%) and 2 clusters of novel genotype 6 variants (2.1%). HCV genotype 1 predominated in Vietnam (60%, n = 169/282) but the proportion of infections attributable to genotype 1 varied between regions and risk groups and, in the Southern part of Vietnam, genotype 6 viruses dominated in dialysis and multi-transfused patients (73.9%). This study confirms a high prevalence of HCV infection in Vietnamese IDUs and, notably, reveals high levels of HCV infection associated with dialysis and blood transfusion.

Molecular Epidemiological Evaluation of the Recent Resurgence in Mumps Virus Infections in Ireland

ABSTRACT Mumps is a vaccine-preventable disease; however, outbreaks have been reported in a number of countries with childhood immunization programs, particularly among young adults at the tertiary stage of education. We have retrospectively investigated the epidemiological, virological, and serological factors associated with mumps cases identified in Ireland from 2004 to 2009. Genetic analysis of mumps virus strain variability demonstrated that a single genotype, genotype G, was circulating, and it was also detected in cerebrospinal fluid samples obtained from patients with meningitis. We observed that younger individuals were disproportionately affected with neurological sequelae following mumps virus infection, and the average age of patients with mumps virus RNA detected in cerebrospinal fluid was 19.25 years (median, 19 years; range, 14 to 24 years). Our analysis showed a 4-fold rise in mumps cases in 2008-2009 and an increased incidence in infection in those ≥30 years of age. Over a 6-year period (2004 to 2009), a total of 7,805 serum samples were investigated; of this number, 1,813 (23%) were positive for mumps virus-specific IgM. We observed a strong bias for acute mumps virus infection in males compared to females (P < 10−32) that was independent of vaccination status.

Human Cytomegalovirus UL144 Is Associated with Viremia and Infant Development Sequelae in Congenital Infection

ABSTRACT Human cytomegalovirus (HCMV) strains may be genotyped based on polymorphisms that exist within the UL144 gene, which is one of 19 viral genes lost in attenuated laboratory strains. In the present study, UL144 genotypes in congenitally infected babies (congenital cytomegalovirus [cCMV]) were determined, and the relationship between the genotype, viral load, cytokine profile, and patient developmental outcome was investigated. All cCMV infections identified during 2006 and 2007 were included (n = 29). A portion of the infants were clinically assessed at birth and at 12 to 18 months postinfection for cCMV clinical sequelae (n = 18/29). The plasma viral load (PVL) was requested for 23/29 patients, and the UL144 genotype was determined (n = 27/29). The cytokine profile in patient plasma or serum was assessed (n = 20/29). UL144 genotypes A, B, and C were detected within the cCMV population at 33.3%, 29.6%, and 25.9%, respectively. UL144 A and C were associated with a high PVL (P < 0.04). Furthermore, a significant association between the developmental outcome and UL144 A and C was observed (P < 0.04). Only patients infected with UL144 B and A/B were described as having a normal clinical outcome. In addition, a significant correlation between interleukin 10 (IL-10) levels and the PVL was observed (P < 0.04); however, there was no association between the genotype and the cytokine profile. The present study determined that the specific detection of UL144 genotypes A and C was indicative of serious cCMV infection and more likely to lead to long-term cCMV-associated clinical manifestations. The inclusion of HCMV UL144 genotyping along with the recommended PVL monitoring following cCMV diagnosis may aid prediction of the clinical outcome.

Molecular epidemiology of norovirus strains circulating in Ireland from 2003 to 2004.

Since 2002, the burden of norovirus (NoV) infection in Ireland has increased. Outbreaks in institutional settings are the most common causing widespread disruption to health service delivery. This is the first national study of NoV in the Republic of Ireland and its aim was to identify the major NoV strains circulating in Ireland over a 13-month period between November 2003 and November 2004, inclusive. A prospective study screened faecal samples (n = 478) for NoV RNA. Positive samples (n = 116) were further analysed by a second PCR, targeted to the orf1/orf2 junction of the virus. Phylogenetic analysis was based on sequence alignments of this domain. GII/4 viruses represented 92.2% of sequences, 2.7% were GII/2, GII/3 and GGIIB cluster-like strains. The remaining 5.2% were of GI origin. NoV was detectable throughout the study period, although two peaks of infection were observed. The majority of infections were caused by a range of closely related GII/4 NoV strains.

Injection of new psychoactive substance snow blow associated with recently acquired HIV infections among homeless people who inject drugs in Dublin, Ireland, 2015.

In February 2015, an outbreak of recently acquired HIV infections among people who inject drugs (PWID) was identified in Dublin, following similar outbreaks in Greece and Romania in 2011. We compared drug and risk behaviours among 15 HIV cases and 39 controls. Injecting a synthetic cathinone, snow blow, was associated with recent HIV infection (AOR: 49; p=0.003). Prevention and control efforts are underway among PWID in Dublin, but may also be needed elsewhere in Europe.

Multiplex real-time PCR for the detection and quantitation of HTLV-1 and HTLV-2 proviral load: addressing the issue of indeterminate HTLV results.

BACKGROUND Routine diagnosis of Human T Lymphotropic virus (HTLV) infection is primarily serologically based; however the proportion of unresolved and indeterminate Western blot results range from 0.02% to 50% in endemic areas. OBJECTIVES To validate a sensitive in-house quantitative multiplex real-time assay (mqRT-PCR), capable of detecting and quantifying HTLV-1 and HTLV-2, and use it to differentiate unresolved serological profiles, and monitor infection in HTLV-1 infected patients. STUDY DESIGN The mqRT-PCR was designed as a single-tube assay. Quantitative results were reported as copy number of HTLV provirus per 10(6) cells and the numbers of cells were calculated based on the quantitative result for albumin, of which there are 2 copies/cell. Assay standards were amplified from HTLV-1 infected MT-2 cells and HTLV-2 transfected CEM cells. Blood samples were obtained from HTLV seropositive former blood donors. RESULTS The mqRT-PCR assay was efficient (98.8-101.2%), reproducible (coefficient of variance<5%) and sensitive to 1 copy for HTLV-1, HTLV-2 and Albumin. The assay resolved the infection profile in 16/17 patients, with undetermined subtype, all of which were reassigned as HTLV-1 infections. In addition, the average PVL detected in patients suffering from HTLV-1 associated HAM/TSP (n=23, 13,450 copies/10(6) cells) was significantly higher than those detected in asymptomatic carriers (n=21, 6665 copies/10(6) cells). CONCLUSIONS We propose a new testing algorithm for the laboratory diagnosis of HTLV infection, which includes HTLV specific mqRT-PCR for resolving HTLV serological results. Furthermore, quantitation of PVL load by real-time PCR may be useful in assessing the link between infection and disease, and in monitoring patients undergoing therapy.

Reactivation of BK polyomavirus in patients with multiple sclerosis receiving natalizumab therapy.

Natalizumab therapy in multiple sclerosis has been associated with JC polyomavirus—induced progressive multifocal leucoencephalopathy. We hypothesized that natalizumab may also lead to reactivation of BK, a related human polyomavirus capable of causing morbidity in immunosuppressed groups. Patients with relapsing remitting multiple sclerosis treated with natalizumab were prospectively monitored for reactivation of BK virus in blood and urine samples, and for evidence of associated renal dysfunction. In this cohort, JC and BK DNA in blood and urine; cytomegalovirus (CMV) DNA in blood and urine; CD4 and CD8 T-lymphocyte counts and ratios in peripheral blood; and renal function were monitored at regular intervals. BK subtyping and noncoding control region sequencing was performed on samples demonstrating reactivation. Prior to commencement of natalizumab therapy, 3 of 36 patients with multiple sclerosis (8.3%) had BK viruria and BK reactivation occurred in 12 of 54 patients (22.2%). BK viruria was transient in 7, continuous in 2 patients, and persistent viruria was associated with transient viremia. Concomitant JC and CMV viral loads were undetectable. CD4:CD8 ratios fluctuated, but absolute CD4 counts did not fall below normal limits. In four of seven patients with BK virus reactivation, transient reductions in CD4 counts were observed at onset of BK viruria: these resolved in three of four patients on resuppression of BK replication. No renal dysfunction was observed in the cohort. BK virus reactivation can occur during natalizumab therapy; however, the significance in the absence of renal dysfunction is unclear. We propose regular monitoring for BK reactivation or at least for evidence of renal dysfunction in patients receiving natalizumab.

Molecular epidemiology of human metapneumovirus in Ireland.

Human metapneumovirus (hMPV) is a cause of respiratory illness ranging from wheezing to bronchiolitis and pneumonia in children. A quantitative real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR) assay was developed for the detection of all four main genetic lineages of hMPV and employed to validate an indirect immunofluorescence (IF) assay to detect hMPV positive specimens. The IF assay detected 24 positives from a screen of 625 randomly selected pediatric respiratory specimens collected (3.8% prevalence). From this cohort of 625 specimens, 229 were also tested by real‐time RT‐PCR assay. This included the 24 IF positive specimens and 205 randomly selected specimens from both study periods. In addition to confirming all the IF positives, the real‐time assay detected an additional six hMPV positive specimens giving rise to a combined prevalence of 4.8%. Phylogenetic analysis showed that hMPV subtypes A2b and B2 to be the most prevalent genotypes circulating in our population and surprisingly no hMPV subgroups A1 or B1 were detected during this study period. Based on this phylogenetic analysis, we propose the existence of sub‐clusters of hMPV genotype B2 present in our population which we term subtypes B2a and B2b. The mean log 10 copies/ml of quantitative RT‐PCR determinations from these 30 hMPV positive respiratory specimens was 6.35 (range = 4.44–8.15). Statistical analysis of quantitative RT‐PCR determinations of viral load from these 30 respiratory specimens suggests that hMPV genotype B specimens have a higher viral load than hMPV genotype A isolates (P < 0.03). J. Med. Virol. 80:510–516, 2008.

Molecular Epidemiology of Circulating Measles Virus in Ireland 2002-2007

The molecular characterization of measles virus (MeV) is a valuable epidemiological tool to monitor virus transmission and to discriminate between imported and endemic infection. There has been significant immigration into Ireland in recent years and many individuals originate from regions of high measles incidence. Ireland has had a number of outbreaks of MeV which appear attributable to sub‐optimal vaccine uptake and possibly imported strains as new genotypes have been identified in recent years. To ascertain any significant changes in circulating measles genotypes we investigated 65 confirmed measles cases between the years 2002 and 2007. The laboratory diagnosis of measles was confirmed by detection of measles‐specific IgM in oral fluid in conjunction with a real‐time polymerase chain reaction assay targeting the MeV hemagglutinin gene. Phylogenetic analysis based on the 3′ hypervariable region of the nucleoprotein gene was performed and three genotypes, all within measles clade D, were found to be circulating during this time period. In 2002 and 2003, genotype D8 (n = 2) was observed whereas genotype D7 was dominant in 2003 (n = 31). A distinct change in the circulating MeV genotype and increased genetic diversity was observed between 2004 and 2007. All cases were within genotype D4 (n = 32) but were phylogenetically distinct from each other. These data provide important epidemiologic baseline information on MeV in Ireland and facilitates detailed examination of measles transmission. J. Med. Virol. 81:125–129, 2009.