Molecular epidemiology of human herpesvirus 8 in patients with HHV-8-related diseases in Ireland.

Human Herpesvirus 8 (HHV-8) has been classified by sequence analysis of open reading frame (ORF) K1, ORF K15, and variable sequence loci within the central constant region. The purpose of this study was to examine the molecular epidemiology of HHV-8 in an Irish population. This retrospective study included 30 patients who had HHV-8 DNA detected in plasma. Nested end-point PCR was used to characterise four regions of the HHV-8 genome, K1, T0.7 (K12), ORF 75, and K15. Sequencing data were obtained for 23 specimens from 19 patients. Phylogenetic analysis of ORF K1 demonstrated that subtypes A, B, C and F were present in 37%, 11%, 47% and 5%, respectively. For T0.7 and ORF 75, sequencing data were obtained for 12 patients. For T0.7, subtypes A/C, J, B, R and Q were present in 58%, 17%, 8%, 8%, and 8%, respectively. For ORF 75, subtypes A, B, C and D were present in 58%, 8%, 25%, and 8%, respectively. K15 sequences were determined for 13 patients. 69% had the P allele and 31% had the M allele. The data generated by this study demonstrate that a broad variety of HHV-8 subtypes are represented in patients exhibiting HHV-8-related disease in Ireland, a low prevalence country. The predominance of C and A K1 subtypes was as expected for a Western European population. The 31% prevalence for K15 subtype M was higher than expected for a Western European population. This may represent the changing and evolving epidemiology in Ireland due to altered migration patterns.

Phylogenetic analysis of the complete genome of 11 BKV isolates obtained from allogenic stem cell transplant recipients in Ireland.

BK polyomavirus (family Polyomaviridae) may cause hemorrhagic cystitis (BKV-HC) in hematopoietic stem cell transplant recipients. Eleven complete BKV genomes (GenBank accession numbers: JN192431-JN192441) were sequenced from urine samples of allogenic hematopoietic stem cell transplant recipients and compared to complete BKV genomes in the published literature. Of the 11 isolates, seven (64%) were subgroup Ib-1, three (27%) isolates belonged to subgroup Ib-2 and a single isolate belonged to subtype III. The analysis of single-nucleotide polymorphisms in this study showed that isolates could be subclassified into subtypes I-IV and subgroups Ib-1 and Ib-2 on the basis of VP1 of the first part of the Large T-antigen (LTag). The non-coding control region (NCCR) of the 11 isolates was also sequenced. These sequences showed that there was consistent sequence homology within subgroups Ib-1 and Ib-2. Two new mutations were described in the isolates, G→C at O(84) in isolate SJH-LG-310, and a deletion at R(2-7) in isolate SJH-LG-309. No known transcription factor is thought to be present at the site of either of these mutations. There were no rearrangements seen in isolates and this may be because the patients were not followed up over time. There were five nucleotide positions at which subgroup Ib-1 isolated differed from subgroup Ib-2 isolates in the NCCR sequence, O(41) , P(18) , P(31) , R(4) , and S(18) . The mutation O(41) is present in the promoter granulocyte/macrophage stimulating factor) gene and the P(31) mutation is present in the NF-1 gene.

Molecular epidemiology of human respiratory syncytial virus subgroups A and B identified in adults with hematological malignancy attending an Irish hospital between 2004 and 2009.

Human respiratory syncytial virus (HRSV) is an important cause of respiratory infection in patients with hematological malignancy, particularly hematopoietic stem cell transplant recipients. This study investigated the genetic variability of the attachment (G) protein gene among HRSV isolates collected from adult patients with hematological malignancy. Between December 2004 and March 2009, 60 samples collected from 58 adults attending an Irish hospital were positive for HRSV by direct immunofluorescence. Nucleotide sequence analysis of the G gene showed a slightly higher frequency of HRSV subgroup A (52%) than HRSV subgroup B (48%). Genetic variability was higher among subgroup A viruses (up to 13% at nucleotide level) than among subgroup B viruses (up to 4%). Phylogenetic analysis revealed two genotypes of HRSV subgroup A, GA2 and GA5, which cocirculated between 2004/2005 and 2007/2008, although GA2 alone was identified in season 2008/2009. Genotype BA was the only genotype of HRSV subgroup B identified. Genotype-specific amino acid substitutions were identified, with two and seven changes for GA2 and GA5, respectively. Furthermore, one to four potential N-glycosylation sites were found among HRSV subgroup A isolates while two to three were identified in HRSV B isolates. Predicted O-glycosylation sites included 25-34 and 40-43 in HRSV subgroups A and B, respectively. The average synonymous mutation-to-non-synonymous mutation ratios (dS/dN) implied neutral selection pressure on both HRSV subgroup isolates. This study provides data for the first time on the molecular epidemiology of HRSV isolates over five successive epidemic seasons among patients attending an Irish hospital.

Evaluation of the Abbott RealTime CT assay with the BD ProbeTec ET assay for the detection of Chlamydia trachomatis in a clinical microbiology laboratory.

The Abbott RealTime CT assay (Abbott Molecular, Des Plaines, IL) was evaluated by testing male urine samples (n = 204) and female urine samples (n = 207) with matched endocervical swabs (n = 207) collected from patients attending the Genito-Urinary Infectious Disease Clinic, St. James's Hospital, Dublin, Ireland. Results were compared with the BD ProbeTec ET assay (Becton Dickinson, Sparks, MD). Both assays were performed within 3 days of specimen collection. Samples positive with 1 or other assay were subjected to discrepant-based analysis using 2 additional assays, an "in house real-time polymerase chain reaction [PCR]" and a "nested PCR with amplicon sequence detection". After resolution of discordant results, the Abbott RealTime CT assay demonstrated greater clinical sensitivity than the BD ProbeTec ET assay for the detection of Chlamydia trachomatis (CT) in all sample types. Both assays demonstrated acceptable analytic sensitivity with detection limits of 22 and 33 cryptic plasmid copies/reaction, respectively. The sensitivity of the Abbott RealTime CT assay combined with automated throughput establishes this assay as a quality diagnostic tool.

Molecular characterization of hepatitis B virus (HBV) isolates, including identification of a novel recombinant, in patients with acute HBV infection attending an Irish hospital.

Hepatitis B virus (HBV) is known to show significant genetic diversity. There are eight HBV genotypes (A-H) characterized by distinct geographical distribution. Mutations in the HBV genome, in particular precore (PC) and basal core promoter (BCP) mutations, may be important factors in the pathogenesis of disease. In this study genetic heterogeneity and phylogenetic analysis of HBV isolates from 32 naïve patients with acute HBV infection was investigated. Eleven patients presented with severe infection, while the remaining 21 had self-limiting illness. Only four isolates from patients with severe HBV infection harbored the G1896A stop codon mutation. One isolate (Irish-13), collected from a patient with acute asymptomatic infection, had a G1896A mutation and a 243 bp deletion of the polymerase gene. A triple mutation, T1753C/A1762T/G1764A was identified in only one isolate (Irish-3) associated with severe infection. The latter also had a mutation, A2339G, in the core gene, not previously reported in severe acute infection caused by genotype D. Variations within the S gene were identified in 6 isolates, including Gly145Ala, associated with vaccine immune escape, Asp144Glu, Ser143Leu and Phe134Leu, each associated with failure to detect HBsAg. Phylogenetic analysis was determined using amplicons of the S gene (678 bp) and distal-X/PC region (672 bp). Genotype A was the most common (75%), followed by genotype D (15.6%), and equal proportions of C, E, F, and H. A novel recombinant of genotypes D and E was identified in an isolate originating from West Africa. Genetic heterogeneity of HBV isolates of HBV isolates from patients with acute infection needs further study of its significance.