Allogeneic hematopoietic stem cell transplantation and norovirus gastroenteritis: a previously unrecognized cause of morbidity.

BACKGROUND: A retrospective study of the clinical, epidemiologic, and virologic features of norovirus gastroenteritis in 12 adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. METHODS: Norovirus infection was diagnosed by reverse-transcriptase polymerase chain reaction. Strains were genotyped by nucleic acid sequence of the most highly conserved region of the norovirus gene encoding the capsid S (shell) domain. RESULTS: Ten of 12 patients presented with vomiting of short duration, but diarrhea was present in all. The median time from onset to norovirus diagnosis was 1 month (range, 0.25-6.0 months). Eleven patients were receiving immunosuppression when norovirus infection was diagnosed: 8 for graft-versus-host disease (GVHD) in an organ other than gut, 1 for previous gut GVHD, and 2 for presumed gut GVHD that proved to be norovirus gastroenteritis. Six patients required enteral or parenteral nutrition for severe weight loss. In 10 patients, diarrhea lasted a median of 3 months (range, 0.5-14 months) and virus was shed at a high level throughout. The remaining 2 patients died after 4 months of diarrhea (one died of unrelated complications, and the other died of malnutrition). The noroviruses found were GII (untyped), GII-3, GII-4, and GII-7 in 1, 1, 9, and 1 patients, respectively. Eleven of the 12 patients had acquired their infection in the community. Phylogenetic analysis of the GII-4 strains demonstrated that all differed. CONCLUSIONS: Noroviruses are a hitherto unsuspected cause of prolonged morbidity and mortality in adults after allogeneic HSCT. The use of reverse-transcriptase polymerase chain reaction to detect high viral load levels in feces distinguishes norovirus gastroenteritis from gut GVHD.

Conservation and microdiversity of the phospholipase A (pldA) gene of Helicobacter pylori infecting dyspeptics from different countries.

Phospholipase activity is important in bacterial pathogenicity and could contribute to the pathogenic role of Helicobacter pylori by degradation of the gastric mucus, and in maintaining long-term colonisation. Our aim was to determine the degree of variation in the phospholipase A gene (pldA) of H. pylori from different geographic locations, and to investigate links between pldA genotype and clinical disease severity, as well as with variation in cagA status and vacA genotypes. PCR-restriction fragment length polymorphism (RFLP) analysis with MboI and HaeIII was used to study 124 isolates from 10 countries that included the two genome-sequenced strains (26695 and J99), as well as Tx30a and NCTC 11637 (type strain). The 925-bp pldA fragment was amplified with a frequency of 90%. The presence of pldA was confirmed in the other strains using an alternative forward primer. Isolates were distinguished by PCR-RFLP analysis with 10 MboI and four HaeIII restriction patterns that combined to give 25 distinct pldA RFLP types. The pldA M2H2 strain genotype was most common (20%) in the UK but similar strains came from several other countries. Microdiversity was evident in pldA sequences of strains representing different RFLP types, and five M2H2 strains each had a distinct pldA sequence type. Intragenic variation was independent of gastric disease severity as well as strain cagA status and vacA genotype, with the exception of eight geographically diverse strains all with the pldA M4H3/cagA+/vacA s1m1 genotype predominantly from peptic ulcer patients. The study indicated a spectrum of genotypic variants and was supportive of a pldA function in H. pylori colonisation and persistence rather than in chronicity of infection.

Enterovirus infections in England and Wales, 2000-2011: the impact of increased molecular diagnostics.

There have recently been significant changes in diagnostic practices for detecting enterovirus (EV) infections across England and Wales. Reports of laboratory-confirmed EV infections submitted by National Health Service (NHS) hospital laboratories to Public Health England (PHE) over a 12-year period (2000-2011) were analysed. Additionally, the PHE Virus Reference Department (VRD) electronic database containing molecular typing data from 2004 onwards was interrogated. Of the 13,901 reports, there was a decline from a peak of 2254 in 2001 to 589 in 2006, and then an increase year-on-year to 1634 in 2011. This increase coincided with increasing PCR-based laboratory diagnosis, which accounted for 36% of reported cases in 2000 and 92% in 2011. The estimated annual incidence in 2011 was 3.9/100,000 overall and 238/100,000 in those aged <3 months, who accounted for almost one-quarter of reported cases (n = 2993, 23%). During 2004-2011, 2770 strains were submitted for molecular typing to the VRD, who found no evidence for a predominance of any particular strain. Thus, the recent increase in reported cases closely reflects the increase in PCR testing by NHS hospitals, but is associated with a lower proportion of samples being submitted for molecular typing. The high EV rate in young infants merits further investigation to inform evidence-based management guidance.

Inter-seasonal diversity of norovirus genotypes: emergence and selection of virus variants.

This study describes a method used to determine the diversity of NoVs co-circulating in the community that consisted of the analysis of a limited number of strains collected from outbreaks occurring at different times of the NoV season. The diversity of twenty NoV strains collected from outbreaks occurring at the beginning of each NoV season (September) was compared to the diversity found in the middle (December) and at the end of the season (March). The method was validated through the characterisation of greater numbers of strains at times when novel genotypes or variants were detected. A total of 864 strains from outbreaks of gastroenteritis from the 2003/04, 2004/05 and 2005/06 seasons were genotyped, with the majority of outbreaks occurring in the UK. There was a greater diversity of NoV genotypes at the beginning of two of the three seasons, 2003/04 and 2005/06, when compared to strains circulating at the end of the seasons, and GII-4 NoV strains predominated (>90%) at the end of each season. Data from this study also identified the co-circulation and differentiation of three major GII-4 variants (v2, v3, and v4). Detailed analysis of a larger number of strains throughout each season confirmed that variants emerged, became the predominant circulating strain and were ultimately replaced with another variant selected from a pool of variants. By June 2006, GII-4 v4 (Hu/NoV/Rhyl440/2005/UK) emerged as the predominant GII-4 strain, usurping the previous GII-4 v3 strain [Hu/NoV/Hunter284E/040/AU] to become the commonest co-circulating strain, in the UK in 2006.

High-resolution genotyping elucidates the epidemiology of group A streptococcus outbreaks.

Streptococcus pyogenes strains were genotyped by a combination of molecular methods for high- resolution epidemiologic studies of disease outbreaks. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the emm gene is reported. Alone or in conjunction with other molecular techniques (16S ribotyping, pulsed-field gel electrophoresis, and detection of exotoxin genes), PCR-RFLP could differentiate outbreak-related strains from contemporaneous background strains of the same M serotype. Three outbreaks were studied: pharyngitis in a boarding school (serotype M5), cross-infection in a hospital burn unit (serotype M76), and severe invasive disease in two elderly care homes (serotype R28). It was possible, for example, to identify within serotype R28 a clone with particular potential for invasive disease. In all cases, the four molecular methods yielded complementary results that were hierarchically related. Strains could be assigned to the outbreak or the background in a precise, reproducible, and rapid manner.

Use of an amplified-fragment length polymorphism technique to fingerprint and differentiate isolates of Helicobacter pylori.

Amplified-fragment length polymorphism (AFLP) analysis is the name given to a genotypic technique in which adapter oligonucleotides are ligated to restriction enzyme fragments and then used as target sites for primers in a PCR amplification process. The amplified fragments are electrophoretically separated to give strain-specific band profiles. We have developed a single-enzyme approach that did not require costly equipment or reagents for the fingerprinting of strains of Helicobacter pylori. The method was assessed with 46 isolates of H. pylori from 28 patients, and the results were compared with those from other genotypic tests. The AFLP profiles derived from HindIII fragments differentiated strains of H. pylori from unrelated individuals and confirmed the common origin of strains in some family members. AFLP analysis was also applied to investigate persistent infection following antibiotic therapy. Overall, the modified technique was relatively rapid and technically simple yet gave reproducible and discriminatory results. AFLP analysis samples variation throughout the genome and is a valuable addition to the existing genotypic fingerprinting methods for H. pylori.

Genetic diversity in Helicobacter pullorum from human and poultry sources identified by an amplified fragment length polymorphism technique and pulsed-field gel electrophoresis.

Helicobacter pullorum was first isolated from the faeces and carcasses of poultry and has been associated with human gastroenteritis. The aim of this study was to examine interstrain genetic diversity within H. pullorum. Two fingerprinting techniques were used: amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoretic (PFGE) analysis. The 20 strains examined were from four countries and comprised 13 human isolates and seven poultry isolates. Their identity was confirmed by a species-specific PCR assay. The human and poultry isolates had distinct genotypes and most strains showed a high degree of genetic diversity. Genotyping also indicated a clonal origin for two strains from the same poultry flock, and established a close relatedness between three chicken carcass isolates from a processing plant. It is concluded that these two genotyping techniques will provide a useful basis for future epidemiological investigations of H. pullorum in poultry, and may provide a link with its possible causal role in human gastrointestinal infections.

Surveillance and clinical relevance of vacA genotypes of Helicobacter pylori infecting dyspeptic patients in mid-Essex.

The Helicobacter pylori vacuolating cytotoxin is a putative pathogenicity factor encoded by vacA, a mosaic gene with a global distribution. The vacA type prevalence and diversity of H. pylori isolated from antral gastric biopsies of 360 dyspeptic patients in mid-Essex, and of 79 patients from other locations, were investigated in order to test for links with disease severity. Mid (m)-region genotyping and subtyping by vacA HaeIII RFLP (restriction fragment length polymorphism) analysis showed that the m1 and m2 alleles were diverse, with 191 different subtypes. Variation in 44% of strains was accounted for by ten subtypes of which subtype v-1 represented a conserved core (33%) of the m1 form. Prevalence rates for combined mid and signal (s)-region genotypes were 40% for s1/m1, 46% for s1/m2, and 11% for s2/m2. Overall, vacA genotyping provided high typability and discrimination, but no specific RFLP markers could reliably predict a clinically significant presentation due to an H. pylori infection.

Development of a scheme for genotyping Helicobacter pylori based on allelic variation in urease subunit genes.

Helicobacter pylori urease subunit genes in 383 isolates from 10 countries were investigated by PCR-restriction fragment length polymorphism (HaeIII) analysis. Eighty-two different ureAB profiles were documented by reference to known sequences. Variation among 51% of strains was accounted for by 10 predominant patterns, which provided a unique framework for categorizing isolates with geographically diverse origins.