David Cubitt

VIRAL GASTROENTERITIS ABOARD A CRUISE SHIP

A 32-nm small round structured virus (SRSV), possibly related to the Snow Mountain agent (SMA), was implicated as the cause of recurrent outbreaks of gastroenteritis on a cruise ship. There was no identifiable relation to food or water consumption, but the risk of gastroenteritis among passengers who had shared toilet facilities was twice that of those who had a private bathroom and the rate of illness was related to the number of passengers sharing a communal restroom (ie, with one or more toilets): contaminated bathrooms may be an important vehicle for person-to-person spread of this enteric agent. In each cabin, index patients who had vomited in their cabins were more likely to have had cabinmates who subsequently became ill than were index patients who had not vomited. These epidemiological findings implicate vomitus in the transmission of viral gastroenteritis and they are consistent with the transmission of viral agents by airborne droplets or person-to-person contact. New strategies for prevention of viral gastroenteritis should include protection against environmental contamination by viruses in airborne droplets or vomitus.

Improved outcome for children with disseminated adenoviral infection following allogeneic stem cell transplantation

Adenovirus (AdV) infections are a frequent cause of morbidity and mortality following allogeneic stem cell transplantation (SCT), and disseminated infection is associated with high mortality, particularly in paediatric SCT. Here, we describe an approach to reduce mortality from adenoviraemia by combining prospective monitoring for the occurrence of adenoviraemia using a sensitive polymerase chain reaction method, early antiviral therapy and prompt withdrawal of immunosuppression. A total of 155 consecutive paediatric SCT procedures were prospectively monitored, of which 113 (73%) transplants involved donors other than matched siblings and 126 (83%) employed T‐cell depletion. Adenoviraemia was detected in 26/155 (17%) transplants and developed exclusively in patients who had received T‐cell‐depleted grafts. Withdrawal of immunosuppression coupled with early antiviral therapy led to resolution of adenoviraemia in 19/26 (81%) patients with only five patients succumbing to disseminate AdV infection. Survival from adenoviraemia was associated with lymphocyte recovery to above 0·3 × 109/l. Mortality was closely linked with the absence of lymphocyte recovery because of profound T‐cell depletion of the graft with CD34+ magnetic‐activated cell sorting. Mortality from disseminated AdV infection was 5/26 (19%) in this study, which is significantly lower than previously reported.

Asymptomatic and symptomatic excretion of noroviruses during a hospital outbreak of gastroenteritis

ABSTRACT During an investigation of a hospital outbreak of norovirus gastroenteritis identified as being caused by a recombinant genogroup II (rGII-3a) strain, fecal specimens collected from asymptomatic staff and patients were tested by nested PCR. A GII-4 norovirus strain, the predominant strain associated with outbreaks in hospitals over the last few years, was detected in 26 and 33% of asymptomatic staff and patients, respectively. No rGII-3a (Harrow/Mexico) norovirus strains were detected in the samples of asymptomatic staff or patients.

Amino Acid Substitution within the VP7 Protein of G2 Rotavirus Strains Associated with Failure To Serotype

ABSTRACT Rotavirus strains collected in the United Kingdom during the 1995-1996 season and genotyped as G2 by reverse transcription-PCR failed to serotype in enzyme-linked immunosorbent assays using three different G2-specific monoclonal antibodies. The deduced amino acid sequences of the antigenic regions A (amino acids 87 to 101), B (amino acids 142 to 152), and C (amino acids 208 to 221) of VP7 revealed that a substitution at position 96 (Asp→Asn) correlated with the change in ability to serotype these G2 strains.

Phylogenetic analysis of the caliciviruses

A phylogenetic portrait of the genus Calicivirus in the family Caliciviridae was developed based upon published sequences and newly characterized calicivirus (CV) strains, including additional Sapporo‐like HuCV strains in pediatric diarrhea stool specimens from South Africa, the United Kingdom, and the United States. Distance and parsimony methods were applied to nucleotide and amino acid sequences of human and animal calicivirus 3D RNA‐dependent RNA polymerase (∼470nt) and capsid hypervariable regions (∼1,200nt) to generate phylogenetic trees. Pairwise amino acid identity in the 3D region among the Sapporo‐like strains ranged from 61%; to 100%. Human and animal caliciviruses (HuCVs and AnCVs) separated into five genogroups: small round‐structured viruses (SRSV), Sapporo‐like, and hepatitis E virus (HEV)‐like HuCVs and rabbit‐, and vesicular exanthema of swine virus (VESV)‐like AnCVs, each with a distinct genome organization. Each genogroup, including the Sapporo‐like HuCVs, subdivided further into subgenogroups. The capsid region trees had higher levels of confidence than the 3D region trees and limited conclusions about genogroups could be drawn from the 3D region analyses. This analysis suggested that CVs include five potential virus subfamilies. J. Med. Virol. 52:419–424, 1997.

Increased incidence of EBV-related disease following paediatric stem cell transplantation with reduced-intensity conditioning

The incidence of Epstein–Barr virus (EBV) viraemia and lymphoproliferative disease (LPD) was studied in a consecutive cohort of 128 paediatric patients undergoing stem cell transplantation (SCT) with reduced‐intensity conditioning (RIC; n = 65) or conventional‐intensity conditioning (CIC; n = 68). Following CIC, six of 68 (8%) developed viraemia; all remained asymptomatic. EBV viraemia (23 of 65 patients = 35%, P < 0·001) and LPD (10 of 65 = 15%, P < 0·001) were significantly more frequent following RIC. Of the 23 RIC patients who developed viraemia, eight remained asymptomatic, five had symptomatic viraemia (fever ± rash), and 10 patients developed LPD, two of whom died. An absolute lymphocyte count of <0·3 × 109/l at the time of onset of viraemia was strongly predictive of development of LPD (P < 0·05) in this group. The incidence of viraemia was significantly higher in patients receiving serotherapy with antithymocyte globulin (ATG; 15 of 43, 35%) than Campath (12 of 73, 16·4%, P < 0·05). Primary immunodeficiency and acute graft‐versus‐host disease were associated with EBV viraemia in univariate analysis, but were not independent risk factors. In conclusion, EBV viraemia and LPD appear to be significantly more common in children following RIC SCT, particularly with selective depletion of recipient T cells relative to B cells following the use of ATG. This probably reflects the profound immunosuppression following RIC SCT, together with the incomplete ablation of recipient‐derived B cells.

Characterisation of rotavirus G9 strains isolated in the UK between 1995 and 1998.

G9P[6] and G9P[8] rotavirus strains were identified during 1995/96 through the molecular epidemiological surveillance of rotavirus strains circulating in the UK between 1995 and 1998. An increase in the incidence and spread of sporadic infections with rotavirus genotype G9P[8] across the UK was detected in the two following seasons. Partial sequencing of the VP7 gene showed that all the UK strains shared a high degree of homology and were related very closely to G9 strains from the US and from symptomatic infections in India (≥96% homology). The UK strains were related more distantly to the apathogenic Indian strain 116E (85–87.8% homology). Phylogenetic analysis revealed clustering of the UK strains into 3 different lineages (I to III) and into two sub‐lineages within lineage I. There were correlations between VP7 sequence clustering, the P type and the geographical origin of the G9 strains. Partial sequencing of the VP4 gene showed high degree of homology (>98%) among all the P[6] strains, and the sequences obtained from the P[8] strains clustered into 2 of the 3 global lineages described for P[8] strains associated with other G types. These data suggest that G9 strains may be a recent importation into the UK, and that G9P[8] strains may have emerged through reassortment in humans between G9P[6] strains introduced recently and the more prevalent cocirculating G1, G3 and G4 strains that normally carry VP4 genes of P[8] type. J. Med. Virol. 61:510–517, 2000.

EBV-related disease following haematopoietic stem cell transplantation with reduced intensity conditioning

The use of reduced intensity regimens has decreased early mortality following stem cell transplantation. However, the increased immunosuppression following these protocols results in profound and often prolonged lymphopenia, resulting in an increased incidence of viral reactivation. We and others have observed a high incidence of EBV viraemia and post-transplant lymphoproliferative disease (PTLD) following reduced-intensity conditioning regimens, reflecting the delayed recovery of EBV-specific immunity after such transplants. The clinical and histological features at presentation are similar to that seen after conventional intensity conditioning. Given the increasing use of reduced intensity conditioning (RIC) transplants, we review the risk factors for EBV related disease following transplantation with RIC, the potential for pre-emptive therapy of PTLD based on monitoring EBV viraemia and management options in such patients.

Capture and generation of adenovirus specific T cells for adoptive immunotherapy

Adenoviral infections represent a major cause of morbidity and mortality following haematopoietic stem cell transplantation. Current anti‐viral agents are virostatic and it is evident that elimination of adenovirus (ADV) infection is only achieved by recovery of cellular immunity. Using an interferon‐gamma (IFN‐γ) secretion and capture assay to isolate ADV‐specific T cells, followed by a 2 week expansion and restimulation protocol, we generated ADV T cells that may be used for cellular immunotherapy. In contrast to virus‐specific T cells for cytomegalovirus or Epstein‐Barr virus, the ADV response was dominated by CD4+ T cells and the majority of captured cells exhibited an effector/memory immunophenotype. Highly specific antigen responses were demonstrated by intracellular IFN‐γ expression and cytotoxicity assays when the expanded cells underwent restimulation with ADV‐pulsed target cells. Although T cells were initially generated in response to ADV species C, the expanded populations also showed strong activity against ADV species B, suggesting cross‐reactivity across ADV species; a finding that has important clinical consequences in the paediatric setting, where the majority of infections are caused by ADV type B and C. The protocols can be readily translated to generate ADV‐specific T cells suitable for clinical use and offer an effective immunotherapeutic strategy to control ADV infection.

Stool elastase as a diagnostic test for pancreatic function in children with cystic fibrosis

pancreatic insufficient. The remaining three children with cystic fibrosis had normal elastase levels and were pancreatic sufficient; two of them had one copy each of the 3849+10kbC→T mutation; the full genotype of the third child was undetermined. The 3849+10kbC→T mutation had already been associated with less severe disease and may result in the presence of small amounts of normal cystic fibrosis transmembrane conductance regulator (CFTR). Cystic fibrosis is a heterogenous disorder and the rare milder mutations associated with pancreatic sufficiency will not necessarily be detected by elastase testing. The samples of meconium passed by three neonates following surgery for meconium ileus all showed zero activity. These children had longitudinal testing over the subsequent 3 weeks during which period the concentration of elastase remained consistently zero. All three neonates were homozygous for the ∆F508 mutation. A survey of 145 meconium stools in normal neonates showed a mean elastase concentration of 63·9 (7·2) μg and demonstrated that levels rose rapidly to above 200 μg during the first month of life. Others have shown that stool elastase levels in premature and term neonates reached normal adult reference concentration by 2 weeks of age. This study has shown the measurement of stool elastase to be a simple and non-invasive test for exocrine pancreatic function in children with cystic fibrosis across a broad range of genotypes. Faecal pancreatic elastase is stable for a week at room temperature and for months at 4oC, making handling and postage of a small stool sample straightforward. The observation of zero activity in three meconium samples of children subsequently shown to have cystic fibrosis by genotyping highlights the potential role that this assay may play as a screen test in neonates.