An unusual outbreak of rotavirus genotype G2P[6] during the 2005-2006 epidemic season in Philadelphia.

Most rotavirus gastroenteritis is caused by G1P[8] strains. When G2 infections are encountered, the P type has most often been reported to be P[4]. The purpose of our study was to describe an unusual outbreak of G2P[6] cases. Children presenting to The Children's Hospital of Philadelphia with acute gastroenteritis have been monitored for rotavirus antigen in stool by ELISA (with G-typing if ELISA positive) since 1994-1995. Compared to the last 12 rotavirus seasons before the 2006 introduction of a new rotavirus vaccine, the 2005-2006 season had by far the highest number of evaluable rotavirus infections [n = 275 from September 2005 through June 2006, of which 261 (95%) were G typed] and the greatest number of G2 cases (n = 101, 39% of typed strains). Only 16% of G2 strains were associated with P[4], whereas genotype G2P[6] was responsible for 83% of the G2 infections. Eighty-eight percent of the 84 G2P[6] cases occurred in the 60% of patients who were African-Americans, most of whom were urban residents. Among 157 African-American patients, G2 cases (n = 80; 52%) predominated, including 74 due to G2P[6]. Children <6 months old accounted for 27% of cases overall, but 36% of the G2P[6] cases. G2 rotaviruses caused over a third of the community-acquired rotavirus cases in children presenting to CHOP in 2005-2006, attesting to the potential impact of G2 strains during some epidemics. The large majority of G2 strains had the rare P[6] genotype. Urban African-American children under 6 months of age were disproportionately affected.

Decline in cases of rotavirus gastroenteritis presenting to The Children's Hospital of Philadelphia after introduction of a pentavalent rotavirus vaccine.

A pentavalent rotavirus vaccine for infants became available in the United States in February 2006. By 2007, vaccination rates nationwide were estimated to be approximately 50%. We studied the effectiveness of the vaccine in a real-world setting outside of a clinical trial. All children presenting to The Children's Hospital of Philadelphia with acute gastroenteritis have been monitored for the presence of rotavirus antigen in the stool by enzyme-linked immunosorbent assay (ELISA [followed by genotyping if ELISA positive]) since the 1994-1995 epidemic season, presenting a unique opportunity to assess the impact of the recently introduced vaccine. The annual number of community-acquired cases over the preceding 13 years had approached or exceeded 100, with 271 cases in 2005 to 2006 and 167 cases in 2006 to 2007. In the 2007-2008 season, only 36 community-acquired cases were identified, representing an 87% reduction from the same period in 2005 to 2006. G3 was the predominant serotype, accounting for 15 community cases (42%). Our study is limited by its observational design using historical comparisons. Nonetheless, the abrupt decline in rotavirus gastroenteritis cases during the 2007-2008 season likely resulted from vaccination. Because protection rates appeared to have exceeded vaccination rates, herd immunity may have contributed to some degree to the effectiveness of the vaccine.

Enzyme-linked immunosorbent assay for measuring antibodies to pneumococcal polysaccharides for the PNEUMOVAX 23 vaccine: assay operating characteristics and correlation to the WHO international assay.

The Merck pneumococcal (Pn) enzyme-linked immunosorbent assays (ELISAs) for measuring antibodies to 12 serotypes (serotypes 1, 3, 4, 6B, 7F, 8, 9V, 12F, 14, 18C, 19F, and 23F) were validated in 1999. Merck Laboratories developed the Pn assays using 10 microg/ml C polysaccharide, 100 microg/ml Pn polysaccharide (PnPs) 25, and 100 microg/ml PnPs 72 for preadsorption of samples, standards, and controls in order to improve the specificity to the Pn serotypes in the vaccine. The Pn assays utilize postimmunization sera obtained from subjects immunized with PNEUMOVAX 23 as standards for measuring immunoglobulin G concentrations in sera obtained from vaccine clinical trials with adults and infants. This material was calibrated to the Pn reference standard serum, 89SF, subjected to the Merck Pn ELISA adsorbants. Comparisons were made between the Merck Pn assay and the international Pn assay, showing moderate agreement between the two assay formats. This work describes the test procedures and operating characteristics of the Merck Pn assays and the results of experiments performed to compare the Merck Pn ELISAs to the international Pn ELISAs.

Novel rotavirus VP7 typing assay using a one-step reverse transcriptase PCR protocol and product sequencing and utility of the assay for epidemiological studies and strain characterization, including serotype subgroup analysis.

Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in approximately 92% of samples (3, 17, 19).