Solid organ transplant recipients are at risk of morbidity from human papillomavirus (HPV)-related diseases. Quadrivalent HPV vaccine is recommended for posttransplant patients but there are no data on vaccine immunogenicity. We determined the immunogenicity of HPV vaccine in a cohort of young adult transplant patients. Patients were immunized with three doses of quadrivalent HPV vaccine containing viral types 6, 11, 16 and 18. Immunogenicity was determined by type-specific viral-like protein ELISA. Four weeks after the last dose of vaccine, a vaccine response was seen in 63.2%, 68.4%, 63.2% and 52.6% for HPV 6, 11, 16 and 18, respectively. Factors that led to reduced immunogenicity were vaccination early after transplant (p = 0.019), having a lung transplant (p = 0.007) and having higher tacrolimus levels (p = 0.048). At 12 months, there were significant declines in antibody titer for all HPV types although the number of patients who remained seropositive did not significantly differ. The vaccine was safe and well tolerated. We show suboptimal immunogenicity of HPV vaccine in transplant patients. This is important for counseling patients who choose to receive this vaccine. Further studies are needed to determine an optimal HPV vaccine type and schedule for this population.

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10 2 to 10 3 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water Vibrio vulnificus is a gram-negative halophilic bacterium commonly found in warm coastal waters throughout the world (37, 44). In the United States, this microorganism is indigenous to Gulf of Mexico water (gulf water) and thrives during the warmer months. V. vulnificus is known to cause gastroenteritis and, in some cases, septicemia when it is ingested in raw or poorly cooked oysters (25,26). Infection by V. vulnificus often results in fatal consequences, and the mortality rate is up to 60%, primarily in individuals who are immunocompromised (10,11,17,18,28,31).)Since infection by V. vulnificus is one of the leading causes of seafood-related illnesses in the United States, the Interstate Shellfish Sanitation Conference (ISSC) has proposed that illnesses caused by V. vulnificus due to consumption of shellfish must be reduced by at least 60% by the year 2007 (21). In order to achieve this goal, the ISSC suggests that consumable oysters should not contain more than 3 CFU of V. vulnificus per g of oyster meat (21). Therefore, to meet this objective, a rapid, reliable, and sensitive method for detection of this pathogen is as important as the methods of treatment for reducing the numbers of V. vulnificus in oysters to an acceptable level. Detection of V. vulnificus by conventional biochemical and microbiological culture methods, such as the most-probablenumber method or the use of selective agar media, are timeconsuming and require several days to obtain confirmatory results (42). A genetically based colorimetric colony hybridization method targeting the V. vulnificus-specific hemolysin gene, vvh, has been described previously and has been recommended as an alternative method for detection of this microorganism in shellfish (24,33,52). Although the DNA-DNA colony hybridization method in which the vvh gene segment is used as a probe is reliable and specific for detection of this pathogen, it takes at least 3 days to complete. A conventional PCR method for detection of V. vulnificus and other microbial pathogens in shellfish has also been shown to be effective (8,22). However, this PCR approach requires analysis of the amplified DNA in an agarose gel or by DNA-DNA hybridization to confirm the results, which again is time-consuming and laborious.Recently, introduction of a real-time PCR amplification method in which SYBR Green I fluorescent dye is used has made detection of microbial pathogens such as Legionella (46), Escherichia coli (23), Vibrio parahaemolyticus (7), and Campylobacter (29) rapid and cost-effective and the analysis of results simple. The SYBR Green I fluorescent dye binds to the minor grooves of the amplified DNA during the primer annealing and extension steps of each PCR cyc...

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10 2 ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10 3 cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.Vibrio parahaemolyticus is a natural inhabitant of coastal waters worldwide (6,25). This organism is a halophilic, gramnegative bacterium that causes gastroenteritis in humans. Infection results from the consumption of contaminated seafood, particularly raw shellfish. A significant increase in the number of cases of V. parahaemolyticus infections was reported in 1996. A unique clone of V. parahaemolyticus O3:K6 is responsible for many of the recent V. parahaemolyticus outbreaks, including epidemics in India, Russia, Southeast Asia, Japan, and North America (9,13,17,23). Strains of the O3:K6 serovar emerged in Calcutta, India, in 1996 and have accounted for 50 to 80% of V. parahaemolyticus infections annually since then. The same serovar was isolated from patients in various Southeast Asian countries, and it was found that the O3:K6 strains isolated since 1996 are all derived from a single clone (20). In 1997, an outbreak caused by V. parahaemolyticus occurred in the Pacific Northwest, resulting in one death and 209 illnesses. Each of the infected persons had consumed raw oysters (8). V. parahaemolyticus O3:K6 was involved in outbreaks from contaminated oysters harvested from Oyster Bay, N.Y., and Galveston Bay, Tex., in 1998 (8, 12). The V. parahaemolyticus outbreak in Galveston Bay was the largest reported ...

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan

In 2005-2006, about 40% of females and 20% of males had serological evidence of exposure to ≥1 of 9 HPV types. Seroprevalence of all type categories, especially HPV 31/33/45/52/58 among females, varied by race/ethnicity.

Reliable antibody based-assays are needed to evaluate the immunogenicity of current vaccines, impact of altered dosing schemes or of new vaccine formulations. An ideal assay platform would allow multiplex type-specific detection with minimal sample requirement. We used the Meso Scale Discovery (MSD) electrochemiluminescence based detection platform to develop a multiplex direct virus-like particle (VLP) ELISA to detect antibodies to HPV 6, 11, 16, and 18 with a protocol developed for detection using the SI 6000 imager (M4ELISA). MSD prepared the plates in the 7-spot/well format, using the purified VLPs (4 spots) and PBS + BSA pH 7.4 (3 blank spots). Three-point titrations and the parallel line method were used to calculate antibody levels. Dynamic range, precision, and stability of pre-printed plates were determined using a panel of previously characterized sera. Cut-off values using children’s sera were established using 99% RLU limits based on the 4-parameter Johnson Su best fit curve. Results of the M4ELISA were compared to competitive Luminex Immunoassay (cLIA) on n = 4454 sera from a predominantly unvaccinated cohort. Using a VLP coating concentration of 80 μg/ml with BSA provided the most robust RLU signal for all types. The dynamic range of the assay was about 1000 fold, with assay variability under 25% for each of the four vaccine types. Long-term stability of the plates extended to about 7 months from the time plates was received in the laboratory after printing. There was moderate agreement (κ = 0.38–0.54) between M4ELISA and cLIA, with antibody detection for each of the 4 types more frequent with M4ELISA. Quantitative analysis however showed a good correlation between concordant samples by both assays (ρ ≥ 0.6). The MSD platform shows promise for simultaneous quantitation of the antibody responses to four HPV vaccine types in a high-throughput manner.