Multiplex immunoassay to measure antibody response to nine HPV vaccine types.

Well-characterized HPV serology assays are required to evaluate performance of biosimilar candidate vaccines, reduced dosing schedules and novel administration methods. We report characterization of an expanded assay, M9ELISA, that detects antibodies to HPV virus-like particles (VLP) of nine types using direct IgG ELISA on the Meso Scale Discovery (MSD) electrochemiluminescence platform. The method is based on the previously published M4ELISA which detects antibodies to HPV6,11,16, and 18. It has been modified to add detection of antibodies to HPV31,33,45,52 and 58, and to streamline assay and reduce background. The M9ELISA plates were prepared with purified type specific L1 + L2 VLPs coated on 10-spot/well standard MSD microplates. Results of ELISA on three serial dilutions of serum were read on MSD imager, and titers calculated using the parallel line method. Evaluations included dynamic range, assay reproducibility, and stability over time. We compared M9ELISA results to those from a pseudovirion-based neutralization assay in sera from a mixed cohort of unvaccinated and vaccinated individuals (n = ~116) and to competitive Luminex immunoassay (cLIA) results in sera from a predominantly unvaccinated cohort (n = 4426). The linear range of the assay extended over 5 logs, with inter-assay reproducibility coefficient of variation ≤25% for all types. The pre-coated plates were stable for at least 2 years. Spearman correlation of antibody titers showed excellent correlation with PBNA (r = 0.86-0.97) and moderate correlation (r = 0.52-0.68) with cLIA. Thus, the M9ELISA can serve as a useful platform for high-throughput, sensitive and simultaneous quantitation of the antibody responses to nine HPV vaccine types.

Seroprevalence of HPV 6, 11, 16 and 18 and correlates of exposure in unvaccinated women aged 16-64 years in Puerto Rico.

BACKGROUND: To understand risk factors for HPV exposure in Puerto Rican women, we evaluated HPV 6, 11, 16, and 18 serology in women aged living in the San Juan metropolitan area. METHODS: As part of a cross-sectional study, a population-based sample of 524 HPV unvaccinated Hispanic women ages 16-64 years completed face-to-face and computer assisted interviews and provided blood and self-collected anal and cervical specimens. Serology used multiplex virus-like particle based-IgG ELISA and HPV DNA was detected with L1-consensus PCR. RESULTS: 32% and 47% were seropositive to HPV types included in the bivalent (16/18) and quadrivalent (6/11/16/18) vaccines, respectively. Type-specific seroprevalence was HPV6 - 29%, HPV11 - 18%, HPV16 - 23%, and HPV18 - 17%; seroprevalence was high in the youngest age-group (16-19: 26-37%). HPV seropositivity was associated with having ≥ 3 lifetime sexual partners (OR=2.5, 95% CI=1.7-3.9) and detection of anogenital HPV DNA (OR=1.8, 95% CI=1.2-2.6). CONCLUSIONS: The high cumulative exposure of HPV vaccine types 6/11/16/18 in this Hispanic population was influenced by factors related to HPV exposure through sexual behavior. High seroprevalence in the youngest age-group indicates early age of exposure to HPV in Puerto Rico, highlighting the need for HPV vaccination starting prior to age 16.

Clinical presentation, management, and outcomes in the Indian Heart Rhythm Society-Atrial Fibrillation (IHRS-AF) registry.

AIM: A national atrial fibrillation (AF) registry was conducted under the aegis of the Indian Heart Rhythm Society (IHRS), to capture epidemiological data-type of AF, clinical presentation and comorbidities, current treatment practices, and 1-year follow-up outcomes. METHODS: A total of 1537 patients were enrolled from 24 sites in India in the IHRS-AF registry from July 2011 to August 2012. Their baseline characteristics and follow-up data were recorded in case report forms and subsequently analyzed. RESULTS: The average age of Indian AF patients was 54.7 years. There was a marginal female preponderance - 51.5% females and 48.5% males. At baseline, 20.4% had paroxysmal AF; 33% had persistent AF; 35.1% had permanent AF and 11% had first AF episode. At one-year follow-up, 45.6% patients had permanent AF. Rheumatic valvular heart disease (RHD) was present in 47.6% of patients. Hypertension, heart failure, coronary artery disease, and diabetes were seen in 31.4%, 18.7%, 16.2%, and 16.1%, respectively. Rate control was the strategy used in 75.2% patients, digoxin and beta-blockers being the most frequently prescribed rate-control drugs. Oral anticoagulation (OAC) drugs were used in 70% of patients. The annual mortality was 6.5%, hospitalization 8%, and incidence of stroke 1%. CONCLUSIONS: In India, AF patients are younger and RHD is still the most frequent etiology. Almost two-third of the patients have persistent/permanent AF. At one-year follow-up, there is a significant mortality and morbidity in AF patients in India.

Development and evaluation of multiplexed immunoassay for detection of antibodies to HPV vaccine types.

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 pH7.4 (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.

Cross-sectional study of HPV-16 infection in a population-based subsample of Hispanic adults.

OBJECTIVE: This study aimed to estimate the prevalence and correlates of seropositivity to human papillomavirus (HPV)-16 in a subsample of adults who participated in the parent study Epidemiology of Hepatitis C in the adult population of Puerto Rico (PR). SETTING: The parent study was a population-based household survey aimed to estimate the seroprevalence of hepatitis C and other viral infections (hepatitis A, hepatitis B, HIV, and herpes simplex type 2) in PR (n=1654) between 2005 and 2008. PARTICIPANTS: A subsample of the last 450 consecutive adults aged 21-64 years, recruited between February 2007 and January 2008, who participated in the parent study and agreed to participate in HPV testing. PRIMARY AND SECONDARY OUTCOME MEASURES: The samples were tested by ELISA for HPV-16 viral-like particle-specific immunoglobulin G. Information on sociodemographic, health, and lifestyle characteristics was collected. Logistic regression modelling was used to estimate the prevalence odds ratio (POR) to assess factors associated to HPV-16 seropositivity. RESULTS: Prevalence of seropositivity to HPV-16 was 11.3%. Seroprevalence was higher in women (15.8%) than men (5.6%; p=0.001). After adjusting for age and sex, ever smokers (POR 2.06, 95% CI 1.08 to 3.92) and participants with at least five lifetime sexual partners (POR 2.91, 95% CI 1.24 to 6.81) were more likely to be HPV-16 seropositive. CONCLUSIONS: HPV-16 seropositivity is similar to that reported in the USA (10.4%) for NHANES 2003-2004 participants, although different assays were used in these studies. While future studies should evaluate HPV seroprevalence using a larger population-based sample, our results highlight the need to further understand the burden of HPV infection and HPV-related malignancies in PR, population with a low vaccine uptake.

Immunogenicity of quadrivalent human papillomavirus vaccine in organ transplant recipients.

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.

Choosing pacemakers appropriately.

The range of implantable cardiac pacing devices has expanded, with the advances in available technology. Indications for cardiac pacing devices, that is pacemakers, implantable cardioverter defibrillators (ICDs) and cardiac resynchronisation therapy devices (CRTs), have expanded for the treatment, diagnosis and monitoring of bradycardia, tachycardia and heart failure. While the need for pacemakers is increasing, not all patients who require pacemakers are receiving them, especially in the Asia-Pacific region. There is a need to be more critical in advising the use of more expensive devices like ICDs and CRT/CRT-D devices, since most patients in the Asia-Pacific region pay out of pocket for these therapies. The AHA-ACC guidelines need not be blindly followed, since they are too wide-sweeping and are often based on the intention-to-treat basis of trials rather than on the parameters of the patients actually enrolled.

Initial experience with sirolimus and mycophenolate mofetil for renal rescue from cyclosporine nephrotoxicity after heart transplantation.

Calcineurin inhibitors (CNIs) have become the cornerstone of immunosuppressive regimens following heart transplantation, but their use is associated with nephrotoxicity. We evaluated a CNI elimination protocol in 14 patients with renal impairment at 48.3 +/- 36.0 months after heart transplantation. The mean serum creatinine was 321 +/- 107 micromol/L; cyclosporine (n=13) or tacrolimus (n=1) was discontinued with sirolimus commenced immediately, initially aiming for a target trough level of 16 (12 to 20) ng/mL. If patients were not receiving mycophenolate (MMF) this was initiated at 1 g bid. The transfer period was covered with a tapering course of corticosteroids. In addition to monitoring clinical status, hematology, biochemistry, and sirolimus levels, graft function was assessed by echocardiography, ECG, and, where indicated, endomyocardial biopsy. Renal function improved in 12 patients (with 6 having a greater than 40% decrease in serum creatinine), remained unchanged in 1, and deteriorated in 1. Two patients who were converted at 15 and 139 months after transplantation experienced grade 3A rejection. One patient experienced a fall in ejection fraction without histologic evidence of rejection. Sirolimus was discontinued in three patients because of side effects: bone marrow suppression, presumed lymphocytic pneumonitis, and generalized acneform rash complicated by an axillary abcess; 50% of patients continue on sirolimus. In conclusion, withdrawal of CNIs after heart transplantation resulted in an improvement in renal function in most patients: 43% experienced a substantial improvement. CNI elimination protocols need to be refined to reduce the risk of breakthrough rejection and to minimize side effects while protecting renal function after heart transplantation.