Qualitative and quantitative detection of SARS-CoV-2 antibodies from dried blood spots.

INTRODUCTION: Dried blood spot (DBS) sampling is a minimally invasive method for specimen collection with potential multifaceted uses, particularly for serosurveillance of previous SARS-CoV-2 infection. In this study, we assessed DBS as a potential specimen type for assessing IgG and total (including IgG and IgM) antibodies to SARS-CoV-2 in vaccinated and naturally infected patients. METHODS: Six candidate buffers were assessed for eluting blood from DBS cards. The study utilized one hundred and five paired plasma specimens and DBS specimens from prospectively collected SARS-CoV-2 vaccinated individuals, remnants from those with PCR confirmed SARS-CoV-2 infections, or remnants from those without history of infection or vaccination. All specimens were tested with the Siemens SARS-CoV-2 total assay (COV2T) or IgG assay (sCOVG). RESULTS: The lowest backgrounds were observed with water and PBS, and water was used for elution. Relative to plasma samples, DBS samples had a positive percent agreement (PPA) of 94.4% (95% CI: 94.9-100%) for COV2T and 79.2 (68.4-87.0) for sCOVG using the manufacturer's cutoff. The NPA was 100 % (87.1-100.0 and 85.13-100) for both assays. Dilution studies revealed 100% (95% CI: 90.8-100%) qualitative agreement between specimen types on the COV2T assay and 98.0% (88.0-99.9%) with the sCOVG using study defined cutoffs. CONCLUSION: DBS specimens demonstrated high PPA and NPA relative to plasma for SARS-CoV-2 serological testing. Our data support feasibility of DBS sampling for SARS-CoV-2 serological testing.

Perceptions of Seasonal Influenza Vaccine Among U.S. Army Civilians and Dependents in the Kaiserslautern Military Community: A Mixed-Methods Survey.

INTRODUCTION: Influenza is a globally occurring viral respiratory infection that can lead to hospitalizations and death. An influenza outbreak can interfere with combat readiness in a military setting, as the infection can incapacitate soldiers. Vaccination remains the most effective tool to prevent and mitigate seasonal influenza. Although influenza vaccinations for U.S. Army soldiers can be monitored through military health systems, those systems cannot capture DoD civilians and Army dependents who may not use military health services. This study aims to gauge flu vaccine uptake and perceptions in U.S. Army civilians and dependents. MATERIALS AND METHODS: An online survey was e-mailed to civilian and dependent enrollees of Landstuhl Regional Medical Center. The survey contained 24 questions pertaining to demographics, vaccine history, history of the flu, and beliefs toward vaccines. Chi-square tests, t-tests, and logistic regressions were performed to investigate the association between demographic, behavior, and belief factors with vaccine uptake. Free-text answers were coded and categorized by themes. RESULTS: Over 70% of respondents were vaccinated for the flu. There were differences between vaccinated and unvaccinated respondents regarding their perceptions of barriers to vaccination, benefits of the flu vaccine, severity of flu symptoms, and personal risk of getting ill with the flu. After controlling for confounders, flu vaccination in the previous season and healthcare worker status were associated with increased vaccine uptake, while perceived barriers to influenza vaccination were associated with decreased vaccine uptake. CONCLUSIONS: Flu vaccine uptake may be increased by increasing access to vaccination, promoting vaccination and addressing concerns at the provider level, and engaging positively framed public messaging. Increasing flu vaccine uptake is of particular importance as the flu season approaches during the COVID-19 (Coronavirus disease 2019) pandemic.

Microfluidic genotyping by rapid serial PCR and high-speed melting analysis.

BACKGROUND: Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-a-chip disposables to analyze a few targets. In genetics, multiple variants need to be analyzed, and different work flows that rapidly analyze multiple loci in a few targets are attractive. METHODS: We used a microfluidic platform tailored to rapid serial PCR and high-speed melting (HSM) to genotype 4 single nucleotide variants. A contiguous stream of master mix with sample DNA was pulsed with each primer pair for serial PCR and melting. Two study sites each analyzed 100 samples for F2 (c.*97G>A), F5 (c.1601G>A), and MTHFR (c.665C>T and c.1286A>C) after blinding for genotype and genotype proportions. Internal temperature controls improved melting curve precision. The platform's liquid-handling system automated PCR and HSM. RESULTS: PCR and HSM were completed in a total of 12.5 min. Melting was performed at 0.5 °C/s. As expected, homozygous variants were separated by melting temperature, and heterozygotes were identified by curve shape. All samples were correctly genotyped by the instrument. Follow-up testing was required on 1.38% of the assays for a definitive genotype. CONCLUSIONS: We demonstrate genotyping accuracy on a novel microfluidic platform with rapid serial PCR and HSM. The platform targets short turnaround times for multiple genetic variants in up to 8 samples. It is also designed to allow automatic and immediate reflexive or repeat testing depending on results from the streaming DNA. Rapid serial PCR provides a flexible genetic work flow and is nicely matched to HSM analysis.

Human papillomavirus profile of women in Belize City, Belize: correlation with cervical cytopathologic findings.

Cervical carcinoma is the most common cancer among Belizean women; however, data regarding the frequency of human papillomavirus (HPV) genotypes and their association with cervical cancer are nonexistent. We therefore included HPV genotyping as part of a week-long cervical cancer screening campaign conducted in Belize City in 2007. Conventional Papanicolaou smears with Hybrid Capture (HC) 2 HPV testing were performed on 463 women. All HC2-positive samples were genotyped using a developmental GP5+/GP6+ polymerase chain reaction-coupled Luminex assay for 2 low-risk and 18 high-risk HPV types. The prevalence of high-risk HPV was 15.6% in the total population, 10.1% in those with normal cytologic findings, and 93.3% in women with a high-grade squamous intraepithelial lesion. Of patients with HPV infections, 35% had multiple types (5.4% of the total group). Of all women and of women with normal cytologic findings, 5.2% and 2.8%, respectively, had HPV16 or 18. For all women, HPV16, 18, 56, and 52 were present in decreasing order of frequency. HPV11 was present in only one patient, and none had HPV6. HPV16 was found in 47% of high-grade squamous epithelial lesions; however, no case of HSIL had HPV18 or 45. HPV35 and HPV58 were the next most common types in high-grade squamous intraepithelial lesion, each occurring in 20% of cases of high-grade squamous intraepithelial lesion, followed by HPV31 in 13.3%. Although women younger than 25 years old were underrepresented, these data suggest that the HPV profile of this cohort of Belizean women differs somewhat from that in the region. In addition, these data are of importance with regard to the development of HPV vaccines that will be used in less developed countries, where care should be taken not to implement vaccination at the cost of basic screening and diagnostic services.

Impact of 6-month frozen storage of cervical specimens in alkaline buffer conditions on human papillomavirus genotyping.

The impact of 6-month storage of cervical specimens under alkaline conditions that occurs as the result of Hybrid Capture 2 testing on human papillomavirus (HPV) genotyping is not well documented. To examine this issue, 143 frozen hc2-positive specimens in specimen transport medium were selected at random from each of the following groups: specimens stored for 6 months, 4 months, and 2.5 months under alkaline pH (pH 12-13) and specimens stored 1 month at neutral pH (pH 6-7) as controls. Specimens were tested in a masked fashion for 20 HPV genotypes (HPV6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) using a prototype, research-use-only GP5+/6+ L1 consensus PCR method and multiplex hybridization using Luminex xMAP for detection of specific HPV genotypes One control specimen had missing test results. There were no statistical differences in the number of HPV genotypes detected, number of carcinogenic HPV genotypes detected, or in the signal strength among HPV-positive results across groups. Six-month frozen storage of cervical specimens at alkaline pH had little impact on testing for HPV genotypes among hc2-positive women using this HPV genotyping method.