A pyrosequencing protocol for rapid identification of SARS-CoV-2 variants.

Next-generation sequencing (NGS) is the primary method used to monitor the distribution and emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants around the world; however, it is costly and time-consuming to perform and is not widely available in low-resourced geographical regions. Pyrosequencing has the potential to augment surveillance efforts by providing information on specific targeted mutations for rapid identification of circulating and emerging variants. The current study describes the development of a reverse transcription (RT)-PCR-pyrosequencing assay targeting >65 spike protein gene (S) mutations of SARS-CoV-2, which permits differentiation of commonly reported variants currently circulating in the United States with a high degree of confidence. Variants typed using the assay included B.1.1.7 (Alpha), B.1.1.529 (Omicron), B.1.351 (Beta), B.1.375, B.1.427/429 (Epsilon), B.1.525 (Eta), B.1.526.1 (Iota), B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.621 (Mu), P1 (Gamma), and B.1.1 variants, all of which were confirmed by the NGS data. An electronic typing tool was developed to aid in the identification of variants based on mutations detected by pyrosequencing. The assay could provide an important typing tool for rapid identification of candidate patients for monoclonal antibody therapies and a method to supplement SARS-CoV-2 surveillance efforts by identification of circulating variants and novel emerging lineages.

Intrinsically accurate sensing with an optomechanical accelerometer.

We demonstrate a microfabricated optomechanical accelerometer that is capable of percent-level accuracy without external calibration. To achieve this capability, we use a mechanical model of the device behavior that can be characterized by the thermal noise response along with an optical frequency comb readout method that enables high sensitivity, high bandwidth, high dynamic range, and SI-traceable displacement measurements. The resulting intrinsic accuracy was evaluated over a wide frequency range by comparing to a primary vibration calibration system and local gravity. The average agreement was found to be 2.1 % for the calibration system between 0.1 kHz and 15 kHz and better than 0.2 % for the static acceleration. This capability has the potential to replace costly external calibrations and improve the accuracy of inertial guidance systems and remotely deployed accelerometers. Due to the fundamental nature of the intrinsic accuracy approach, it could be extended to other optomechanical transducers, including force and pressure sensors.

Comparison of human papillomavirus detections in urine, vulvar, and cervical samples from women attending a colposcopy clinic.

While urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P = 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.

Reproducibility of linear array for human papillomavirus genotyping.

We conducted a Linear Array test/retest analysis using cytologic specimens from 198 women. A total of 67.2% of samples had the same human papillomavirus (HPV) types detected in both tests (type-specific positive agreement was 83.3% overall [Kappa = 0.9] and 86.8% for carcinogenic types [Kappa = 0.92]). Discordance was highest with a low hybridization signal strength. Overall, Linear Array was highly reproducible.

The role of co-factors in the progression from human papillomavirus infection to cervical cancer.

OBJECTIVE: Co-factors for cervical cancer, including oral contraceptive (OC) use, smoking and multiparity have been identified; however, the stage at which they act in cervical carcinogenesis is not clear. We compared established risk factors among women with CIN2 and CIN3 to evaluate the heterogeneity of these factors in precancer and also assessed their role during cervical carcinogenesis. METHODS: The current analysis included 2783 women with various stages of cervical disease who were enrolled in the Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED) and the Biopsy Study. Associations of co-factors within cervical precancer and at different stages of cervical carcinogenesis were estimated using logistic regression. RESULTS: Long-term OC use (10+years vs. never: OR=2.42, 95% CI: [1.13-5.15]), multiparity (3+ births vs. nulliparous: OR=1.54 [1.04-2.28]), smoking (ever vs. never: OR=1.95 [1.48-2.58]), and no Pap test in the previous five years (2.05 [1.32-3.17]) were positively associated with CIN3 compared to CIN2. We observed that long-term OC use, parity and smoking were associated with an increased risk of CIN3 compared to

The differential diagnosis of pilocytic astrocytoma with atypical features and malignant glioma: an analysis of 16 cases with emphasis on distinguishing molecular features.

Rare pilocytic astrocytomas (PA) have atypical histologic and clinicoradiologic features that raise the differential diagnosis of glioblastoma. Whether ancillary studies can supplement histopathologic examination in placing these cases accurately on the spectrum of WHO Grade I PA to higher-grade glioma is not always clear, partly because these cases are not common. Here, ten PAs with atypical clinicoradiologic and histologic features and six pediatric glioblastoma multiforme (pGBMs) were analyzed for BRAF V600E, IDH1, IDH2, and TP53 mutations. Ki-67, p53, and p16 protein expression were also examined by immunohistochemistry. BRAF-KIAA1549 fusion status was assessed in the PA subgroup. The rate of BRAF-KIAA1549 fusion was high in these PAs (5/7 tumors) including four extracerebellar examples. A single BRAF V600E mutation was identified in the fusion-negative extracerebellar PA of a very young child who succumbed to the disease. TP53 mutations were present only in malignant gliomas, including three pGBMs and one case designated as PA with anaplastic features (with consultation opinion of pGBM). IDH1 and IDH2 were wild type in all cases, consistent with earlier findings that IDH mutations are not typical in high-grade gliomas of patients ≤14 years of age. Immunohistochemical studies showed substantial overlap in Ki-67 labeling indices, an imperfect correlation between p53 labeling and TP53 mutation status, and complete p16 loss in only two pGBMs but in no PAs. These results suggest that (a) BRAF-KIAA1549 fusion may be common in PAs with atypical clinicoradiologic and histologic features, including those at extracerebellar sites, (b) BRAF V600E mutation is uncommon in extracerebellar PAs, and (c) TP53 mutation analysis remains a valuable tool in identifying childhood gliomas that will likely behave in a malignant fashion.

Human papillomavirus load measured by Linear Array correlates with quantitative PCR in cervical cytology specimens.

Carcinogenic human papillomavirus (HPV) infections are necessary causes of most anogenital cancers. Viral load has been proposed as a marker for progression to cancer precursors but has been confirmed only for HPV16. Challenges in studying viral load are related to the lack of validated assays for a large number of genotypes. We compared viral load measured by Linear Array (LA) HPV genotyping with the gold standard, quantitative PCR (Q-PCR). LA genotyping and Q-PCR were performed in 143 cytology specimens from women referred to colposcopy. LA signal strength was measured by densitometry. Correlation coefficients and receiver operating characteristic (ROC) analyses were used to evaluate analytical and clinical performance. We observed a moderate to strong correlation between the two quantitative viral load measurements, ranging from an R value of 0.61 for HPV31 to an R value of 0.86 for HPV52. We also observed agreement between visual LA signal strength evaluation and Q-PCR. Both quantifications agreed on the disease stages with highest viral load, which varied by type (cervical intraepithelial neoplasia grade 2 [CIN2] for HPV52, CIN3 for HPV16 and HPV33, and cancer for HPV18 and HPV31). The area under the curve (AUC) for HPV16 Q-PCR at the CIN3 cutoff was 0.72 (P = 0.004), and the AUC for HPV18 LA at the CIN2 cutoff was 0.78 (P = 0.04). Quantification of LA signals correlates with the current gold standard for viral load, Q-PCR. Analyses of viral load need to address multiple infections and type attribution to evaluate whether viral load has clinical value beyond the established HPV16 finding. Our findings support conducting comprehensive studies of viral load and cervical cancer precursors using quantitative LA genotyping data.

Laser-based comparison calibration of laboratory standard microphones.

A precision laser-based comparison calibration method for laboratory standard microphones is described that uses reference microphones calibrated by the pressure reciprocity method. Electrical drive current and diaphragm velocity are measured while the microphones are driven as transmitters/sources of sound; the diaphragm velocity is measured using scanning laser Doppler vibrometry. Sensitivities determined using this method display very good agreement with those determined directly by reciprocity for seven such test microphones at 250 and 1000 Hz. At these frequencies, the expanded (coverage factor k = 2) uncertainties of this comparison calibration method for these microphones are ±0.05 dB.

MEMS Young's Modulus and Step Height Measurements With Round Robin Results.

This paper presents the results of a microelectromechanical systems (MEMS) Young's modulus and step height round robin experiment, completed in April 2009, which compares Young's modulus and step height measurement results at a number of laboratories. The purpose of the round robin was to provide data for the precision and bias statements of two \ related Semiconductor Equipment and Materials International (SEMI) standard test methods for MEMS. The technical basis for the test methods on Young's modulus and step height measurements are also provided in this paper. Using the same test method, the goal of the round robin was to assess the repeatability of measurements at one laboratory, by the same operator, with the same equipment, in the shortest practical period of time as well as the reproducibility of measurements with independent data sets from unique combinations of measurement setups and researchers. Both the repeatability and reproducibility measurements were done on random test structures made of the same homogeneous material. The average repeatability Young's modulus value (as obtained from resonating oxide cantilevers) was 64.2 GPa with 95 % limits of ± 10.3 % and an average combined standard uncertainty value of 3.1 GPa. The average reproducibility Young's modulus value was 62.8 GPa with 95 % limits of ± 11.0 % and an average combined standard uncertainty value of 3.0 GPa. The average repeatability step height value (for a metal2-over-poly1 step from active area to field oxide) was 0.477 µm with 95 % limits of 7.9 % and an average combined standard uncertainty value of 0.014 µm. The average reproducibility step height value was 0.481 µm with 95 % limits of ± 6.2 % and an average combined standard uncertainty value of 0.014 µm. In summary, this paper demonstrates that a reliable methodology can be used to measure Young's modulus and step height. Furthermore, a micro and nano technology (MNT) 5-in-1 standard reference material (SRM) can be used by industry to compare their in-house measurements using this methodology with NIST measurements thereby validating their use of the documentary standards.

Grading the severity of cervical neoplasia based on combined histopathology, cytopathology, and HPV genotype distribution among 1,700 women referred to colposcopy in Oklahoma.

Diagnosis and treatment of cervical cancer precursors rely on colposcopic biopsy, which is sometimes hampered by incorrect biopsy placement and the unclear prognostic value of poorly reproducible diagnoses such as cervical intraepithelial neoplasia (CIN) Grade 1 and 2. Searching for discrete disease categories that incorporate the value of cytology and that reflect the causal role of particular HPV types, we analyzed histology, cytology and HPV genotype distributions in the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED). This cross-sectional study comprises approximately 1,700 women referred to colposcopy or treatment for the spectrum of cervical disease, including 439 women with

Multiple human papillomavirus genotype infections in cervical cancer progression in the study to understand cervical cancer early endpoints and determinants.

Determining the causal attribution of human papillomavirus (HPV) genotypes to cervical disease is important to estimate the effect of HPV vaccination and to establish a type spectrum for HPV-based screening. We analyzed the prevalence of HPV infections and their attribution to cervical disease in a population of 1,670 women referred to colposcopy for abnormal cytology at the University of Oklahoma. HPV genotyping was performed from cytology specimens using the Linear Array assay that detects 37 HPV genotypes. We used different methods of type attribution to revised cervical disease categories. We found very high prevalence of multiple HPV infections with up to 14 genotypes detected in single specimens. In all disease categories except for cancers, there was a significant trend of having more infections at a younger age. We did not see type interactions in multiple genotype infections. HPV16 was the most frequent genotype at all disease categories. Based on different attribution strategies, the attribution of vaccine genotypes (6, 11, 16, 18) ranged from 50.5 to 67.3% in cancers (n = 107), from 25.6 to 74.8% in CIN3 (n = 305), from 15.2 to 52.2% in CIN2 (n = 427), and from 6.6 to 26.0% in

Human papillomavirus cofactors by disease progression and human papillomavirus types in the study to understand cervical cancer early endpoints and determinants.

Human papillomavirus (HPV) cofactors for cervical cancer include smoking, multiparity, and oral contraceptive use, but their mechanisms of action are not fully understood. It is also unknown whether cofactors vary by HPV genotypes. The Study to Understand Cervical Cancer Early Endpoints and Determinants (SUCCEED) is a cross-sectional study comprising women referred to the University of Oklahoma from November 2003 to September 2007 for abnormal cervical screening results. Detailed questionnaire data and liquid cytology specimens were collected and the latter was genotyped for HPV using the LINEAR ARRAY HPV Genotyping Test. The present analysis includes women with both questionnaire and HPV data and diagnosed with

Controlled formation and resistivity scaling of nickel silicide nanolines.

We demonstrate a top-down method for fabricating nickel mono-silicide (NiSi) nanolines (also referred to as nanowires) with smooth sidewalls and line widths down to 15 nm. Four-probe electrical measurements reveal that the room temperature electrical resistivity of the NiSi nanolines remains constant as the line widths are reduced to 23 nm. The resistivity at cryogenic temperatures is found to increase with decreasing line width. This finding can be attributed to electron scattering at the sidewalls and is used to deduce an electron mean free path of 6.3 nm for NiSi at room temperature. The results suggest that NiSi nanolines with smooth sidewalls are able to meet the requirements for implementation at the 22 nm technology node without degradation of device performance.

Using the EPIDEM Model of Quality Improvement to Increase Value of BCR-ABL1 Tests.

Objectives: As pathologists and laboratorians, we can enhance patient care by promoting the appropriate ordering of diagnostic tests. Our goal was to improve the ordering of BCR-ABL1 tests by using the EPIDEM model of quality improvement. Methods: We applied the EPIDEM model, which emphasizes understanding local context, culture, and resources, to explore inappropriate BCR-ABL1 ordering, promote and implement a new reflexive testing strategy in-house, document and evaluate effectiveness, and make stepwise modifications. Results: Multiple quality improvement interventions correlated with cost savings and decreased total errors and incorrect orders for both BCR-ABL1 major and minor positive patients. Furthermore, our laboratory built stronger collaborative relationships with colleagues within and outside of pathology. Conclusions: Our molecular pathology laboratory successfully used the EPIDEM model of quality improvement to improve the ordering of BCR-ABL1 tests and promote better patient care by focusing on educational efforts and modification of laboratory workflow.

DNA extraction: an understudied and important aspect of HPV genotyping using PCR-based methods.

Testing for the group of approximately 15 carcinogenic human papillomavirus (HPV) genotypes is an important adjunct to cytology. Because carcinogenic strengths of HPV types differ greatly, assays that permit identification of individual HPV genotypes are being introduced. Most HPV genotyping systems proposed for clinical use are PCR-based, depending heavily for validity on careful attention to numerous details. One understudied detail is the effect of different sample preparation methods including DNA extraction. This study examines the influence of DNA extraction on performance of a new PCR-based genotyping kit, the Roche LINEAR ARRAY HPV assay. When volume of sample extracted, DNA extraction methods and/or amount of DNA tested were varied, the HPV type results were reproducible for strong viral bands but not weak ones. Moreover, although the experiments were not comprehensive, they showed that the manufacturer-approved DNA extraction method might not be the best method for use in this assay. Because different "front end" protocols introduce variability into genotyping results, the authors urge laboratories not to vary methods for this assay without due consideration. The results suggest that companies carefully optimize DNA extraction methods prior to commercial introduction of their PCR-based genotyping assays destined for widespread clinical use.

A common 1317TC polymorphism in MTHFR can lead to erroneous 1298AC genotyping by PCR-RE and TaqMan probe assays.

Multiple polymorphisms of the methylenetetrahydrofolate reductase gene (MTHFR) have been documented, and some are associated with decreased enzyme activity. One polymorphism, 677CT, is commonly tested in the context of thrombosis. Recently, consideration has also been extended to 1298AC, which is also associated with reduced catalytic activity. This report describes problems arising during the development of a PCR restriction enzyme assay for 1298AC. In the process of validating a PCR-MboII assay, it was realized that a nearby 1317TC polymorphism rendered a restriction fragment length polymorphism (RFLP) pattern that was virtually indistinguishable from a 1298A allele. An alternate approach, involving primer mutagenesis and Fnu4HI digestion, resolved the problem. To validate the latter assay, samples were obtained from a CLIA-approved facility that had developed a multiplexed real-time PCR using TaqMan probes for simultaneous assessment of 677CT and 1298AC. Interlaboratory results concurred for 10 out of 11 samples; however, one sample was consistently heterozygous by PCR-Fnu4HI and homozygous 1298CC by real-time PCR. Bidirectional sequencing confirmed that the sample was a compound 1298AC/1317TC heterozygote. It is likely that the 1317C variant, residing with 1298A on one chromosome, disrupted primer annealing in the TaqMan assay, leading to preferential amplification of the 1298C/1317T chromosome and hence an aberrant homozygous 1298CC genotype. This validation exercise emphasizes the need for comprehensive appraisal and continual reassessment of the optimal performance of molecular diagnostic assays. It is hoped that laboratories offering MTHFR 1298AC testing are cognizant of some of the inherent problems in published methods.

Relationship of the 1793G-A and 677C-T polymorphisms of the 5,10-methylenetetrahydrofolate reductase gene to coronary artery disease.

Numerous studies have investigated the relationship between polymorphisms, in particular 677C-T and 1298A-C, of the methylene-tetrahydrofolate reductase (MTHFR) gene and coronary artery disease (CAD) with conflicting results. This study investigates the potential association of two point mutations in MTHFR, 677C-T and 1793G-A, along with other risk factors, with CAD. This is the first hospital-based study to investigate 1793G-A in this context. Genotype analysis was performed on 729 Caucasians and 66 African Americans undergoing coronary angiography using a novel PCR-based assay involving formation of Holliday junctions. Allelic frequencies for 677C-T were 66.2% C and 33.8% T for Caucasians and 90.9% C and 9.1% T for African Americans. With respect to the 1793G-A polymorphism, allelic frequencies were 94.7% G and 5.3% A for Caucasians and 99.2% G and 0.8% A for African Americans. Disease associations were examined in the Caucasian patients due to their greater genotype variability and larger number in the patient cohort. Results suggest that neither 677CT heterozygotes (OR-1.36; 95% CI 0.95 to 1.96) nor mutant homozygotes (OR-0.73; 95% CI 0.44 to 1.20) have either an increased or decreased risk for CAD compared to the 677CC genotype. Likewise, the 1793GA genotype did not demonstrate a statistically significant association with CAD compared to 1793GG patients (OR-0.79; 95% CI 0.47 to 1.33). Mean homocysteine levels (mumol/L) increased from normal to mutant for 677C-T (677CC: 10.2; 677CT: 11.0; 677TT: 11.6) and normal to heterozygous in 1793G-A (1793GG: 10.7; 1793GA: 11.5). These MTHFR polymorphisms did not contribute to the prediction of clinically defined CAD in Caucasians.

Abnormal melt curve profile during prothrombin 20210G --> A analysis due to the 20209C --> T variant.

The common factor II 20210G --> A mutation, located in the 3'-untranslated region, is an important risk factor for the development of thromboembolic disorders, especially in Caucasians. A number of methods are employed for clinical laboratory diagnosis of this mutation, some of which are capable of detecting adjacent 3'-end sequence variations. We present results from an African deep vein thrombosis patient tested for the 20210G --> A mutation by real-time polymerase chain reaction and melt-curve analysis using hybridization probes that incidentally detected an adjacent 3'-untranslated region variant. The patient sample was tested using the Factor II (Prothromobin) G20210A Kit (Roche Diagnostics, Indianapolis, Indiana, USA), in conjunction with the Roche LightCycler. A polymerase chain reaction fragment from the 3'-end of the F2 gene was subsequently sequenced for identification of the variant. Melt-curve analysis revealed a normal 20210*G peak and an unknown aberrant allelic peak. Following sequence analysis, the patient was determined to be heterozygous for 20209C --> T. The presence of the 20209C --> T variant in the current patient and in eight other reported individuals of African descent, most with thrombosis-associated complaints, suggests that this rare variant poses a potential increased risk for thromboembolic disease in this ethnic group.

RM 8111: Development of a Prototype Linewidth Standard.

Staffs of the Semiconductor Electronics Division, the Information Technology Laboratory, and the Precision Engineering Laboratory at NIST, have developed a new generation of prototype Single-Crystal CD (Critical Dimension) Reference (SCCDRM) Materials with the designation RM 8111. Their intended use is calibrating metrology instruments that are used in semiconductor manufacturing. Each reference material is configured as a 10 mm × 11 mm silicon test-structure chip that is mounted in a 200 mm silicon carrier wafer. The fabrication of both the chip and the carrier wafer uses the type of lattice-plane-selective etching that is commonly employed in the fabrication of micro electro-mechanical systems devices. The certified CDs of the reference features are determined from Atomic Force Microscope (AFM) measurements that are referenced to high-resolution transmission-electron microscopy images that reveal the cross-section counts of lattice planes having a pitch whose value is traceable to the SI meter.

Adenocarcinoma of the cervix involving the fallopian tube mucosa: report of a case.

BACKGROUND: Fallopian tube involvement by cervical carcinoma has rarely been documented, with literature reports focusing primarily on squamous cell carcinoma. CASE PRESENTATION: In this report, we present the case of a 50 year old woman who presented with an abnormal Pap test with atypical squamous and glandular cells. A loop electrosurgical excision procedure (LEEP) was performed and led to the diagnosis of stage IB1 endocervical adenocarcinoma. Subsequent radical hysterectomy, bilateral salpingo-oophorectomy, and bilateral pelvic lymph node dissection showed a well-differentiated endocervical adenocarcinoma of usual type with superficial spread to the endometrium and right fallopian tube. The patient received no adjuvant therapy and has remained without evidence of disease. CONCLUSIONS: While the advent of more extensive fallopian tube sampling has led to increased discovery and discussion of fallopian tube involvement by metastatic carcinoma, its impact on treatment and prognosis remains to be seen.