Complete Concordance Research Articles

One-step colorimetric isothermal detection of COVID-19 with AI-assisted automated result analysis: A platform model for future emerging point-of-care RNA/DNA disease diagnosis

Colorimetric loop-mediated DNA isothermal amplification-based assays have gained momentum in the diagnosis of COVID-19 owing to their unmatched feasibility in low-resource settings. However, the vast majority of them are restricted to proprietary pH-sensitive dyes that limit downstream assay optimization or hinder efficient result interpretation. To address this problem, we developed a novel dual colorimetric RT-LAMP assay using in-house pH-dependent indicators to maximize the visual detection and assay simplicity, and further integrated it with the artificial intelligence (AI) operated tool (RT-LAMP-DETR) to enable a more precise and rapid result analysis in large scale testing. The dual assay leverages xylenol orange (XO) and a newly formulated lavender green (LG) dye for distinctive colorimetric readouts, which enhance the test accuracy when performed and analyzed simultaneously. Our RT-LAMP assay has a detection limit of 50 viral copies/reaction with the cycle threshold (Ct) value ≤ 39.7 ± 0.4 determined by the WHO-approved RT-qPCR assay. RT-LAMP-DETR exhibited a complete concordance with the results from naked-eye observation and RT-qPCR, achieving 100% sensitivity, specificity, and accuracy that altogether render it suitable for ultrasensitive point-of-care COVID-19 screening efforts. From the perspective of pandemic preparedness, our method offers a simpler, faster, and cheaper (∼$8/test) approach for COVID-19 testing and other emerging pathogens with respect to RT-qPCR.

Next-day detection of viable Listeria monocytogenes by multiplex reverse transcriptase real-time PCR

Listeria monocytogenes continues to be a major challenge for the food industry due to its ubiquity and difficulty to be eliminated from processing facilities. The DNA amplification-based methods can overcome limitations of culture-based methods; however, due to the stability of the DNA, false positive results may occur, associated to the presence of harmless, dead microorganisms. The incorrect assessment of the results will have a huge impact on the producers in terms of stopping the production/distribution, leading to economic losses. In the current study, a multiplex RT-qPCR method was developed. The detection of mRNA allows for the specific detection of live bacteria. Additionally, two genetic targets (hly and actA) were co-amplified for improved specificity, along with an Internal Amplification Control to rule out false negative results due to reaction inhibition. The optimized methodology was evaluated in smoked salmon samples inoculated with different combinations and concentrations of live and dead bacteria. The method demonstrated high sensitivity (LOD50/LOD95 of 1.2/5.1 cfu/25 g). The performance of the method was compared against the reference standard ISO 11290, for which a Cohen's k of 0.94 was obtained, being interpreted as “almost complete concordance” among both methods. In addition, other parameters evaluated included the relative sensitivity, specificity, accuracy, as well as the positive and negative predictive values, all being above 90%. This next-day methodology can significantly reduce the time of analysis of culture-based methods and can specifically detect live L. monocytogenes.

Investigation of genes and pathways involved in breast cancer subtypes through gene expression meta-analysis

BackgroundMolecular-based studies have revealed heterogeneity in Breast cancer BC while also improving classification and treatment. However, efforts are underway to distinguish between distinct subtypes of breast cancer. In this study, the results of several microarray studies were combined to identify genes and pathways specific to each BC subtype. MethodsMeta-analysis of multiple gene expression profile datasets was screened to find differentially expressed genes (DEGs) across subtypes of BC and normal breast tissue samples. Protein-protein interaction network and gene set enrichment analysis were used to identify critical genes and pathways associated with BC subtypes. The differentially expressed genes from meta-analysis was validated using an independent comprehensive breast cancer RNA-sequencing dataset obtained from the Cancer Genome Atlas (TCGA). ResultsWe identified 110 DEGs (13 DEGs in all and 97 DEGs in each subtype) across subtypes of BC. All subtypes had a small set of shared DEGs enriched in the Chemokine receptor bind chemokine pathway. Luminal A specific were enriched in the translational elongation process in mitochondria, and the enhanced process in luminal B subtypes was interferon-alpha/beta signaling. Cell cycle and mitotic DEGs were enriched in the basal-like group. All subtype-specific DEG genes (100%) were successfully validated for Luminal A, Luminal B, ERBB2, and Normal-like. However, the validation percentage for Basal-like group was 77.8%. ConclusionIntegrating researches such as a meta-analysis of gene expression might be more effective in uncovering subtype-specific DEGs and pathways than a single-study analysis. It would be more beneficial to increase the number of studies that use matched BC subtypes along with GEO profiling approaches to reach a better result regarding DEGs and reduce probable biases. However, achieving 77.8% overlap in basal-specific genes and complete concordance in specific genes related to other subtypes can implicate the strength of our analysis for discovering the subtype-specific genes.

Pooled testing of sputum with Xpert MTB/RIF and Xpert Ultra during tuberculosis active case finding campaigns in Lao People’s Democratic Republic

IntroductionActive case finding (ACF) of individuals with tuberculosis (TB) is a key intervention to find the 30% of people missed every year. However, ACF requires screening large numbers of individuals...

High-resolution melting PCR analysis for genotyping the gene polymorphism of TNF-α, TGF-β1, IL-10, and IFN-γ in lung transplant recipients

High-resolution melting (HRM) analysis is a genotyping method which has the advantages of simple, rapid, low-cost and closed-tube operation.This study evaluated HRM analysis as an option for detecting the single nucleotide polymorphism (SNP) of cytokine, and profiled the distribution of cytokine gene polymorphism in the lung transplant recipients (LTRs).High-resolution melting-polymerase chain reaction (HRM-PCR) assays for genotyping tumor necrosis factor alpha (TNF-α) (-308 A/G), tumor growth factor beta 1 (TGF-β1) (+869 T/C), interleukin 10 (IL-10) (-592 C/A, -819 T/C, -1082 G/A), and interferon gamma (IFN-γ) (+874 T/A) SNPs were developed on the LightCycler® 480. The SNPs of the aforementioned cytokine genes in 322 LTRs and 266 normal controls were detected using HRM-PCR approach. To confirm the accuracy of the HRM-PCR assay, we randomly selected 100 samples from the LTRs and detected the aforementioned SNPs with sequence-specific primer-polymerase chain reaction (SSP-PCR) method, using a commercial kit.The data show that the HRM-PCR assay can distinguish all the cytokine SNPs, and the results of HRM-PCR analysis are in complete concordance to the genotyping results obtained using a commercial kit (κ = 1.0). Our data also show that the allele and genotype frequencies of the abovementioned cytokine are not significantly different between the LTRs and the control groups (p > 0.05). In addition, we found the genotypes of TGF-β1 +869 associated with high expression phenotype were prevalent in the LTRs. On the contrary, for TNF-α -308, IL-10 and IFN-γ, the genotypes associated with low expression phenotype were most common in the LTRs.In this study, we described a rapid, low-cost and high-throughput HRM-PCR technology for genotyping cytokine SNPs. Our data may be utilized for future studies examining the associations of cytokine gene polymorphisms with the prognosis of the LTRs.

The inconsistency between two major aneuploidy-screening platforms\u2014single-nucleotide polymorphism array and next-generation sequencing\u2014in the detection of embryo mosaicism

BackgroundIn preimplantation genetic testing for aneuploidy (PGT-A), appropriate evaluation of mosaic embryos is important because of the adverse implications of transferring embryos with high-level mosaicism or discarding those with low-level mosaicism. Despite the availability of multiple reliable techniques for PGT-A, data comparing the detection of mosaicism using these techniques are scarce. To address this gap in the literature, we compared the detection ability of the two most commonly used PGT-A platforms, next-generation sequencing (NGS) and the single-nucleotide polymorphism (SNP) array, for mosaic embryos.ResultsWe retrospectively reviewed the data of PGT-A or preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR) conducted at our center from January 2018 to October 2020, and selected blastocysts that underwent aneuploidy screening with both an SNP array and NGS. Trophectoderm biopsy, multiple displacement amplification (MDA), and aneuploidy screening with an SNP array were conducted on the enrolled blastocysts. When the SNP array indicated mosaicism, NGS was performed on the corresponding MDA product for verification. Among the 105 blastocysts diagnosed with mosaicism with the SNP array, 80 (76.19%) showed mosaicism in NGS, with complete and partial concordance rates of 47.62% (50/105) and 18.10% (19/105), respectively. The complete discordance rate of the two platforms was 34.29% (36/105). That is, 10.48% (11/105) of the blastocysts were diagnosed with completely different types of mosaicism with the two platforms, while 13.33% (14/105) and 10.48% (11/105) of the embryos diagnosed as showing mosaicism with SNP were detected as showing aneuploidy and euploidy with NGS, respectively.ConclusionsThe consistency of NGS and the SNP array in the diagnosis of embryo mosaicism is extremely low, indicating the need for larger and well-designed studies to determine which platform is more accurate in detecting mosaic embryos.

Application of Interphase Fluorescent in Situ Hybridization: a Screening Tool for the Diagnosis of Microdeletion Syndrome.

Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy between the results of interphase and metaphase, we compared the quantitative results of FISH for 5 kinds of microdeletion syndrome and gender determination disorders (SDD). A total of 282 (135 for DiGeorge syndrome, 20 for Kalmann syndrome, 7 for Miller-Dieker syndrome, 38 for Prader Willi/Angelman syndrome, 62 for Williams syndrome, and 20 for SDD (SRY FISH)) were enrolled. For SRY FISH, we artificially mixed fresh blood of male and female with various ratios and then compared the results of metaphase and interphase SRY FISH. Using a bio-cell chip, we performed interphase FISH in 168 patients with microdeletion syndromes and compared the results with manual interphase. The concordance rate between the results of metaphase and interphase was 100% in microdeletion syndrome. In the disorders of gender development, SRY FISH showed 100% concordance between interphase and metaphase when we counted 50 metaphase cells and 100 interphase cells. Comparison with mixtures of male and female blood at various ratios also showed 100% concordance. The results of bio-cell chip showed 100% concordance between previous interphase FISH results. Considering the complete concordance between interphase and metaphase in microdeletion syndrome, the application of interphase FISH without performing metaphase FISH can be a screening test for microdeletion syndrome. Confirmation by metaphase FISH can be performed only in cases with abnormal results by interphase FISH.

Correlation between Surgical Outcomes of Primary Hyperparathyroidism with Neck Ultrasound and Parathyroid Scan-Tc99m/MIBI Localization Studies in Dubai Hospital

Objective: The aim of this study is to assess the correlation between the findings in preoperative localization studies and biochemical parameters with the surgical outcome in patients with primary hyperparathyroidism. Design and Methods: In patients with hyperparathyroidism, preoperative localization imaging studies are considered important for their role in surgical cure, operative length, and extent of surgical incision. Sometimes discordant imaging studies lead to further extensive diagnostic work-up to avoid operative risks and may result in delay to surgical referral. In this single-center retrospective study at a tertiary referral center in Dubai, we evaluated the concordance of presurgical imaging modalities in primary hyperparathyroidism with the surgical outcome after parathyroidectomy. Results: This study included n = 59 patients. 74.6% were female, and the mean age was 52.59 years (SD = 13.6). Preoperative parathyroid sestamibi scans were done on n = 50 patients with 68% having a positive result. A radiology department neck ultrasound was done on n = 42 patients, 52% of whom had a positive finding for a parathyroid adenoma, while n = 17 patients required an endocrinology department neck ultrasound scan with 71% having a positive finding. Complete concordance of findings with the postoperative pathology result was found in 78% of patients who had a preoperative parathyroid sestamibi, 78% of patients who had a preoperative radiology department neck ultrasound, and 86% of patients with a preoperative endocrinology department ultrasound. There was no correlation between the preoperative laboratory values of calcium and PTH with the scan findings based on our data. Conclusion: Our results showed a high degree of complete concordance between the usual imaging modalities and surgical findings.

Reproducibility of Cytomorphological Diagnosis and Assessment of Risk of Malignancy of Thyroid Nodules Based on the Bethesda System for Reporting Thyroid Cytopathology: A Tertiary Cancer Center Perspective.

The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was introduced for unifying the terminology and morphologic criteria along with the corresponding risk of malignancy, leading to more consistent management approaches. The aim of this study was to study the utility and reproducibility of TBSRTC in reporting thyroid cytology in a referral cancer center. The fine-needle aspiration (FNA) of all thyroid nodules were included for a period of 5 years, from January 2016 to December 2021, in this cancer center. They were retrospectively reviewed and recategorized according to TBSRTC by two experienced pathologists. Cytohistopathological correlation was done for the cases which underwent surgical resection. 522 fine-needle aspiration cytology (FNAC) of thyroid swellings were evaluated and categorized according to TBSRTC. There was agreement in the cytological diagnosis of 512 cases, of which 260 (50.78%) were benign lesions, 189 (36.91%) were malignant, 5 (0.97%) were unsatisfactory/nondiagnostic, 41 (8.01%) were follicular neoplasm/suspicious for neoplasm, 13 (2.53%) were suspicious for malignancy, and 4 (0.78%) cases were reported as atypia of undetermined significance. Two cytopathologists were in agreement in 512 cases (98%) of cases. Almost complete concordance was noted in the malignant (99%) and benign categories (98%). Disagreement was seen in 10 cases. Histological follow-up was available in 201 cases with an overall malignancy rate of 62.68% (126/201). TBSRTC proved to be a very simple and effective reporting system for thyroid FNAC, especially in the setting of a cancer center. This enables proper triaging of cases with thyroid masses into those who require surgical intervention and those who can avoid it, thereby preventing unnecessary morbidity.

A Comparative Study of Squash Smear Cytology Diagnosis and Radiological Diagnosis with Histopathology in Central Nervous System Lesions.

Background:Space occupying lesions (SOLs) of central nervous system (CNS) constitutes important cause of neurological morbidity and mortality. Squash cytology is technically a simple and rapid intraoperative diagnostic tool. Radiology is supportive of histopathological diagnosis.Objectives:To enumerate the histopathological patterns of various central nervous system (CNS) lesions, to correlate cytopathological diagnosis with histopathological diagnosis, and to correlate radiological diagnosis with histopathological diagnosis.Materials and Methods:It was a retrospective study of CNS lesion cases from January 2015 to August 2018. Cytological-histopathological concordance and radiological-histopathological concordance were calculated. Chi-square test was the statistical tool used for statistical analysis.Results:Histopathological diagnosis of 50 cases included neoplastic lesions (42 cases [84%]) and non-neoplastic lesions (8 cases [16%]). Correct diagnosis was achieved by squash cytology in 36 cases (72%) and radiological diagnosis in 25 cases (50%) by complete concordance. However, diagnostic accuracy of squash and radiology improved considerably by 90% and 76%, respectively, after applying partial concordance criteria. For the detection of neoplastic lesions, squash cytology had 98% and radiology had 80% diagnostic efficacy.Conclusion:Preoperative radiological investigation and intraoperative squash smear cytology are complementary to each other. A multidisciplinary approach is necessary for the management of patients.

Анализ конкордантности результатов преимплантационного генетического тестирования на анеуплоидии методом Next Generation Sequencing на платформе Illumina в клетках разных областей трофэктодермы

Study Objective: To study trophoblast (TB) cells taken from various sections of the embryo using Next Generation Sequencing (NGS) on Illumina platform, and to compare data in order to identify the degree of discordance between various samples from one embryo. Study Design: Comparative study. Materials and Methods. For the study, we used human embryos at early development stages, which originated from artificial insemination of germ cells taken from healthy donors with normal karyotype within the scope of the in vitro fertilisation program. We selected 14 human embryos originating from insemination of oocytes of 10 donors aged 20 to 32 years old with sperm taken from 9 donors from the semen bank of the International Centre for Reproductive Medicine. Two embryos underwent degradation during defrosting. For 12 embryos, we performed a repeated TB cells biopsy from two independent sections: one biopsy from TB adjacent to inner cell mass (ICM) cells and the other TB biopsy from blastocyte pole opposite to the embryoblast. Study Results. A comparison of molecular karyotype of TB cells taken from various sections of blastocyte in 12 embryos, 36 samples (3 sample for each embryo), demonstrated partial discordance only in one observation. In initial study, molecular karyotype of an embryo showed trisomy 16 syndrome: Seq(16)x3,(XY)x1. In the follow-up study, we found an additional deletion in the form of mosaicism in chromosome 7 section adjacent to ICM of the embryo: Seq(16)x3,(7q21.3 -> 7q36.3)x[0.5]). All other results demonstrated complete concordance irrespective of a TB section in question or a laboratory where sequencing was performed. Conclusion. It may be concluded that pre-implantation aneuploidy genetic testing of 5-day-old embryos using Next Generation Sequencing on Illumina platform allows obtaining reliable information on chromosomal abnormalities and can be successfully used to identify aneuploidy in pre-implantation embryos. Keywords: pre-implantation aneuploidy genetic testing, Next Generation Sequencing, in vitro fertilisation, aneuploidy, embryo mosaicism, chromosomal pathology.

Feasibility and Accuracy of Menstrual Blood Testing for High-risk Human Papillomavirus Detection With Capture Sequencing

High-risk human papillomavirus (hrHPV) persistent infection is the major etiology of cervical precancer and cancer. Noninvasive self-sampling HPV testing is a promising alternative cervical cancer screening for avoiding stigma and improving patient willingness to participate. To investigate the feasibility and accuracy of menstrual blood (MB) hrHPV capture sequencing in hrHPV detection. This cohort study collected 137 sanitary pads from 120 women who were premenopausal and had hrHPV as detected by cervical HPV GenoArray testing. Patients were recruited from September 1, 2020, to April 1, 2021, at Central Hospital of Wuhan, China. Target capture sequencing was performed to determine hrHPV genotypes in MB. Sanger sequencing was performed as the criterion standard for detecting hrHPV genotypes among enrolled women. Data were analyzed from April 1 through June 1, 2021. Complete concordance, incomplete concordance, and discordance of MB hrHPV capture sequencing and conventional HPV testing were defined according to genotype overlapping levels. Concordance of the 2 detection methods and comparative power of MB hrHPV capture sequencing during different menstrual cycle days (MCDs) were the main outcomes. A total of 120 enrolled women with hrHPV (mean [SD; range] age, 33.9 [6.9; 20.0 -52.0] years) provided 137 sanitary pads. The overall concordance rate of MB hrHPV capture sequencing and cervical HPV testing was 92.7% (95% CI, 88.3%-97.1%), with a κ value of 0.763 (P < .001). Among 24 samples with incomplete concordance or discordant results, 11 samples with additional hrHPV genotypes (45.8%), 5 true-negative samples (20.8%), and the correct hrHPV genotypes of 2 samples (8.3%) were correctly identified by MB hrHPV capture sequencing. MB hrHPV detection of hrHPV was equivalent on different MCDs, with an MB hrHPV-positive rate of 27 of 28 patients (96.4%) for MCD 1, 52 of 57 patients (91.2%) for MCD 2, 27 of 28 patients for MCD 3, 4 of 4 patients (100%) for MCD 4, and 3 of 3 patients (100%) for MCD 5 (P = .76). The sensitivity of the MB hrHPV capture sequencing was 97.7% (95% CI, 95.0%-100%). These findings suggest that MB hrHPV capture sequencing is a feasible and accurate self-collected approach for cervical cancer screening. This study found that this method is associated with superior performance in identification of HPV genotypes and true-negative events compared with cervical HPV testing.

High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

Background: High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. Methods: Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of KEL*01 and KEL*02, JK*01 and JK*02, FY*01, FY*02, and FY*02 N.01, DI*01 and DI*02, GYPB*03 and GYPB*04, RHCE*E and RHCE*e, and DO*01 and DO*02. Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. Results: The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. Conclusions: We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

New Insight regarding Legionella Non-Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene.

ABSTRACTThe identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes.IMPORTANCE Legionella spp. are a widely spread bacteria that cause a fatal form of pneumonia. While traditional laboratory techniques have provided valuable systems for Legionella pneumophila identification, the amplification of the mip gene has been recognized as the only useful tool for Legionella non-pneumophila species identification both in clinical and environmental samples. Several studies focused on the mip gene classification scheme showed its limitations and the need to improve the classification scheme, including other genes. Our study provides significant advantages on Legionella identification, providing a reproducible new rpoB gene classification scheme that seems to be more accurate than mip gene sequencing, bringing out greater genetic variation on Legionella species. In addition, the combined use of both the mip and rpoB genes allowed us to identify presumed new Legionella species, improving epidemiological investigations and acquiring new understanding on Legionella fields.

Results of a study of remote reporting over virtual applications and using visually calibrated monitors

Background and Objectives The purpose of this study is to examine differences in image quality, discrepancy rates, productivity and user experience between remote reporting over Virtual Application (VA) using visually calibrated monitors, and reporting using diagnostic grade workstations in hospital premises. Methods Three specialist accredited radiologists examined and provisionally reported outpatient CT and MR studies over PACS delivered as a VA, using visually calibrated monitors from their homes. They then proceeded to view the same studies within hospital premises and issue a final report. Surveys were filled out for each imaging study. Discrepancies were reviewed and assigned RADPEER scores. Results A total of 51 outpatient CT and MRIs were read. Relative to hospital premise reporting, on a Likert scale of 5 (the higher the better), average image quality was 3.9, speed of loading and image manipulation was 4.4 and productivity was 4.1. Remote reporting user experience did not differ significantly between CT versus MRI studies. Complete concordance rate was 80.4% (41/51) and only one of the studies had a significant discrepancy, which may have been due to extra time given to interpretation. All three radiologists reported factors influencing image display and quality as the top factor impacting remote reporting throughput. Conclusions Remote reporting over VA with visually calibrated monitors for CT and MR can be useful in periods of staffing difficulty to augment on-site radiologists, though attention must be paid to its limitations and policies defined by local leadership with reference to relevant national position

E6-E7 based nested multiplex PCR assay for genital HPV detection and simultaneous typing of 15 high and low-risk HPV types

PurposeDue to a wide range of Human Papillomavirus (HPV) types associated with genital cancers; HPV genotyping remains important for the introduction of an appropriate vaccine, disease diagnosis, follow-up and epidemiological surveys. Currently, available molecular genotyping assays are not only expensive but also requires dedicated and expensive equipment which is not feasible in the majority of low-and-middle-socioeconomic countries. The purpose of the study was to develop and evaluated a cost-effective nested-multiplex polymerase chain reaction (NM-PCR) assay for HPV genotyping. MethodsHPV-DNA containing plasmids and cervical scrapings from histologically confirmed cervical cancer cases were used to evaluate the NM-PCR. In the first round PCR, a set of consensus primers were used to amplify 38 mucosal HPV types. HPV Type-specific primers were used in the second-round polymerase chain reaction (PCR) to amplify 15 HPV types in three multiplex cocktails. The assay sensitivity was determined with the control panel containing one to 1010genome equivalents (GE). DNA sequencing was done to confirm the PCR results. ResultsThe assay was able to amplify all HPV types and detected as few as 50GE per reaction. A total of 23 endo-cervical samples obtained from healthy, HPV negative subjects and 52 histologically confirmed cervical scrapings were processed for HPV genotyping by NM-PCR. HPV DNA was detected in all histologically confirmed samples. DNA sequencing results showed complete concordance with PCR results. ConclusionsThe designed nested PCR based assay had good concordance with clinical histology and sequencing results and appears to be a promising tool for HPV genotyping especially in resource-constrained settings.

Genomic medicine year in review: 2021

Genomic medicine year in review: 2021

Clinical Utility of Circulating Tumor DNA in Advanced Rare Cancers.

PurposePatients with advanced/relapsed rare cancers have few treatment options. Analysis of circulating tumor DNA in plasma may identify actionable genomic biomarkers using a non-invasive approach.Patients and MethodsRare cancer patients underwent prospective plasma-based NGS testing. Tissue NGS to test concordance was also conducted. Plasma DNA alterations were assessed for incidence, functional impact, therapeutic implications, correlation to survival, and comparison with tissue NGS.ResultsNinety-eight patients were analyzed. Diseases included soft-tissue sarcoma, ovarian carcinoma, and others. Mean turn-around-time for results was 9.5 days. Seventy-six patients had detectable gene alterations in plasma, with a median of 2.8 alterations/patient. Sixty patients had a likely pathogenic alteration. Five received matched-therapy based on plasma NGS results. Two developed known resistance mutations while on targeted therapy. Patients with an alteration having VAF ≥5% had a significantly shorter survival compared to those of lower VAF. Tissue NGS results from eleven of 22 patients showed complete or partial concordance with plasma NGS.ConclusionPlasma NGS testing is less invasive and capable of identifying alterations in advanced rare cancers in a clinically meaningful timeframe. It should be further studied as a prospective enrollment assay in interventional studies for patients with rare advanced stage cancers.Clinical Registration[https://www.umin.ac.jp/ctr/index-j.htm], identifier UMIN000034394.

Degree of concordance on the management of patients with gastroesophageal reflux disease. A study among specialists that are members of the Sociedad Latinoamericana de Neurogastroenterología (SLNG)

Introduction and aimsDue to its different clinical manifestations, gastroesophageal reflux disease (GERD) requires diverse diagnostic and therapeutic interventions. The aim of the study was to evaluate the degree of agreement among Latin American specialists, with respect to the management of GERD. Materials and methodsA cross-sectional study was conducted through a survey with 42 statements (22 related to diagnosis and 20 to treatment) applied to 56 specialists from Latin America. There were 4 possible statement responses: in complete agreement, in partial agreement, in partial disagreement, and in complete disagreement. Reproducibility, level of agreement, and concordance were measured through the Kappa statistic. ResultsThe response rate was 81% (47/56). General concordance was low, given that there was complete concordance in only 12 statements (28.6%). There was partial concordance in 22 statements (52.4%) and no concordance in 8 (19%). The following themes had the most disagreement: the performance of endoscopy before beginning treatment, the use of proton pump inhibitors (PPIs) in patients with extraesophageal symptoms and with no typical symptoms, and the combined use of PPIs and prokinetics. ConclusionsIn the present study, we found that there was agreement among the Latin American specialists for the diagnosis and management of GERD in less than one-third of the recommendations considered standard. The low concordance could be related to the fact that the availability of diagnostic tools and medications, as well as the prevalence of GERD phenotypes, is different in each country.

A Clinically Validated Targeted Capture Panel to Identify Translocations, Copy Number Abnormalities, and Mutations in Multiple Myeloma

A Clinically Validated Targeted Capture Panel to Identify Translocations, Copy Number Abnormalities, and Mutations in Multiple Myeloma