Polymerase Chain Reaction-based Assay Research Articles

COVID-19 Hospitalization in Hawai'i and Patterns of Insurance Coverage, Race and Ethnicity, and Vaccination.

The people of Hawai'i have both high rates of health insurance and high levels of racial and ethnic diversity, but the degree to which insurance status and race and ethnicity contribute to health outcomes in COVID-19 remains unknown. To evaluate the associations of insurance coverage, race and ethnicity (using disaggregated race and ethnicity data), and vaccination with outcomes for COVID-19 hospitalization. This retrospective cohort study included hospitalized patients at a tertiary care medical center between March 2020 and March 2022. All patients hospitalized for acute COVID-19, identified based on diagnosis code or positive results on polymerase chain reaction-based assay for SARS-CoV-2, were included in analysis. Data were analyzed from May 2022 to May 2023. COVID-19 requiring hospitalization. Electronic medical record data were collected for all patients. Associations among race and ethnicity, insurance coverage, receipt of at least 1 COVID-19 vaccine, intensive care unit (ICU) transfer, in-hospital mortality, and COVID-19 variant wave (pre-Delta vs Delta and Omicron) were assessed using adjusted multivariable logistic regression. A total of 1176 patients (median [IQR] age of 58 [41-71] years; 630 [54%] male) were hospitalized with COVID-19, with a median (IQR) body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) of 30 (25-36) and Sequential Organ Failure Assessment score of 1 (0-2). The sample included 16 American Indian or Alaska Native patients, 439 Asian (not otherwise specified) patients, 15 Black patients, 66 Chinese patients, 246 Filipino patients, 76 Hispanic patients, 107 Japanese patients, 10 Korean patients, 299 Native Hawaiian patients, 523 Pacific Islander (not otherwise specified) patients, 156 Samoan patients, 5 Vietnamese patients, and 311 White patients (patients were able to identify as >1 race or ethnicity). When adjusting for age, BMI, sex, medical comorbidities, and socioeconomic neighborhood status, there were no differences in either ICU transfer (eg, Medicare vs commercial insurance: odds ratio [OR], 0.84; 95% CI, 0.43-1.64) or in-hospital mortality (eg, Medicare vs commercial insurance: OR, 0.85; 95% CI, 0.36-2.03) as a function of insurance type. Disaggregation of race and ethnicity revealed that Filipino patients were more likely to die in the hospital (OR, 1.79; 95% CI, 1.04-3.03; P = .03). When considering variant waves, mortality among Filipino patients was highest during the pre-Delta time period (OR, 2.72; 95% CI, 1.02-7.14; P = .04), when mortality among Japanese patients was lowest (OR, 0.19; 95% CI, 0.03-0.78; P = .04); mortality among Native Hawaiian patients was lowest during the Delta and Omicron period (OR, 0.35; 95% CI, 0.13-0.79; P = .02). Patients with Medicare, compared with those with commercial insurance, were more likely to have received at least 1 COVID-19 vaccine (OR, 1.85; 95% CI, 1.07-3.21; P = .03), but all patients, regardless of insurance type, who received at least 1 COVID-19 vaccine had reduced ICU admission (OR, 0.40; 95% CI, 0.21-0.70; P = .002) and in-hospital mortality (OR, 0.42; 95% CI, 0.21-0.79; P = .01). In this cohort study of hospitalized patients with COVID-19, those with government-funded insurance coverage (Medicare or Medicaid) had similar outcomes compared with patients with commercial insurance, regardless of race or ethnicity. Disaggregation of race and ethnicity analysis revealed substantial outcome disparities and suggests opportunities for further study of the drivers underlying such disparities. Additionally, these findings illustrate that vaccination remains a critical tool to protect patients from COVID-19 mortality.

Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Survival rates in non-small cell lung cancer (NSCLC) are low. Detection of circulating tumor DNA in liquid biopsy (plasma) is increasingly used to identify targeted therapies for clinically actionable mutations, including EGFR mutations in NSCLC. The cobas® EGFR Mutation Test v2 (cobas EGFR test) is FDA-approved for EGFR mutation detection in tissue or liquid biopsy from NSCLC. Standard K2EDTA tubes require plasma separation from blood within 4 to 8 hours; however, Roche Cell-Free DNA (cfDNA) Collection Tubes (Roche cfDNA tube) enable whole blood stability for up to 7 days prior to plasma separation. This analysis assessed performance of Roche cfDNA tubes with the cobas EGFR test for the detection of EGFR mutations in plasma from healthy donors or patients with NSCLC. Overall, test performance was equally robust with either blood collection tube, eg, regarding limit of detection, linearity, and reproducibility, making Roche cfDNA tubes suitable for routine clinical laboratory use in this setting. Importantly, the Roche cfDNA tubes provided more flexibility for specimen handling versus K2EDTA tubes, eg, in terms of tube mixing, plasma separation, and sample stability, and do not require processing of blood within 8 hours thereby increasing the reach of plasma biopsies in NSCLC.

Asymptomatic norovirus infection during outbreaks in China: A systematic review and meta-analysis.

Acute gastroenteritis outbreaks may be caused by the excretion of norovirus (NoV) from asymptomatic individuals. Despite numerous studies involving asymptomatic NoV infection during outbreaks in China, a comprehensive assessment of its role has not been conducted, which is critical for emergency management. Our objective was to assess the prevalence of asymptomatic NoV infection during outbreaks in China. We conducted a comprehensive search of multiple databases, including PubMed, Web of Science, Cochrane Library, China National Knowledge Infrastructure, China Wanfang, and China Weipu, between January 1, 1997 and June 19, 2023. The retrieved articles and their references underwent screening, which utilized polymerase chain reaction-based assays for the detection of NoV in asymptomatic individuals during outbreaks that occurred in China. The primary summary data were the prevalence of asymptomatic NoV infection in outbreaks. We generated pooled estimates of asymptomatic prevalence in the population as a whole and in subgroups by using random-effect models. Of the 97 articles included, the pooled asymptomatic prevalence of NoV among 5117 individuals in outbreaks was 17.6% (95% confidence interval [CI]: 14.1-21.3). The asymptomatic prevalence of NoV GII (17.1%, 95% CI: 12.9-21.5) was similar to that of NoV GI (22.0%, 95% CI: 12.8-32.4). However, the proportion of asymptomatic individuals involved in NoV GII (57.44%) was significantly higher than that of NoV GI (5.12%), and NoV GII (75.26%) was reported much more frequently than NoV GI (14.43%) in the included articles. Meta-regression analysis of 11 possible influencing factors (geographic region, setting, season, sample type, genotype, transmission route, occupation, age, per capita income, study quality, and cases definition) showed that the source of heterogeneity might be related to the outbreak settings, per capita income, and study quality (p = 0.037, 0.058, and 0.026, respectively). Of particular note was the asymptomatic prevalence peaked in preschoolers (27.8%), afterward, it fell into trough in elementary and junior school children (10.5%), before the second peak located in adults (17.8%), and the elderly (25.2%). Prevalent genotypes reported include GII.4, followed by GII.17, GII.2, GII.3, GII.6, and so forth. The estimated asymptomatic prevalence of NoV during outbreaks in China was as high as 17.6%, with NoV GII dominating. In addition, genetic subtypes of NoV in outbreaks should be detected whenever possible. The role of asymptomatic individuals in NoV outbreaks cannot be ignored. This knowledge will help governments develop public health policies and emergency response strategies for outbreaks, assess the burden, and develop vaccines.

Longitudinal post-radiotherapy plasma Epstein-Barr virus DNA trends inform on optimal risk stratification in endemic nasopharyngeal carcinoma

ObjectivesTo characterize longitudinal changes in Epstein-Barr virus (EBV) DNA post-radiotherapy in nasopharyngeal carcinoma (NPC) patients, and investigate whether an early (0–2 weeks) or delayed (8–12 weeks) EBV DNA result better predicts for disease-free survival (DFS). Materials and MethodsHistologically-confirmed NPC patients with ≥1 EBV DNA test quantified using the harmonized BamHI-W polymerase chain reaction-based assay at 0–2 and 8–12 weeks post-radiotherapy were included. ResultsWe identified 302 patients with EBV DNA measured at 0–2 weeks post-radiotherapy; of which, 110 (36.4 %) underwent a repeat test at 8–12 weeks post-treatment. Patients harboring a detectable EBV DNA at 0–2 weeks experienced an inferior DFS (adjusted HR1-264 copies 1.72 [95 %CI: 1.05–2.83], P = 0.031; AHR≥265 copies 4.39 [95 %CI: 1.68–11.44], P = 0.002 relative to 0 copies/mL). At 8–12 weeks, we observed substantial shifts in EBV DNA readings from 0 to 2 weeks; 76/110 (69.1 %) and 34/110 (30.9 %) patients at 0–2 weeks versus 90/110 (81.8 %) and 20/110 (18.2 %) at 8–12 weeks recorded undetectable and detectable EBV DNA, respectively. Positive EBV DNA at 8–12 weeks was strongly associated with relapse (73.3 % [11/15] for 1–264; 80.0 % [4/5] for ≥265 subgroups had relapses versus 15.6 % [14/90] for 0 copies/mL). Area under receiver operating curve values for 2-year relapse rates were 0.817 (95 %CI: 0.725–0.909) for stage + EBV DNA8-12w versus 0.654 (95 %CI: 0.542–0.765) for stage + EBV DNA0-2w. ConclusionEBV DNA is dynamic post-radiotherapy, and delayed EBV DNA testing better enriched for higher-risk NPC patients. This implicates trials investigating adjuvant chemotherapy intensification based on early EBV DNA testing.

Engaging students in a genetics course-based undergraduate research experience utilizing Caenorhabditis elegans in hybrid learning to explore human disease gene variants.

Genetic analysis in model systems using bioinformatic approaches provides a rich context for a concrete and conceptual understanding of gene structure and function. With the intent to engage students in research and explore disease biology utilizing the nematode Caenorhabditis elegans model, we developed a semester-long course-based undergraduate research experience (CURE) in a hybrid (online/in-person) learning environment-the gene-editing and evolutionary nematode exploration CURE (GENE-CURE). Using a combination of bioinformatic and molecular genetic tools, students performed structure-function analysis of disease-associated variants of uncertain significance (VUS) in human orthologs. With the aid of a series of workshop-style research sessions, students worked in teams of two to six members to identify a conserved VUS locus across species and design and test a polymerase chain reaction-based assay for targeted editing of a gene in the nematode and downstream genotyping. Research session discussions, responsible conduct of research training, electronic laboratory notebook, project reports, quizzes, and group poster presentations at a research symposium were assessed for mastery of learning objectives and research progress. Self-reflections were collected from students to assess engagement, science identity, and science efficacy. Qualitative analysis of these reflections indicated several gains suggesting that all students found many aspects of the GENE-CURE rewarding (learning process of research, self-confidence in research and science identity, and personal interest) and challenging (iterative research and failure, time management, COVID-19 pandemic, and life issues).

Pattern of Primary Resistance of Helicobacter pylori to Clarithromycin among Pediatric Patients from North-Eastern Romania.

Helicobacter pylori antibiotic resistance has increased worldwide and affects the effectiveness of current therapies. The recommended first-line empiric treatment should be tailored to the local clarithromycin resistance rate. This study aimed to determine the pediatric patient profile and rate of clarithromycin resistance for patients diagnosed with Helicobacter pylori by gastric biopsy. We studied 84 positive gastric samples for Helicobacter pylori. Positive results were confirmed by a rapid urease test and histopathological examination, with the type of gastritis established according to the Sydney System. Gastric biopsy samples were stored in RNA saver. Clarithromycin resistance was determined by a real-time polymerase chain reaction-based molecular assay after RNA-DNA extraction. Of the 84 biopsy samples analyzed, 35 (41.6%) were resistant to clarithromycin. Clarithromycin resistance was found mainly in girls (80%) with a mean age of 15 years (range 6-17 years). The history of prior exposure to clarithromycin was 91.6%. The concordance between the histopathological examination and the PCR test was 100%. One in 2.4 children infected with Helicobacter pylori had a strain resistant to clarithromycin. This resistant strain may be a reason for treatment failure in Romanian children, yet this is uninvestigated. The high rate of bacterial resistance to this antibiotic among children indicates the need for susceptibility testing before therapy.

Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Stewart’s vascular wilt and leaf blight of sweet corn is caused by the Gram-negative enteric bacterium Pantoea stewartii subsp. stewartii. Stewart’s wilt results in substantial yield losses worldwide warranting rapid and accurate disease diagnosis. Recombinase polymerase amplification (RPA) is an isothermal technique that is tolerant to host plant-derived inhibitors and is, therefore, ideally suited for rapid in-field detection vis-à-vis traditional polymerase chain reaction-based molecular assays. An RPA assay coupled with a Lateral Flow Device (LFD) was developed for rapid, accurate, and sensitive real-time detection of P. stewartii subsp. stewartii directly from the infected host offering in-field pathogen detection, timely disease management, and satisfying quarantine and phytosanitary requirements. Twelve novel primer sets were designed against conserved genomic regions of P. stewartii subsp. Stewartii; however, only the primers for amplification of the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB were discernibly unique and adequate for unambiguous identification of P. stewartii subsp. stewartii. The P. stewartii subsp. stewartii-specific primers were further validated in a simplex RPA assay for specificity against twenty-six bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii subsp. stewartii with an empirical detection limit of 0.0005 pg μL−1 bacterial DNA and 1 × 102 CFU mL−1 (app. two bacterial cells used per RPA reaction) in minimally processed samples for accurate, low-cost, and point-of-need diagnosis of the quarantine pathogen P. stewartii subsp. stewartii.

Pitfalls of using polymerase chain reaction-based assays for JAK2 and CALR exon 9 variant testing in myeloproliferative neoplasms: Knowing when to go the extra mile!

The BCR::ABL1 negative myeloproliferative neoplasms are sequentially tested for JAK2 p.V617F, followed by CALR exon 9 pathogenic variants. Historically, these variants were thought to be mutually exclusive. However, recent reports indicate coexisting JAK2 p.V617F and CALR exon 9 somatic variants. Analysis of JAK2 p.V617F and CALR exon 9 variant was performed by polymerase chain reaction (PCR)-based assays. Subsequent testing was performed on the Genexus integrated sequencer (ThermoFisher) using the Oncomine myeloid assay GX v2. CALR exon 9 variants were positive in 3 cases, while 2 were positive for JAK2 p.V617F on PCR-based assays. Next-generation sequencing confirmed the JAK2 P.V617F status in all cases. CALR variants resulting in in-frame deletions were identified in 2 cases at a variant allele frequency of 52.16% and 50.91%, while the third case had an intronic CALR variant c.-48G>A at a variant allele frequency of 51.1%. Thus, CALR variants in all 3 cases were interpreted as potentially germline. Of the 228 cases that underwent JAK2 p.V617F and CALR cotesting in the past 2 years, only these 2 cases were positive for both JAK2 p.V617F and CALR exon 9 variants. These cases highlight the importance of understanding the pitfalls of molecular techniques in current practice.

Limitations of Dual-Single Guide RNA CRISPR Strategies for the Treatment of Central Nervous System Genetic Disorders.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a toxic gain-of-function CAG expansion in the first exon of the huntingtin (HTT) gene. The monogenic nature of HD makes mutant HTT (mHTT) inactivation a promising therapeutic strategy. Single nucleotide polymorphisms frequently associated with CAG expansion have been explored to selectively inactivate mHTT allele using the CRISPR/Cas9 system. One of such allele-selective approaches consists of excising a region flanking the first exon of mHTT by inducing simultaneous double-strand breaks at upstream and downstream positions of the mHTT exon 1. The removal of the first exon of mHTT deletes the CAG expansion and important transcription regulatory sites, leading to mHTT inactivation. However, the frequency of deletion events is yet to be quantified either in vitro or in vivo. Here, we developed accurate quantitative digital polymerase chain reaction-based assays to assess HTT exon 1 deletion in vitro and in fully humanized HU97/18 mice. Our results demonstrate that dual-single guide RNA (sgRNA) strategies are efficient and that 67% of HTT editing events are leading to exon 1 deletion in HEK293T cells. In contrast, these sgRNA actively cleaved HTT in HU97/18 mice, but most editing events do not lead to exon 1 deletion (10% exon 1 deletion). We also showed that the in vivo editing pattern is not affected by CAG expansion but may potentially be due to the presence of multiple copies of wildtype (wt)/mHTT genes HU97/18 mice as well as the slow kinetics of AAV-mediated CRISPR/Cas9 delivery.

Correlation between KRAS and NRAS mutational status and clinicopathological features in 414 cases of metastatic colorectal cancer in Morocco: the largest North African case series

BackgroundAdvances in molecular biology have improved understanding of the molecular features of carcinogenesis and progression of colorectal cancer. It is clear that the efficacy of anti-EGFR depends upon the RAS mutational status, since any mutation in RAS is associated with resistance to anti-EGFR therapy. The aim of this study is to report the largest North African description of KRAS and NRAS status in metastatic colorectal cancer and to describe the association of these mutations with clinicopathological characteristics.MethodsThis is a prospective study of all consecutive unselected metastatic colorectal cancer samples, collected from the Laboratory of Pathology at the National Institute of Oncology of Rabat, Morocco, from January 1st 2020 to December 31st 2021. The molecular analysis was performed on the Idylla™ platform (fully automated real-time polymerase chain reaction-based assay) for KRAS and NRAS mutations in exons 2, 3 and 4. These mutations were correlated to gender, primary tumor site, histological type and degree of differentiation of tumor using adequate statistical methods.ResultsFour hundred fourteen colorectal tumors were screened for KRAS and NRAS mutations. These mutations occurred in 51.7% of tumors for KRAS (mainly in exon 12) and in 3% of tumors for NRAS. There was a significant correlation between NRAS mutation and age of colorectal patients in this study. The low rate of invalid RAS tests (1.7% for KRAS and 3.1% for NRAS) was certainly obtained due to the strict respect of pre-analytical factors such as cold ischemia time and formalin fixation.ConclusionWe report the largest North African analysis of NRAS and KRAS status in colorectal metastatic patients. This study showed the ability in low middle income countries to perform a high rate of valid tests and the unusual trend towards older patients for NRAS mutations.

Prevalence of mismatch repair deficiency in a large prospective prostate cancer cohort.

5031 Background: Mismatch repair (MMR) deficiency and microsatellite instability (MSI) are predictive biomarkers for immunotherapy response. The best approach to identify patients with such tumors is unclear in prostate cancer. Methods: This study included men diagnosed with primary prostate cancer during prospective follow-up of the Health Professionals Follow-up Study (HPFS) and Physicians’ Health Study (PHS). The highest-grade/index lesions of tumor tissue from radical prostatectomy (95%) or transurethral resections of the prostate were mounted in triplicate on tissue microarrays. Immunohistochemistry for the MMR proteins MLH1, MSH2, MSH6, and PMS2 was performed, with scoring as MMR-deficient requiring a visible staining in a non-tumor internal positive control of the same case. For validation, a polymerase chain reaction-based MSI assay (Idylla MSI Test, Biocartis) was performed on tumor DNA of MMR-deficient cases and a selection of MMR-intact cases. Results: The study included 1016 men with prostate cancer. Tumor stage was predominantly pathologically localized (71% stage pT1/2, 18% T3a/b) with a full distribution of Gleason scores, including 20% Gleason score 6 (grade group 1), 36% 3+4 (grade group 2), 23% 4+3 (grade group 3), 8% 8 (grade group 4) and 13% 9-10 (grade group 5). MMR tumor scoring could be performed for MLH1 in 747 cases (75% of those with tumor tissue), for MSH2 in 903 cases (90%), for MSH6 in 708 cases (74%), and for PMS2 in 703 cases (72%). The remaining tumors were unevaluable due to lack of non-tumor tissue necessary as an internal positive control. Of the 903 tumors evaluable for MSH2 protein loss, 4 tumors had loss of MSH2 (prevalence 0.4%, 95% confidence interval [CI] 0.2–1.1%), and 3 of 708 evaluable tumors had concomitant loss of MSH6 (prevalence 0.4%, 95% CI 0.1–1.2%). No tumor had loss of MLH1 or PMS2. The MMR-deficient tumors had Gleason scores of 3+4, 8 (two cases), and 9–10. Tumor DNA of 1 of the 4 MMR-deficient tumors met the manufacturer’s cut-off for MSI-high; two additional MMR-deficient cases had non-zero repeats, with good reproducibility between tumor cores and technical replicates. One MSH2-deficient tumor and 6 DNA samples from 3 MMR-intact cases had repeat scores of zero. Conclusions: In this nationwide prospective study, MMR deficiency was rare in primary, surgically treated prostate cancer. This low prevalence contrasts with hospital-based studies of MMR deficiency and MSI that may have represented tumors with certain clinical features. The low prevalence and the need for an internal positive control for reliable scoring calls into question to what extent immunohistochemistry-based screening for MMR deficiency on limited tissue specimens, such as prostate biopsies, is routinely feasible.

Evaluation of SARS-CoV-2 antibody levels on hospital admission as a correlate of protection against mortality.

Millions of people have now been vaccinated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, it is still unclear which antibody levels provide protection against mortality. It is further unknown whether measuring antibody concentrations on hospital admission allows for identifying patients with a high risk of mortality. To evaluate whether anti-SARS-CoV2-spike antibodies on hospital admission predict in-hospital mortality in patients with coronavirus disease 2019. We conducted a prospective, multicentre cohort study on 1152 hospitalized patients who tested positive for SARS-CoV-2 with a polymerase chain reaction-based assay. Patients were classified by vaccination status. Anti-SARS-CoV-2 spike antibodies were determined on hospital admission. The investigated end point was in-hospital mortality for any cause. Spike antibodies on hospital admission were significantly lower in non-survivors in both non-vaccinated (73U/ml, 95%CI 0-164 vs. 175U/ml, 95%CI 124-235, p=0.002) and vaccinated patients (1056U/ml, 95%CI 701-1411 vs. 1668U/ml, 95%CI 1580-1757, p<0.001). Further, spike antibodies were significantly lower in fully vaccinated and boostered patients who died compared to those who survived (mean 883U/ml, 95%CI 406-1359 vs. 1292U/ml, 95%CI 1152-1431, p=0.017 and 1485U/ml, 95%CI 836-2133 vs. 2050U/ml, 95%CI 1952-2149, p=0.036). Patients infected with the currently prevailing Omicron variant were three times more likely to die if spike antibodies were <1200U/ml (OR 3.458, 95%CI 1.562-7.656, p=0.001). After adjusting for potential confounders, this value increased to an aOR of 4.079 (95%CI 1.809-9.198, p<0.001). Anti-SARS-CoV2 spike-antibody levels on hospital admission are inversely associated with in-hospital mortality. Hospitalized patients with lower antibody levels have a higher risk of mortality.

Highly Sensitive Detection of Complicated Mutations of Drug Resistance in Mycobacterium tuberculosis Using a Simple, Accurate, Rapid, and Low-Cost Tailored-Design Competitive Wild-Type Blocking Assay.

Establishing simple, rapid, and highly sensitive molecular assays is crucial for timely diagnosis and effective treatment of drug-resistant tuberculosis. However, current genotypic drug susceptibility testing (DST) still encounters enormous challenges including lower sensitivity than phenotypic DST and insufficient accuracy. Herein, a simple, low-cost, multiplex real-time polymerase chain reaction-based assay is established to achieve highly sensitive detection of low-abundant mutants through competitive wild-type blocking (COWTB). Analytical performance of the COWTB assay can achieve 1% or even 0.1% mutants under background of 10000 wild-type genomes/test. Furthermore, clinical practice feasibility is evaluated to identify resistance to rifampicin (RIF), isoniazid (INH), and streptomycin (SM) on 92 actual clinical samples, its sensitivity is 93.8% for RIF and 100% for INH and SM, and specificity is 100% each for RIF, INH, and SM when using DNA sequencing as the reference standard. In comparison, the sensitivity of reverse dot blotting assay commonly used in clinics is 93.8%, 90.0%, and 84.6%, and the specificity is 96.1%, 98.6%, and 100% for RIF, INH, and SM, respectively. Importantly, the COWTB assay can also be applicable for other drug-resistant mutations and pave a promising detection strategy to fill the gap between phenotypic and genotypic DST for detecting low-abundant drug-resistant M. tuberculosis.

Diagnostic performance of fecal Helicobacter pylori antigen test in Uganda

We evaluated the diagnostic performance of a qualitative stool antigen test (SAT) in individuals with dyspepsia in rural Uganda using the polymerase chain reaction-based 16S ribosomal RNA assay (16S rRNA) for nucleotide sequences for two common H. pylori-associated genes as the reference standard. We enrolled 150 adults with dyspepsia with no self-reported use of antibiotic and/or antiacid medication medications within a fortnight. We performed blinded SAT on fecal specimens and 16S rRNA tests on gastric specimens. Using nonlinear mixed models, SAT had a sensitivity of 85⋅1% (95%CI 76⋅54%, 93⋅6%), and specificity of 97⋅6% (95%CI 94⋅3, 100). Twelve individuals with dyspepsia need to be tested to correctly diagnose 10 with H. pylori infection using SAT. The SAT is a robust diagnostic test to improve the diagnosis of H. pylori infection in people with dyspepsia in resource-limited settings.

Analysis of Circulating Tumor DNA to Predict Risk of Recurrence in Patients With Esophageal and Gastric Cancers.

Circulating tumor DNA (ctDNA) analyses allow for postoperative risk stratification in patients with curatively treated colon and breast cancers. Use of ctDNA in esophagogastric cancers (EGC) is less characterized and could identify high-risk patients who have been treated with curative intent. In this retrospective analysis of real-world data, ctDNA levels were analyzed in the preoperative, postoperative, and surveillance settings in patients with EGC using a personalized multiplex polymerase chain reaction-based next-generation sequencing assay. Plasma samples (n = 943) from 295 patients at > 70 institutions were collected before surgery, postoperatively, and/or serially during routine clinical follow-up from September 19, 2019, to February 21, 2022. ctDNA detection was annotated to clinicopathologic features and recurrence-free survival. A total of 295 patients with EGC were analyzed, and 212 patients with stages I-III disease were further explored. Pretreatment ctDNA was detected in 96% (23/24) of patients with preoperative time points. Postoperative ctDNA was detected in 23.5% (16/68) of patients with stage I-III EGC within 16 weeks (molecular residual disease window) after surgery without receiving systemic therapy. ctDNA detection at any time point after surgery (hazard ratio [HR], 23.6; 95% CI, 10.2 to 66.0; P < .0001), within the molecular residual disease window (HR, 10.7; 95% CI, 4.3 to 29.3; P < .0001), and during the surveillance period (HR, 17.7; 95% CI, 7.3 to 50.7; P < .0001) was associated with shorter recurrence-free survival. In multivariable analysis, ctDNA status and clinical stage of disease were independently associated with outcomes. Using real-world data, we demonstrate that postoperative tumor-informed ctDNA detection in EGC is feasible and allows for enhanced patient risk stratification and prognostication during curative-intent therapy.

Development of a Multiplex Polymerase Chain Reaction-Based DNA Lateral Flow Assay as a Point-of-Care Diagnostic for Fast and Simultaneous Detection of MRSA and Vancomycin Resistance in Bacteremia.

To reduce high mortality and morbidity rates, timely and proper treatment of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection is required. A multiplex polymerase reaction (mPCR)-based DNA lateral flow assay (MBDLFA) was developed as a point-of-care diagnostic for simultaneous identification of S. aureus, methicillin resistance, and vancomycin resistance directly from blood or blood cultures. A mPCR was developed to detect nuc, mecA, and vanA/B; its sensitivity, specificity, and limit of detection (LOD) were determined. The developed reaction was further modified for use in MBDLFA and its sensitivity for detection of target genes from artificially inoculated blood samples was checked. The optimized mPCR successfully detected nuc, mecA, and vanA/B from genomic DNA of bacterial colonies with LODs of 107, 107, and 105 CFU/mL, respectively. The reaction was sensitive and specific. The optimized mPCR was used in MBDLFA that detected nuc, mecA, and vanA/B with LODs of 107, 108, and 104 CFU/mL, respectively, directly from artificially inoculated blood. The developed MBDLFA can be used as a rapid, cheap point-of-care diagnostic for detecting S. aureus, MRSA, and vancomycin resistance directly from blood and blood cultures in ~2 h with the naked eye. This will reduce morbidity, mortality, and treatment cost in S. aureus bacteremia.

Detection of FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) Mutations from Marrow Tissues in Patients with Acute Myeloid Leukaemia

INTRODUCTION: Acute myeloid leukaemia is a haematological malignancy with diverse cytogenetic abnormalities and molecular mutations. Amongst the important mutations are FMS-related tyrosine kinase 3 (FLT3) and nucleophosmin 1 (NPM1) gene mutations. These mutations have been shown to be of prognostic significance. A cross-sectional study to examine the frequency of these mutations and their association with the haematological and cytogenetic characteristics of the cases was carried out in Kuantan, Pahang, Malaysia. MATERIALS AND METHODS: A total of 43 cases were included in the study. Polymerase chain reaction-based assays were employed for mutation detection from the retrieved trephine biopsy tissue blocks. Mutation positivity was subsequently validated by Sanger DNA sequencing. RESULTS: Six of the 43 cases (14.0%) of the acute myeloid leukaemia were positive for FLT3-type internal tandem duplications (FLT3-ITD) and a similar proportion (6/43, 14.0%) were positive for NPM1 mutations. FLT3 mutations at codon D835 (FLT3-D835) mutation was identified in three of the cases (7.0%) while concurrent mutations of NPM1 and FLT3-ITD were seen in two of the mutation-positive cases (4.7%). The total white cell count was found to be significantly higher in patients with FLT3 mutations (p=0.001). Other haematological parameters and the cytogenetic results did not reveal any significant association with the mutational status. CONCLUSION: The frequency of FLT3-ITD, FLT3-D835, and NPM1 mutations among AML patients were 14%, 7%, and 14% respectively. Follow-up studies to include the clinical parameters and the treatment outcomes are advocated.

Circulating Tumor DNA After Neoadjuvant Chemotherapy in Breast Cancer Is Associated With Disease Relapse.

Detection of circulating tumor DNA (ctDNA) after neoadjuvant chemotherapy in patients with early-stage breast cancer may allow for early detection of relapse. In this study, we analyzed ctDNA using a personalized, tumor-informed multiplex polymerase chain reaction-based next-generation sequencing assay. Plasma samples (n = 157) from 44 patients were collected before neoadjuvant therapy (baseline), after neoadjuvant therapy and before surgery (presurgery), and serially postsurgery including a last follow-up sample. The primary end point was event-free survival (EFS) analyzed using Cox regression models. Thirty-eight (86%), 41 (93%), and 38 (86%) patients had baseline, presurgical, and last follow-up samples, respectively. Twenty patients had hormone receptor-positive/human epidermal growth factor receptor 2-negative, 13 had triple-negative breast cancer, and 11 had human epidermal growth factor receptor 2-positive disease. Baseline ctDNA detection was observed in 22/38 (58%) patients and was significantly associated with Ki67 > 20% (P = .036) and MYC copy-number gain (P = .0025, false discovery rate = 0.036). ctDNA detection at presurgery and at last follow-up was observed in 2/41 (5%) and 2/38 (5%) patients, respectively. Eight relapses (seven distant and one local) were noted (median follow-up 3.03 years [range, 0.39-5.85 years]). After adjusting for pathologic complete response (pCR), ctDNA detection at presurgery and at last follow-up was associated with shorter EFS (hazard ratio [HR], 53; 95% CI, 4.5 to 624; P < .01, and HR, 31; 95% CI, 2.7 to 352; P < .01, respectively). Association between baseline detection and EFS was not observed (HR, 1.4; 95% CI, 0.3 to 5.9; P = .67). The presence of ctDNA after neoadjuvant chemotherapy is associated with relapse in early-stage breast cancer, supporting interventional trials for testing the clinical utility of ctDNA monitoring in this setting.

Developing a novel Bifidobacterium phage quantitative polymerase chain reaction-based assay for tracking untreated wastewater

Microbial source tracking (MST) tools provide insights on fecal pollution levels in aquatic environments using predominantly quantitative PCR (qPCR) assays that target host-associated molecular marker genes. Existing wastewater-associated marker genes have shown limited or significant cross-reactions with non-human fecal samples. In this study, we mined the current Gut Phage Database (GPD) and designed a novel untreated wastewater-specific Bifidobacterium phage qPCR assay (i.e., Bifi assay). The sensitivity and specificity of the Bifi marker genes were assessed by collectively analyzing untreated (n = 33) and treated (n = 15) wastewater and non-human fecal samples (i.e., Rabbit, mouse, cow, horse, pig, chicken, sheep, dog, deer, kangaroos; n = 113) in Shenzhen, Guangdong Province, China and Brisbane, Australia. Bifi assay revealed 100% host-specificity against non-human fecal samples collected from Shenzhen and Brisbane. Furthermore, this marker gene was also detected in all untreated and treated wastewater samples, whose concentrations ranged from 5.54 to 6.83 log10 GC/L. In Shenzhen, the concentrations of Bifi marker gene were approximately two orders of magnitude lower than Bacteroides (HF183/BacR287 assay) and CrAssphage (CPQ_56 assay). The concentration of Bifi marker gene in untreated wastewater from Brisbane was 1.35 log10 greater than those in Shenzhen. Our results suggest that Bifi marker gene has the potential to detect and quantify the levels of human fecal pollution in Shenzhen and Brisbane. If additional detection sensitivity is required for environmental studies, Bifi marker gene should be paired with either CrAssphage or HF183/BacR287 marker genes.

Artificial aptamer that inhibits interleukin-23/interleukin-23 receptor interaction discovered via SELEX

Interaction between the pro-inflammatory cytokine interleukin-23 (IL-23) and IL-23 receptor (IL-23R) is related to the development of inflammatory autoimmune diseases such as psoriasis, inflammatory bowel disease, and Crohn's disease. In this study, we conducted systematic evolution of ligands by exponential enrichment (SELEX) for in vitro selection against human IL-23 and observed RNA sequence enrichment in the final SELEX round. IL-23-pull-down assay by chemiluminescence detection and fluorescence imaging demonstrated that SELEX-enriched RNA clone bound to IL-23. Quantitative polymerase chain reaction-based pull-down assay using the IL-23 alpha (IL-23A) subunit, a component of the IL-23 heterodimer, indicated that the RNA clone bound to IL-23A, which is favorable for autoimmune disease treatment. We also observed that the novel IL-23-binding RNA aptamer inhibited interaction between IL-23 and IL-23R. Thus, the novel IL-23-binding RNA aptamer can be used for IL-23 studies and has potential to be used for IL-23 diagnosis and IL-23-related inflammatory autoimmune disease treatment.