VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance.

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs.

Detection of gonadotropin-releasing hormone and its analogues in equine and canine urine by high-resolution data-independent acquisition.

Gonadotropin-releasing hormone (GnRH) and its synthetic analogues are considered banned substances by the racing industry. GnRH is used as a pharmaceutical to regulate the female oestrous cycle, but the hormone is also thought to increase the production of testosterone in male animals. Using liquid chromatography in conjunction with high-resolution mass spectrometry (LC-HRMS) and data-independent acquisition (DIA), a method is presented for the detection of intact and truncated peptides of GnRH and its analogues down to the low picogram level in equine urine. The study of the catabolism of GnRH and analogues in plasma, combined with the analysis of urine from administration studies, reveals a common pattern of peptide catabolites that can be used to guide the design of MS-based screens for this class of drugs. This culminated in the successful detection of the peptide in two out-of-competition canine urine samples.

The prognostic value of the neutrophil-to-lymphocyte ratio in patients with muscle-invasive bladder cancer treated with neoadjuvant chemotherapy and radical cystectomy.

INTRODUCTION: The neutrophil-to-lymphocyte ratio (NLR) is an attractive marker because it is derived from routine bloodwork. NLR has shown promise as a prognostic factor in muscle invasive bladder cancer (MIBC) but its value in patients receiving neoadjuvant chemotherapy (NAC) before radical cystectomy (RC) is not yet established. Since NLR is related to an oncogenic environment and poor antitumor host response, we hypothesized that a high NLR would be associated with a poor response to NAC and would remain a poor prognostic indicator in patients receiving NAC. METHODS: A retrospective analysis was performed on patients with nonmetastatic MIBC (cT2-4aN0M0) who received NAC prior to RC between 2000 and 2013 at 1 of 19 centers across Europe and North America. The pre-NAC NLR was used to split patients into a low (NLR ≤ 3) and high (NLR > 3) group. Demographic and clinical parameters were compared between the groups using Student's t test, chi-squared, or Fisher's exact test. Putative risk factors for disease-specific and overall survival were analyzed using Cox regression, while predictors of response to NAC (defined as absence of MIBC in RC specimen) were investigated using logistic regression. RESULTS: Data were available for 340 patients (199 NLR ≤ 3, 141 NLR > 3). Other than age and rate of lymphovascular invasion, demographic and pretreatment characteristics did not differ significantly. More patients in the NLR > 3 group had residual MIBC after NAC than the NLR ≤ 3 group (70.8% vs. 58.3%, P = 0.049). NLR was the only significant predictor of response (odds ratio: 0.36, P = 0.003) in logistic regression. NLR was a significant risk factor for both disease-specific (hazard ratio (HR): 2.4, P = 0.006) and overall survival (HR:1.8, P = 0.02). CONCLUSION: NLR > 3 was associated with a decreased response to NAC and shorter disease-specific and overall survival. This suggests that NLR is a simple tool that can aid in MIBC risk stratification in clinical practice.

Adductor Canal Nerve Versus Femoral Nerve Blockade for Pain Control and Quadriceps Function Following Anterior Cruciate Ligament Reconstruction With Patellar Tendon Autograft: A Prospective Randomized Trial.

PURPOSE: To compare femoral nerve blockade (FNB) versus adductor canal nerve blockade (ACB) for postoperative pain control and quadriceps muscle function in patients undergoing anterior cruciate ligament (ACL) reconstruction with patellar tendon autograft. METHODS: A randomized therapeutic trial of 90 patients undergoing ACL reconstruction with patellar tendon autograft was conducted comparing ACB versus FNB at 24 hours, 2 and 4 weeks, and 6 months postsurgery. Early outcome measures included average pain score and morphine equivalent units (milligrams) consumed, quadriceps surface electromyography, straight leg raise, and ability to ambulate without assistive devices. The 6-month outcome measures included knee range of motion (ROM), isokinetic knee extension peak torque, single-leg squat, and single-leg hop performance. Complications were recorded throughout the study for the development of anterior knee pain, knee extension ROM loss, deep vein thrombosis, and graft failure. Mixed-model analysis of variance and Mann-Whitney U tests were performed using an alpha of .05. RESULTS: Quadriceps surface electromyography deficits were higher for FNB at 24 hours (P < .001) and 2 weeks (P < .001) when compared with the ACB group. There were no between-groups difference for subjective pain (P = .793) or morphine consumption (P = .358) within the first 24 hours of surgery. A higher percentage of patients in the ACB group met the full ambulation criteria at 4 weeks compared with the FNB group (100% vs 84.2%, P < .001). No between-group differences were observed at 6 months; however, the rate of knee extension ROM loss was higher for the FNB group versus the ACB group (21.1% vs 5.0%, P = .026), respectively. CONCLUSIONS: ACB was as effective as FNB at providing pain control while eliciting fewer quadriceps muscle activation deficits and fewer postoperative complications. Based on previous evidence and the results of this study, we recommend the use of ACB over FNB for the analgesic management of patients undergoing ACL reconstruction with patellar tendon autograft. LEVEL OF EVIDENCE: Level I, prospective randomized controlled trial.

Genetic architecture of wild soybean (Glycine soja) response to soybean cyst nematode (Heterodera glycines).

The soybean cyst nematode (SCN) is one of the most destructive pathogens of soybean plants worldwide. Host-plant resistance is an environmentally friendly method to mitigate SCN damage. To date, the resistant soybean cultivars harbor limited genetic variation, and some are losing resistance. Thus, a better understanding of the genetic mechanisms of the SCN resistance, as well as developing diverse resistant soybean cultivars, is urgently needed. In this study, a genome-wide association study (GWAS) was conducted using 1032 wild soybean (Glycine soja) accessions with over 42,000 single-nucleotide polymorphisms (SNPs) to understand the genetic architecture of G. soja resistance to SCN race 1. Ten SNPs were significantly associated with the response to race 1. Three SNPs on chromosome 18 were localized within the previously identified quantitative trait loci (QTLs), and two of which were localized within a strong linkage disequilibrium block encompassing a nucleotide-binding (NB)-ARC disease resistance gene (Glyma.18G102600). Genes encoding methyltransferases, the calcium-dependent signaling protein, the leucine-rich repeat kinase family protein, and the NB-ARC disease resistance protein, were identified as promising candidate genes. The identified SNPs and candidate genes can not only shed light on the molecular mechanisms underlying SCN resistance, but also can facilitate soybean improvement employing wild genetic resources.

Genome-Wide Association Study of Resistance to Soybean Cyst Nematode (Heterodera glycines) HG Type 2.5.7 in Wild Soybean (Glycine soja).

Soybean cyst nematode (SCN) is the most destructive soybean pest worldwide. Host plant resistance is the most environmentally friendly and cost-effective way of mitigating SCN damage to soybeans. However, overuse of the resistant soybean [Glycine max (L.) Merr.] cultivars from limited genetic resources has resulted in SCN race shifts in many soybean-growing areas. Thus, exploration of novel sources of SCN resistance and dissection of the genetic basis are urgently needed. In this study, we screened 235 wild soybean (Glycine soja Sieb. & Zucc.) accessions to identify genotypes resistant to SCN HG Type 2.5.7 (race 5), a less investigated type but is prevalent in the southeastern US. We also dissected the genetic basis of SCN resistance using a genome-wide association study with SNPs genotyped by SoySNP50k iSelect BeadChip. In total, 43 resistant accessions (female index < 30) were identified, with 10 SNPs being significantly associated with SCN HG 2.5.7 resistance in this wild species. Furthermore, four significant SNPs were localized to linked regions of the known quantitative trait locus (QTL) rhg1 on chromosome 18. The other four SNPs on chromosome 18 and two SNPs on chromosome 19 are novel. Genes encoding disease resistance-related proteins with a leucine-rich region, a mitogen-activated protein kinase (MAPK) on chromosome 18, and a MYB transcription factor on chromosome 19 were identified as promising candidate genes. The identified SNPs and candidate genes will benefit future marker-assisted breeding and dissection of the molecular mechanisms underlying the soybean-SCN interaction.

What is the impact of age on reoperation rates for femoral neck fractures treated with internal fixation and hemiarthroplasty? A comparison of hip fracture outcomes in the very elderly population.

METHODS: A retrospective comparative study was performed at a level 1 trauma center at which electronic medical records and digital radiographs were reviewed for 949 femoral neck fractures. For the primary outcome of reoperation based on age, Kaplan-Meier models were built and analysis applied. RESULTS: A total of 334 fractures were nondisplaced treated with closed reduction and percutaneous pinning (CRPP), and 615 were displaced managed with hemiarthroplasty (HA). Overall, 98 patients (10.33%) required reoperation. Increasing reoperation rates for CRPP was seen with each subsequent age group. The opposite was seen with HA in which increasing age groups showed lower reoperation rates.

Effects of Immunonutrition for Cystectomy on Immune Response and Infection Rates: A Pilot Randomized Controlled Clinical Trial.

UNLABELLED: After radical cystectomy (RC), patients are at risk for complications including infections. The expansion of myeloid-derived suppressor cells (MDSCs) after surgery may contribute to the lower resistance to infection. Immune response and postoperative complications were compared in men consuming either specialized immunonutrition (SIM; n=14) or an oral nutrition supplement (ONS; n=15) before and after RC. MDSC count (Lin- CD11b+ CD33+) was significantly different between the groups over time (p=0.005) and significantly lower in SIM 2 d after RC (p<0.001). MDSC count expansion from surgery to 2 d after RC showed a weak association with an increase in infection rate 90 d after surgery (p=0.061). Neutrophil:lymphocyte ratio was significantly lower in SIM compared with ONS 3h after the first incision (p=0.039). Participants receiving SIM had a 33% reduction in postoperative complication rate (95% confidence interval [CI], 1-64; p=0.060) and a 39% reduction in infection rate (95% CI, 8-70; p=0.027) during late-phase recovery. The small sample size limits the study findings. PATIENT SUMMARY: Results show that the immune response to surgery and late infection rates differ between radical cystectomy patients receiving specialized immunonutrition versus oral nutrition supplement in the perioperative period. TRIAL REGISTRATION: ClinicalTrials.gov NCT01868087.

Multicenter assessment of neoadjuvant chemotherapy for muscle-invasive bladder cancer.

BACKGROUND: The efficacy of neoadjuvant chemotherapy (NAC) for muscle-invasive bladder cancer (BCa) was established primarily with methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC), with complete response rates (pT0) as high as 38%. However, because of the comparable efficacy with better tolerability of gemcitabine and cisplatin (GC) in patients with metastatic disease, GC has become the most commonly used regimen in the neoadjuvant setting. OBJECTIVE: We aimed to assess real-world pathologic response rates to NAC with different regimens in a large, multicenter cohort. DESIGN, SETTING, AND PARTICIPANTS: Data were collected retrospectively at 19 centers on patients with clinical cT2-4aN0M0 urothelial carcinoma of the bladder who received at least three cycles of NAC, followed by radical cystectomy (RC), between 2000 and 2013. INTERVENTION: NAC and RC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was pathologic stage at cystectomy. Univariable and multivariable analyses were used to determine factors predictive of pT0N0 and ≤pT1N0 stages. RESULTS AND LIMITATIONS: Data were collected on 935 patients who met inclusion criteria. GC was used in the majority of the patients (n=602; 64.4%), followed by MVAC (n=183; 19.6%) and other regimens (n=144; 15.4%). The rates of pT0N0 and ≤pT1N0 pathologic response were 22.7% and 40.8%, respectively. The rate of pT0N0 disease for patients receiving GC was 23.9%, compared with 24.5% for MVAC (p=0.2). There was no difference between MVAC and GC in pT0N0 on multivariable analysis (odds ratio: 0.89 [95% confidence interval, 0.61-1.34]; p=0.6). CONCLUSIONS: Response rates to NAC were lower than those reported in prospective randomized trials, and we did not discern a difference between MVAC and GC. Without any evidence from randomized prospective trials, the best NAC regimen for invasive BCa remains to be determined. PATIENT SUMMARY: There was no apparent difference in the response rates to the two most common presurgical chemotherapy regimens for patients with bladder cancer.

Modifier 22 on perioperative outcomes of robotic assisted laparoscopic prostatectomy.

INTRODUCTION: Robotic assisted laparoscopic prostatectomy (RALP) is a mainstay in the treatment of prostate cancer. Current procedure terminology (CPT) identifies a case that requires substantially greater effort than usual by using the modifier 22 code (M22). Our objective was to identify the most common etiologies leading to M22 at our institution and determine the effect on perioperative outcomes. MATERIALS AND METHODS: We retrospectively reviewed our prostatectomy database from 2009-2012 to identify patients who underwent RALP with and without M22. Reasons for M22 were determined by review of operative reports. Comparisons were made using Chi-square analysis and independent t-tests for continuous data. RESULTS: Of 579 patients identified from our database, 208 (36%) had a M22. Eighty-six (41%) patients had ≥ 2 documented reasons for M22. Adhesiolysis was the most common reason for M22 followed by large prostate and previous hernia mesh. Body mass index (BMI) (29.8 versus 28), prostate volume (53 g versus 44 g), operative time (259 minutes versus 234 minutes), and discharge from hospital with pelvic drain in place (6.7% versus 3%) were all significantly higher in the M22 group. Final pathological stage and positive margin rate were not increased in those with a M22. Complications were not different between those with and without M22. CONCLUSION: The M22 code is associated with longer operative times, larger prostates, and higher BMI. Adverse effects on final pathological stage, margin status and complications were not found in those with M22. Many patients with a M22 have more than one reason documented as for the explanation of the modifier.

Side effects of perioperative intravesical treatment and treatment strategies for these side effects.

Perioperative intravesical chemotherapy has a well-established role in the treatment of non-muscle invasive bladder cancer. There are multiple agents that can be used in this fashion with varying properties. Although chemical cystitis is the most common side effect and is usually self-limiting, significant toxicity can occur with intravesical chemotherapy. It is imperative that the urologist is aware of the acute and delayed side effects of intravesical chemotherapy and how to manage potential complications. Both local and systemic toxicities are discussed, as well as strategies to minimize and manage them.