The Role of Human Epidermal Growth Factor Receptor 2 (HER2)-Targeted Therapies in Early-Stage Breast Cancer: Current Practices, Treatment De-escalation, and Future Prospects.

Human epidermal growth factor receptor 2 (HER2)-targeted therapy has transformed the treatment paradigm for early-stage HER2-positive breast cancer, providing personalized and effective interventions. This comprehensive review delves into the current state of HER2-targeted therapies, emphasizing pivotal clinical trials that have demonstrated their substantial impact on long-term outcomes. Combination therapies that integrate HER2-targeted agents with chemotherapy exhibit enhanced tumor responses, particularly in neoadjuvant settings. Neoadjuvant chemotherapy (NACT) is explored for its role in tumor downsizing, facilitating breast-conserving surgery (BCS), and incorporating oncoplastic solutions to address both oncologic efficacy and aesthetic outcomes. Innovative axillary management post-NACT, such as targeted axillary dissection (TAD), is discussed for minimizing morbidity. The review further explores the delicate balance between maximal therapy and de-escalation, reflecting recent trends in treatment approaches. The therapeutic landscape of HER2-low breast cancer is examined, highlighting considerations in HER2-positive breast cancer with BReast CAncer gene (BRCA) mutations. Emerging immunotherapeutic strategies, encompassing immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy, are discussed in the context of their potential integration into treatment paradigms. In conclusion, the evolving landscape of HER2-positive early-stage breast cancer treatment, characterized by targeted therapies and multidisciplinary approaches, underscores the need for ongoing research and collaborative efforts. The aim is to refine treatment strategies and enhance patient outcomes in this dynamic and rapidly evolving field.

Analytical and clinical validation of multiplex droplet digital PCR assay for detecting pathogenic fungal infection in lungs.

Pulmonary invasive fungal infection in immunocompromised hosts is difficult to diagnose, and current tools for diagnosis or monitoring of response to antifungal treatments have inherent limitations. Droplet digital PCR (ddPCR) has emerged as a promising tool for pulmonary pathogen detection with high sensitivity. This study presents a novel ddPCR panel for rapid and sensitive identification of pulmonary fungal pathogens. First, a ddPCR method for detecting three fungal genera, including Pneumocystis, Aspergillus, and Cryptococcus, was established and evaluated. Then, the clinical validation performance of ddPCR was compared with that of qPCR using 170 specimens, and the 6 specimens with inconsistent results were further verified by metagenomics next-generation sequencing, which yielded results consistent with the ddPCR findings. Finally, the area under the ROC curve (AUC) was used to evaluate the efficiency of ddPCR. While the qPCR identified 16 (9.41%) cases of Aspergillus and 6 (3.53%) cases of Pneumocystis, ddPCR detected 20 (11.76%) Aspergillus cases and 8 (4.71%) Pneumocystis cases. The AUC for Aspergillus, Cryptococcus, and Pneumocystis was 0.974, 0.998, and 0.975, respectively. These findings demonstrated that the ddPCR assay is a highly sensitive method for identifying pathogens responsible for invasive fungal pulmonary infections, and is a promising tool for early diagnosis. .

Syndromic testing for respiratory pathogens but not National Early Warning Score can be used to identify viral cause in hospitalised adults with lower respiratory tract infections.

BACKGROUND: Community-acquired lower respiratory tract infection (LRTI) is a common reason for hospitalisation. Antibiotics are frequently used while diagnostic microbiological methods are underutilised in the acute setting. OBJECTIVES: We aimed to investigate the relative proportion of viral and bacterial infections in this patient group and explore methods for proper targeting of antimicrobial therapy. METHODS: We collected nasopharyngeal samples prospectively from adults hospitalised with LRTIs during three consecutive winter seasons (2016-2019). Syndromic nasopharyngeal testing was performed using a multiplex PCR panel including 16 viruses and four bacteria. Medical records were reviewed for clinical data. RESULTS: Out of 220 included patients, a viral pathogen was detected in 74 (34%), a bacterial pathogen in 63 (39%), both viral and bacterial pathogens in 49 (22%), while the aetiology remained unknown in 34 (15%) cases. The proportion of infections with an identified pathogen increased from 38% to 85% when syndromic testing was added to standard-of-care testing. Viral infections were associated with a low CRP level and absence of pulmonary infiltrates. A high National Early Warning Score did not predict bacterial infections. CONCLUSIONS: Syndromic testing by a multiplex PCR panel identified a viral infection or viral/bacterial coinfection in a majority of hospitalised adult patients with community-acquired LRTIs.

A novel fast hybrid capture sequencing method for high-efficiency common human coronavirus whole-genome acquisition.

Rapid and accurate sequencing of the entire viral genome, coupled with continuous monitoring of genetic changes, is crucial for understanding the epidemiology of coronaviruses. We designed a novel method called micro target hybrid capture system (MT-Capture) to enable whole-genome sequencing in a timely manner. The novel design of probes used in target binding exhibits a unique and synergistic "hand-in-hand" conjugation effect. The entire hybrid capture process is within 2.5 hours, overcoming the time-consuming and complex operation characteristics of the traditional liquid-phase hybrid capture (T-Capture) system. By designing specific probes for these coronaviruses, MT-Capture effectively enriched isolated strains and 112 clinical samples of coronaviruses with cycle threshold values below 37. Compared to multiplex PCR sequencing, it does not require frequent primer updates and has higher compatibility. MT-Capture is highly sensitive and capable of tracking variants.IMPORTANCEMT-Capture is meticulously designed to enable the efficient acquisition of the target genome of the common human coronavirus. Coronavirus is a kind of virus that people are generally susceptible to and is epidemic and infectious, and it is the virus with the longest genome among known RNA viruses. Therefore, common human coronavirus samples are selected to evaluate the accuracy and sensitivity of MT-Capture. This method utilizes innovative probe designs optimized through probe conjugation techniques, greatly shortening the time and simplifying the handwork compared with traditional hybridization capture processes. Our results demonstrate that MT-Capture surpasses multiplex PCR in terms of sensitivity, exhibiting a thousandfold increase. Moreover, MT-Capture excels in the identification of mutation sites. This method not only is used to target the coronaviruses but also may be used to diagnose other diseases, including various infectious diseases, genetic diseases, or tumors.

Taqman PACMAN: a simple molecular approach for positive rapid antigen test confirmation during periods of low prevalence.

Antigen-based rapid diagnostic tests (Ag-RDTs) were widely deployed to enhance SARS-CoV-2 testing capacity during the COVID-19 pandemic. Consistent with national guidance for low prevalence settings, positive Ag-RDTs were confirmed using nucleic acid amplification tests (NAATs) to avoid false positive results. However, increasing demands for positive Ag-RDT confirmation competed with other testing priorities in clinical laboratories. This work hypothesized that real-time RT-PCR without nucleic acid extraction (NAE) would be sufficiently sensitive to support positive Ag-RDT confirmation. Ag-RDT and NAAT results from community-based asymptomatic testing sites prior to the omicron variant wave were compared to calculate the weekly false positive rate (FPR) and false detection rate (FDR). Real-time RT-PCR was compared with and without NAE using 752 specimens previously tested positive for SARS-CoV-2 using commercial NAATs and 344 specimens from Ag-RDT-positive individuals. The impact of SARS-CoV-2 prevalence on laboratory resources required to sustain Ag-RDT confirmation was modeled for the RT-PCR with and without NAE. Overall, FPR was low [0.07% (222/330,763)] in asymptomatic testing sites, but FDR was high [30.7% (222/724)]. When RT-PCR was compared with and without NAE, 100% concordance was obtained with NAAT-positive specimens, including those from Ag-RDT-positive individuals. NAE-free RT-PCR significantly reduced time to results, human resources, and overall costs. A 30.7% FDR reaffirms the need for NAAT-based confirmation of positive Ag-RDT results during low SARS-CoV-2 prevalence. NAE-free RT-PCR was shown to be a simple and cost-sparing NAAT-based solution for positive Ag-RDT confirmation, and its implementation supported data-driven broader Ag-RDT deployment into communities, workplaces, and households. IMPORTANCE: Rapid antigen testing for SARS-CoV-2 was widely deployed during the COVID-19 pandemic. In settings of low prevalence, national guidance recommends that positive antigen test results be confirmed with molecular testing. Given the high testing burden on clinical laboratories during the COVID-19 pandemic, the high volume of positive antigen tests submitted for confirmatory testing posed challenges for laboratory workflow. This study demonstrated that a simple PCR method without prior nucleic acid purification is an accurate and cost-effective solution for positive rapid antigen test confirmation. Implementing this method allowed molecular confirmatory testing for positive antigen tests to be sustained as antigen testing was expanded into large populations such as workplaces, schools, and households.

Detection of Escherichia coli O157H7 and Campylobacter jejuni in Bovine Carcasses in Two Slaughterhouses in Bio-Bío District, Chile.

Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.

Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

Neq2X7: a multi-purpose and open-source fusion DNA polymerase for advanced DNA engineering and diagnostics PCR.

Thermostable DNA polymerases, such as Taq isolated from the thermophilic bacterium Thermus aquaticus, enable one-pot exponential DNA amplification known as polymerase chain reaction (PCR). However, properties other than thermostability - such as fidelity, processivity, and compatibility with modified nucleotides - are important in contemporary molecular biology applications. Here, we describe the engineering and characterization of a fusion between a DNA polymerase identified in the marine archaea Nanoarchaeum equitans and a DNA binding domain from the thermophile Sulfolobus solfataricus. The fusion creates a highly active enzyme, Neq2X7, capable of amplifying long and GC-rich DNA, unaffected by replacing dTTP with dUTP in PCR, and tolerant to various known PCR inhibitors. This makes it an attractive DNA polymerase for use, e.g., with uracil excision (USER) DNA assembly and for contamination-free diagnostics. Using a magnification via nucleotide imbalance fidelity assay, Neq2X7 was estimated to have an error rate lower than 2 ∙ 10-5 bp-1 and an approximately 100x lower fidelity than the parental variant Neq2X, indicating a trade-off between fidelity and processivity - an observation that may be of importance for similarly engineered DNA polymerases. Neq2X7 is easy to produce for routine application in any molecular biology laboratory, and the expression plasmid is made freely available.

Quality Control of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel.

Isogenic H/N/KRAS-less mouse embryonic fibroblast (MEF) cell lines have been developed to assist in cell-based assays of RAS inhibitors. The quality control assessment of a panel of these isogenic MEFs is described here, with a focus on ensuring the proper insertion of the desired mutant RAS transgene, a determination of gene copy number, and an investigation of potential off-target mutations which could lead to phenotypes which are undesired in downstream experiments. Using this suite of quality control tools, a MEF cell line can be readily validated, and researchers can be assured of the rationale for an observed phenotype.

The Relationship of Pathological Response and Visceral Muscle and Fat Volume in Women With Breast Cancer Who Received Neoadjuvant Chemotherapy.

Objective: Differences in individual muscle/fat volumes may change the effectiveness of chemotherapy. In this study, the relationship between trunkal muscle and fat volume and body mass index (BMI) obtained before receiving neoadjuvant chemotherapy (NCT) in patients with breast cancer and complete pathological response (pCR) was investigated. Materials and Methods: The volumes of psoas, abdominal and paraspinal muscles, and trunkal subcutaneous and visceral fat were calculated using CoreSlicer AI 2.0 opensource program from the F-18 fluorodeoxyglucose positron emission tomography/computed tomography (CT) and CT images before NCT and postoperative pCR rates to NCT were recorded. Muscle/fat volumes and BMI prior to NCT were compared in terms of pathological pCR rates. Patients were followed up regularly for recurrence and survival. Results: Ninety-three patients were included with median (range) values for age, BMI, and body weights of 48 (28-72) years, 27 (16.8-51.6) kg/m2, and 71.94 (43-137) kg, respectively. The median follow-up time was 18.6 (6.7-59.6) months. No significant correlation was found between total muscle or fat volumes of patients with and without pCR. BMI [26.2 (16.8-51.6) kg/m2 vs. 24.6 (20.3-34.3) kg/m2, p = 0.03] and pCR rates in patients with low right-psoas muscle volume [11.74 (7.03-18.51) vs. 10.2 (6.71-13.36), p = 0.025] were significantly greater. A significant relationship was found between right psoas muscle volume and disease-free survival (DFS) (11.74 cm3 (7.03-18.51) vs. 10.2 cm3 (6.71-13.36), p = 0.025). However, no significant relationship was detected between total muscle-fat volume, BMI and overall survival and DFS (p>0.05). Conclusion: This is the first published study investigating the relationship between the pCR ratio and body muscle and fat volume measured by CoreSlicer AI 2.0 in patients with breast cancer who received NCT. No correlation was found between the pCR ratio and total muscle plus fat volume. However, these results need to be validated with larger patient series.

Assessment of TNF-α (-857 C/T) gene polymorphism in oral lichen planus disease: A case-control study.

Background and aims: Oral lichen planus (OLP) is an inflammatory mucocutaneous disorder with an immune-mediated pathogenesis. The tumor necrosis factor-α (TNF-α) level in the serum of OLP patients is significantly higher than in the control group. TNF-α-857 C/T polymorphism can be related to the increased TNF-α level in blood circulation. This study investigated the relationship between TNF-α (-857 C/T) polymorphism and OLP patients in an Iranian population. Methods: Saliva samples were taken from 200 people, including 100 patients with OLP and 100 healthy people who did not have significant differences in age and sex. Then, DNA was extracted from them and the TNF-α (-857 C/T) genotype was identified using the polymerase chain reaction with confronting two-pair primers method. Statistical Package for the Social Sciences version 16 software analyzed the results. Results: The frequency of C/C, C/T, and T/T genotypes of the TNF-α-857 C/T polymorphism in the patient group were 78%, 18%, and 4%, respectively, and in the control group were 72%, 23%, and 5%, respectively. The differences between the two groups regarding allele (χ 2 = 0.97, p = 0.32) and genotype (χ 2 = 0.96, p = 0.62) frequency among the studied population were insignificant. Conclusion: This study showed that the difference in the frequency of single nucleotide polymorphism TNF-α-857 C/T polymorphism in the patient and control group had no significant relationship with the increased OLP incidence. Also, no significant association was observed between allele and genotype frequency of TNF-α (-857 C/T) with OLP subtypes.

Advancing Genetic Alphabet Expansion: Synthesis of 7-(2-Thienyl)-Imidazo[4,5-b]pyridine (Ds) and 4-(4-Pentyne-1,2-diol)-1-Propynyl-2-Nitropyrrole (Diol-Px) for Use in Replicable Unnatural Base Pairs for PCR Applications.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Detection of ectoparasites and investigation of infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul, Brazil.

This study aimed to investigate the presence of ectoparasites and the occurrence of natural infection by Rickettsia spp. and Trypanosoma spp. in bats from Rio Grande do Sul (RS), Brazil. The evaluated animals were obtained from the Instituto de Pesquisas Veterinárias Desidério Finamor, sent by the Centro Estadual de Vigilância Sanitária, to carry out rabies diagnostic tests, during the period from 2016 to 2021. The bats came from 34 municipalities in RS. Of the 109 animals surveyed, 35.8% (39/109) had 385 ectoparasites, with an average of 9.9 parasites per animal. Of these bats, all had insectivorous feeding habits, with 35.9% (14/39) females and 64.1% (25/39) males. The co-parasitism of Chirnyssoides sp., Ewingana inaequalis, and Chiroptonyssus robustipes on Molossus currentium (Mammalia, Chiroptera) was recorded for the first time. All bats surveyed were negative for infection by the protozoan and bacteria. Thus, the expansion of the occurrence of these ectoparasites in insectivorous bats in RS was observed. Furthermore, this study corresponds to the first recorded interspecific associations for the species.

Effectiveness of a standardized quality control management procedure for COVID-19 RT-PCR testing: a large-scale diagnostic accuracy study in China.

BACKGROUND: The study aimed to construct a standardized quality control management procedure (QCMP) and access its accuracy in the quality control of COVID-19 reverse transcriptase-polymerase chain reaction (RT-PCR). METHODS: Considering the initial RT-PCR results without applying QCMP as the gold standard, a large-scale diagnostic accuracy study including 4,385,925 participants at three COVID-19 RT-PCR testing sites in China, Foshan (as a pilot test), Guangzhou and Shenyang (as validation sites), was conducted from May 21, 2021, to December 15, 2022. RESULTS: In the pilot test, the RT-PCR with QCMP had a high accuracy of 99.18% with 100% specificity, 100% positive predictive value (PPV), and 99.17% negative predictive value (NPV). The rate of retesting was reduced from 1.98% to 1.16%. Its accuracy was then consistently validated in Guangzhou and Shenyang. CONCLUSIONS: The RT-PCR with QCMP showed excellent accuracy in identifying true negative COVID-19 and relieved the labor and time spent on retesting.

ddPCR enables rapid detection of bloodstream infections in patients on home parenteral nutrition: A prospective cohort study.

INTRODUCTION: Chronic intestinal failure patients (CIF) require a central venous access device (CVAD) to administer parenteral nutrition. Most serious complication related to a CVAD is a central line-associated bloodstream infection (CLABSI). The golden standard to diagnose a CLABSI are blood cultures, however, they may require 1-5 days before getting a result. Droplet digital polymerase chain reaction (ddPCR) for the detection of pathogen 16S/28S rRNA is a novel culture-independent molecular technique that has been developed to enhance and expedite infection diagnostics within two and a half hours. In this study, we prospectively compared ddPCR with blood cultures to detect pathogens in whole blood. METHODS: We included adult CIF patients with a clinical suspicion of CLABSI in this prospective single-blinded clinical study. Blood cultures were routinely collected and subsequently two central samples from the CVAD and two peripheral samples from a peripheral venous access point. Primary outcome was the sensitivity and specificity of ddPCR. RESULTS: In total, 75 patients with 126 suspected CLABSI episodes were included, with 80 blood samples from the CVAD and 114 from peripheral veins. The central ddPCR samples showed a sensitivity of 91% (95%CI 77-98), and specificity of 96% (95%CI 85-99). Peripheral ddPCR samples had a sensitivity of 63% (95%CI 46-77) and specificity of 99% (95%CI 93-100). CONCLUSION: ddPCR showed a high sensitivity and specificity relative to blood cultures and enables rapid pathogen detection and characterization. Clinical studies should explore if integrated ddPCR and blood culture outcomes enables a more rapid pathogen guided CLABSI treatment and enhancing patient outcomes.

Antimicrobial resistance of avian pathogenic Escherichia coli isolated from broiler, layer, and breeder chickens.

Background and Aim: Antimicrobials are extensively used in poultry production for growth promotion as well as for the treatment and control of diseases, including avian pathogenic Escherichia coli (APEC). Poor selection, overuse, and misuse of antimicrobial agents may promote the emergence and dissemination of antimicrobial resistance (AMR) in APEC. This study aimed to assess antimicrobial susceptibility patterns and detect antibiotic resistance genes (ARGs) in APEC isolated from clinical cases of colibacillosis in commercial broiler, layer, and breeder chickens. Materials and Methods: A total of 487 APEC were isolated from 539 across 300 poultry farms in various regions of Nepal. Antimicrobial susceptibility patterns was determined using the Kirby-Bauer disk diffusion and broth microdilution methods. The index of AMR, such as multiple antibiotic resistance (MAR) index, resistance score (R-score), and multidrug resistance (MDR) profile, were determined. Polymerase chain reaction was employed to detect multiple ARGs and correlations between phenotypic and genotypic resistance were analyzed. Results: The prevalence of APEC was 91% (487/539). All of these isolates were found resistant to at least one antimicrobial agent, and 41.7% of the isolates were resistant against 8-9 different antimicrobials. The antibiogram of APEC isolates overall showed the highest resistance against ampicillin (99.4%), whereas the highest intermediate resistance was observed in enrofloxacin (92%). The MAR index and R-score showed significant differences between broiler and layers, as well as between broiler breeder and layers. The number of isolates that were resistant to at least one agent in three or more antimicrobial categories tested was 446 (91.6%) and were classified as MDR-positive isolates. The ARGs were identified in 439 (90.1%) APEC isolates, including the most detected mobilized colistin resistance (mcr1) which was detected in the highest (52.6%) isolates. Overall, resistance gene of beta-lactam (blaTEM), mcr1, resistance gene of sulphonamide (sul1) and resistance gene of tetracycline (tetB) (in broiler), were detected in significantly higher than other tested genes (p < 0.001). When examining the pair-wise correlations, a significant phenotype-phenotype correlation (p < 0.001) was observed between levofloxacin and ciprofloxacin, chloramphenicol and tetracycline with doxycycline. Similarly, a significant phenotype-genotype correlation (p < 0.001) was observed between chloramphenicol and the tetB, and colistin with blaTEM and resistance gene of quinolone (qnrA). Conclusion: In this study, the current state of APEC AMR in commercial chickens is revealed for the first time in Nepal. We deciphered the complex nature of AMR in APEC populations. This information of molecular surveillance is useful to combat AMR in APEC and to contribute to manage APEC associated diseases and develop policies and guidelines to enhance the commercial chicken production.

Comparison of different screening methods for the selection of Ascochyta blight disease on chickpea (Cicer arietinum L.) genotypes.

Chickpea (Cicer arietinum L.) is the second most important edible food grain legume, widely grown all over the world. However, the cultivation and production of chickpea are mainly affected by the Ascochyta blight (AB) disease, which causes losses of up to 100% in areas with high humidity and warm temperature conditions. Various screening methods are used in the selection of chickpea genotypes for resistance to AB disease. These methods are natural field condition (NFC), artificial epidemic field condition (AEC), marker-assisted selection (MAS), and real-time PCR (RT-PCR). The study was conducted with 88 chickpea test genotypes between the 2014 and 2016 growing seasons. The results of the screening were used to sort the genotypes into three categories: susceptible (S), moderately resistant (MR), and resistant (R). Using MAS screening, 13, 21, and 54 chickpea genotypes were identified as S, MR, and R, respectively. For RT-PCR screening, 39 genotypes were S, 31 genotypes were MR, and 18 genotypes were R. In the AEC method for NFC screening, 7, 17, and 64 genotypes were S, MR, and R, while 74 and 6 genotypes were S and MR, and 8 genotypes were R-AB disease. As a result of screening chickpea genotypes for AB disease, it was determined that the most effective method was artificial inoculation (AEC) under field conditions. In the study, Azkan, ICC3996, Tüb-19, and Tüb-82 were determined as resistant within all methods for Pathotype 1.

Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method.

Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1-15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures.

Effect of dietary chitosan on the growth performance, intestinal histology and growth-related gene expression in stellate sturgeon (Acipenser stellatus) juveniles.

As sturgeon breeding has proliferated, there has been a heightened demand for growth stimulators in their diets. This study aimed to determine the impact of dietary chitosan on growth performance, whole-body proximate composition, growth-related gene expression, and intestinal histology in juvenile Acipenser stellatus. A total of 180 A. stellatus juveniles with an average weight of 31.90 ± 0.73 g were fed with diets containing 0 (control), 1.5, 3.0, 4.5, and 6.0 g chitosan.kg-1 basic diet for eight weeks. The findings revealed a significant enhancement in growth performance with rising chitosan concentrations. Furthermore, chitosan supplementation upregulated the expression of the growth hormone gene in both brain and liver tissues. In liver samples, the most pronounced expression of the insulin-like growth factor-1 gene was noted at 6.0 g chitosan.kg-1, while in brain samples, peak expressions were observed in both the 4.5 and 6.0 g chitosan.kg-1 treatments. While the whole-body proximate composition remained relatively stable, there was a notable decrease in whole-body lipids with the escalation of chitosan dosage. Intestinal villi dimensions, both height and width, were amplified in the chitosan-supplemented groups compared to controls. In summation, chitosan supplementation showed promise in bolstering growth performance, refining intestinal morphology, and enhancing growth-related gene expression. Analysis of the polynomial regression of weight gain and specific growth rate revealed that the optimum dietary chitosan requirements in A. stellatus were 5.32 and 5.21 g chitosan.kg-1, respectively.

Dynamics and prognostic value of plasma cell-free DNA PCR in patients with invasive aspergillosis and mucormycosis.

Aspergillus species and Mucorales agents are the primary etiologies of invasive fungal disease (IFD). Biomarkers that predict outcomes are needed to improve care. Patients diagnosed with invasive aspergillosis and mucormycosis using plasma cell-free DNA (cfDNA) PCR were retested weekly for 4 weeks. The primary outcome included all-cause mortality at 6 weeks and 6 months based on baseline cycle threshold (CT) values and results of follow-up cfDNA PCR testing. Forty-five patients with Aspergillus and 30 with invasive Mucorales infection were retested weekly for a total of 197 tests. Using the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSG) criteria, 30.7% (23/75), 25.3% (19/75), and 38.7% (29/75) had proven, probable, and possible IFD, respectively. In addition, 97.3% (73/75) were immunocompromised. Baseline CT increased significantly starting at week 1 for Mucorales and week 2 for Aspergillus. Aspergillosis and mucormycosis patients with higher baseline CT (CT >40 and >35, respectively) had a nonsignificantly higher survival rate at 6 weeks, compared with patients with lower baseline CT. Mucormycosis patients with higher baseline CT had a significantly higher survival rate at 6 months. Mucormycosis, but not aspergillosis patients, with repeat positive cfDNA PCR results had a nonsignificantly lower survival rate at 6 weeks and 6 months compared with patients who reverted to negative. Aspergillosis patients with baseline serum Aspergillus galactomannan index <0.5 and <1.0 had significantly higher survival rates at 6 weeks when compared with those with index ≥0.5 and ≥1.0, respectively. Baseline plasma cfDNA PCR CT can potentially be used to prognosticate survival in patients with invasive Aspergillus and Mucorales infections. IMPORTANCE: We show that Aspergillus and Mucorales plasma cell-free DNA PCR can be used not only to noninvasively diagnose patients with invasive fungal disease but also to correlate the baseline cycle threshold with survival outcomes, thus potentially allowing the identification of patients at risk for poor outcomes, who may benefit from more targeted therapies.