High performance integrated terahertz sensor for detection of biomolecular processes in solution

The analytical performance of methods for the determination of hydride forming elements has been improved recently by the development of procedures in which the hydride is trapped on the interior surface of a graphite furnace atomizer. The signal for a given concentration increases with increase in sample volume and it is often implied that a decrease in the limit of detection may also be achieved by increasing the sample volume. To evaluate this claim, a simple equation was derived which predicts the relationship between detection limit and sample volume when all the contributions to the blank are proportional to sample volume. A time-based approach to the variation of sample volume was developed to ensure that the analyte introduced from reagent contamination was, in fact, proportional to sample volume. Detection limits were measured for a series of sample volumes between 156 and 1560 µl. As the sample volume was increased, the detection limit improved significantly from 0.3 to around 0.05 µg l–1 up to a volume of about 500 µl. Between 500 and 1000 µl, a further improvement, to around 0.02 µg l–1, was obtained, but for volumes larger than 1000 µl no further significant improvement was obtained. Good agreement between the predicted and experimentally determined variations in detection limit with sample volume was obtained and thus the underlying inverse proportionality of the relationship between detection limit and sample volume was confirmed. This rectangular hyperbolic relationship has practical consequences for the extent to which detection limits can be improved by increasing the sample volume, even when the blank is very low or zero.

OBJECTIVES We conducted this study to determine what sample volume, concentration, and limit of detection (LOD) are adequate for environmental validation of Legionella control. We also sought to determine whether time required to obtain culture results can be reduced compared to spread-plate culture method. We also assessed whether polymerase chain reaction (PCR) and in-field total heterotrophic aerobic bacteria (THAB) counts are reliable indicators of Legionella in water samples from buildings. DESIGN Comparative Legionella screening and diagnostics study for environmental validation of a healthcare building water system. SETTING Veterans Health Administration (VHA) facility water system in central Texas. METHODS We analyzed 50 water samples (26 hot, 24 cold) from 40 sinks and 10 showers using spread-plate cultures (International Standards Organization [ISO] 11731) on samples shipped overnight to the analytical lab. In-field, on-site cultures were obtained using the PVT (Phigenics Validation Test) culture dipslide-format sampler. A PCR assay for genus-level Legionella was performed on every sample. RESULTS No practical differences regardless of sample volume filtered were observed. Larger sample volumes yielded more detections of Legionella. No statistically significant differences at the 1 colony-forming unit (CFU)/mL or 10 CFU/mL LOD were observed. Approximately 75% less time was required when cultures were started in the field. The PCR results provided an early warning, which was confirmed by spread-plate cultures. The THAB results did not correlate with Legionella status. CONCLUSIONS For environmental validation at this facility, we confirmed that (1) 100 mL sample volumes were adequate, (2) 10× concentrations were adequate, (3) 10 CFU/mL LOD was adequate, (4) in-field cultures reliably reduced time to get results by 75%, (5) PCR provided a reliable early warning, and (6) THAB was not predictive of Legionella results. Infect Control Hosp Epidemiol 2018;39:259-266.

Canine parvovirus type 2 (CPV-2) is an important pathogen that causes anorexia, vomiting, acute gastroenteritis, and bloody diarrhea. CPV-2 was divided into three variants (2a–2c) based on residue 426 of the VP2 protein. Recently, a novel Asian CPV-2c isolate was more prevalent in the Asian continent. The diagnostic tools of CPV-2 are characterized into traditional (such as immunochromatography test) and molecular methods based on their pathogen detection mechanisms. However, the low sensitivity of the immunochromatographic tests is supported by their simplicity and rapid ability to provide results for CPV-2. Surprisingly, some immunochromatography tests showed low sensitivity for the detection of novel CPV-2c compared to a real-time polymerase chain reaction (PCR) assay. Sixty rectal swabs were collected from naturally infected dogs with CPV-2 at the Animal Disease Diagnostic Center, National Pingtung University of Science and Technology, Taiwan, from 2016 to 2019. A novel in-house QubeMDx PCR system was used for diagnosis of three CPV-2 variants and compared to real-time PCR diagnosis. This diagnostic assay detected all of CPV-2 variants within 30 min and the sensitivity was identical to a real-time PCR assay using clinical samples. This study provides evidence that this novel, commercially available in-house PCR assay (QubeMDx PCR system) provides useful, simple, sensitive, reliable, and rapid diagnosis of three CPV-2 variants in veterinary clinics.

The diagnosis of cutaneous leishmaniasis (CL) is traditionally based on microscopic demonstration of amastigote forms in tissue biopsies or smears. However, this method usually presents low sensitivity, and in atypical forms, CL may be overlooked because of similarity to other dermal diseases. Thus, it is necessary to apply specific diagnostic methods as polymerase chain reaction (PCR). To evaluate the possible advantage of PCR in the diagnosis and species identification of CL in patients with atypical clinical presentation. Fifty-one patients clinically suspected of CL with positive and negative controls were tested. After microscopic examination, extraction of DNA was performed on their smears and analysed by two specific PCR assays for diagnosis and species identification. For these methods, conserved and variable regions of kinetoplastic DNA (KDNA) of Leishmania species have been amplified, respectively. Atypical forms of CL were evaluated among PCR-positive patients. PCR results were positive in 37 out of 51 cases (72.5%), among whom microscopic examination revealed Leishmania amastigotes in only 3 (5.9%). Among these patients, 10 (27%) had atypical presentation of CL; using species-specific primers, 6 patients had Leishmania major, 3 had Leishmania tropica and 1 patient had no species diagnosis. None of the samples of other dermal diseases revealed positive results (specificity, 100%). All patients were successfully treated by CL-specific drug regimens. The results showed that KDNA PCR methods have a higher sensitivity compared with microscopic method. Moreover, PCR could identify the parasite species for specific therapy. Microscopic method had low sensitivity and less value in chronic and atypical CL cases.

Development and validation of a high throughput direct radioimmunoassay for the quantitative determination of serum and plasma melatonin (N-acetyl-5-methoxytryptamine) in mice

Schistosomiasis (Bilharzia), a neglected tropical disease caused by Schistosoma parasites, afflicts over 240 million people globally, disproportionately impacting Sub-Saharan Africa. Current diagnostic tests, despite their utility, suffer from limitations like low sensitivity. Polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) remain the most common and sensitive nucleic acid amplification tests. Still, the sensitivity of nucleic acid amplification tests is significantly affected by the copy number of amplification targets, resulting in underestimation of true Schistosoma infections, especially in low transmission settings. Additionally, lengthy qPCR run times pose challenges when dealing with large sample volumes and limited resources. In this study, the identical multi-repeat sequences (IMRS) were used as a novel approach to enhance the sensitivity of nucleic acid-based Bilharzia diagnosis. To identify novel genomic repeat regions, we utilized the IMRS algorithm, with modifications to enable larger target region (100-200bp) identification instead of smaller sequences (18-30bp). These regions enabled customised primer-probe design to suit requirements for qPCR assay. To lower the qPCR amplification times, the assay was conducted using fast cycling condition. Regression analysis, and qPCR data visualization was conducted using Python programming. Using Schistosoma mansoni and S. haematobium, we found that IMRS-based qPCR, employing genus-specific primers and TaqMan probes, offers exceptional analytical sensitivity, detecting as little as a single genome copy per microliter within 36 minutes. The lowest concentration of DNA detected using IMRS-based PCR and qPCR represented tenfold improvement over conventional PCR. As part of further development, there is a need to compare IMRS-based qPCR against other qPCR methods for Schistosoma spp. Nonetheless, IMRS-based diagnostics promise a significant advancement in bilharzia diagnosis, particularly in low-transmission settings, potentially facilitating more effective control and treatment strategies.

Background Malaria, a persistent public health issue in Nigeria, particularly among children, is often complicated by misdiagnosis, hindering effective treatment and control. The global adoption of rapid diagnostic tests (RDTs) for malaria has significantly improved management. This study, therefore, compares the diagnostic performance of microscopy, RDT, and polymerase chain reaction (PCR) for Plasmodium falciparum detection in children in Kano state, Nigeria, providing crucial insights for effective control and elimination.MethodsCapillary blood samples and dried blood spots (DBS) were collected from 200 febrile children in a selected health center in Kano and systematically tested using RDT, microscopy, and nested polymerase PCR. The sensitivity and specificity for each diagnostic method were calculated, with nested PCR as the reference to assess diagnostic accuracy. Structured questionnaires were used to obtain information on possible risk factors. Data were analyzed using univariate, bivariate, and multivariate analysis, with p<0.05 considered significant.ResultsOut of the 200 children enrolled in the study, 112 (56%) tested positive by microscopy, 60 (30%) by RDT, and 70 (35%) by PCR. Using PCR (n=70) as the reference test, microscopy demonstrated a sensitivity of 73% and a specificity of 53%, while the RDT exhibited a lower sensitivity of 27% but a higher specificity of 68%. False positivity rates of 22 (31.5%) for RDT and 33 (46.9%) for microscopy were observed. Regarding false negatives, RDT had a much higher rate of 51 (72.9%) than microscopy of 19 (27.1%).ConclusionThe study divulges the limitations of RDT in malaria detection, particularly its low sensitivity. With its higher sensitivity, microscopy also showed a significant false-positive rate. The study suggests a practical solution combining RDT and microscopy for routine malaria diagnosis, which could significantly improve diagnostic accuracy. More ongoing research is needed to develop more suitable RDTs for routine malaria diagnosis in endemic communities.

Chlamydia trachomatis is known as the most common bacterial infection agent to pass with sexual transition. This microorganism is an obligatory intracellular parasite. A variety of infections are caused by C. trachomatis, including trachoma, pneumonias in newborns, genital and urinary tract infections, and lymphogranuloma venereum (LGV), which is caused by LGV strains. The diagnosis of Chlamydia trachomatis can be made by cultures and isolations, antigens and antibodies (direct fluorescence, enzyme immunoassays), hybridization, or polymerase chain reaction (PCR). Each year, infection and diagnosis rates increase in the developed world. Since Chlamydia is mostly asymptomatic, screening, and treatment are a key to detecting cases. Polymerase chain reaction (PCR), ligase chain reaction (LCR), and nucleic acid sequence-based amplification (NASBAa) molecular methods can be used for the detection, low concentration, quantification, and identification of organisms. While the traditional PCR method confirms its existence, it can quantify real-time PCR (RT-PCR). This method (RT-PCR) may have low sensitivity among variants of the same species. Also, PCR scans, which receive urine service, offer great advantages. PCR from initial void urine (FVU) samples is highly sensitive in detecting the organism. Urine Chlamydia screenings are more acceptable in large populations and asymptomatic detections.

We read the article by Rosário et al.1 in the Journal of Medical Virology with great interest. The authors conducted a study in women participating in the Regional Cervical Cancer Screening Program from the Northern Region of Portugal to evaluate the association between the composition of the cervicovaginal microbiome (CVM), high-risk human papillomavirus (hrHPV) status, and cytology results. Although the authors demonstrate interesting bacterial associations with women's age and hrHPV genotypes, we have several concerns regarding the study methods, results, and statistical analyses. The authors state that they investigated the CVM in their study, however, they only performed the characterization by polymerase chain reaction (PCR), targeting specifically bacterial microorganisms associated with the cervicovaginal microenvironment. This PCR-targeted approach has low species resolution and covers a minority of the CVM composition compared with traditional microbiome sequencing methods.2-4 Moreover, the commercial kits used in the study have not been properly investigated and validated for CVM profiling, especially in the context of hrHPV infections and cervical diseases, and thus, the associations described in the study might not be entirely accurate and should be interpreted cautiously. Further validation of these PCR-based kits should include assessing mock cervicovaginal community samples and comparison analyses with 16S rRNA gene sequencing of HPV-negative and HPV-positive samples. Species-level identification is extremely important for microbiome-hrHPV association studies due to the presence of diverse Lactobacillus species and the assortment of this complex in specific microbial communities with species-specific dynamics in health and disease.5 For instance, communities with Lactobacillus iners dominance are at higher risk of dysbiosis and hrHPV infection6 whereas communities with Lactobacillus crispatus dominance exhibit a protective role toward the development of hrHPV-induced cervical abnormalities.5 Since these species were not distinguished in the study due to the limited resolution of the methods, it is not adequate to strongly conclude the associations found with the Lactobacillus genus, challenging the interpretation and comparison of these results with current literature. Another concern is the described correlation of Gardnerella vaginalis, Atopobium vaginae, and Mobilincus species with protection against cervical abnormalities.1 Most microbiome-hrHPV studies have reported opposite results.2, 5, 7-9 In fact, these bacterial genera and species are characteristic of a Lactobacillus-depleted community that has a higher risk for hrHPV acquisition, persistence, and cervical disease development.5 The only described association of this community and species with a “health” stage throughout hrHPV infections is viral clearance,10 where it is been hypothesized that a shift in immune responses causes a temporary change in the CVM composition, thereby allowing the growth of these pathogenic species.10, 11 The authors also attributed these associations to women's age, and therefore, it is unclear whether age or the occurrence of cervical abnormalities is the contributing factor in these correlations.5, 12 Even though the study referred to these contradictory results in the discussion, we believe that this inconsistency should be further clarified. An in-depth analysis of the potential contributing factors in this population, such as age, ethnicity, immunity, hrHPV genotyping, bacterial vaginosis (BV), microbiome continuity and dynamics, viral-induced dysbiosis,6 and the results observed in the study is needed to fully understand these associations in the context of the natural history of HPV infections. As mentioned above, these inconsistent results may be explained by the study's targeted bacterial profiling approach with low resolution and by the statistical analyses. In the univariate logistic regression analyses,1 the authors showed results from a total of 72 statistical tests but according to the study's Methods and absence of q values, we assume that the authors did not correct their analyses for multiple comparisons. We have similar comments for the results obtained with the multivariate logistic regression analyses.1 In summary, the CVM has been the subject of increasing research interest due to its potential impact on the risk of cervical abnormalities and various gynecological conditions, including cervical cancer. While research into the CVM and cervical neoplasia is promising, it is essential to address methodological challenges and employ comprehensive, high-resolution multiomics investigations to advance our understanding and potentially translate these findings into improved gynecological care and cervical cancer preventive strategies, before strong conclusions on a limited set of data can be drawn. All authors contributed equally to this work. The authors declare no conflict of interest.

135-P

Background:SARS-CoV-2 transmission has an impact on education. In this study, we assessed the performance of rapid antigen detection tests (RADTs) versus polymerase chain reaction (PCR) for the diagnosis of SARS-CoV-2 infection in school settings, and RADT use for monitoring exposed contacts.Methods:In this real-world, prospective observational cohort study, high-school students and staff were recruited from 2 high schools in Montréal, Canada, and followed from Jan. 25 to June 10, 2021. Twenty-five percent of asymptomatic participants were tested weekly by RADT (nasal) and PCR (gargle). Class contacts of cases were tested. Symptomatic participants were tested by RADT (nasal) and PCR (nasal and gargle). The number of cases and outbreaks were compared with those of other high schools in the same area.Results:Overall, 2099 students and 286 school staff members consented to participate. The overall specificity of RADTs varied from 99.8% to 100%, with a lower sensitivity, varying from 28.6% in asymptomatic to 83.3% in symptomatic participants. Secondary cases were identified in 10 of 35 classes. Returning students to school after a 7-day quarantine, with a negative PCR result on days 6–7 after exposure, did not lead to subsequent outbreaks. Of cases for whom the source was known, 37 of 51 (72.5%) were secondary to household transmission, 13 (25.5%) to intraschool transmission, and 1 to community contacts between students in the same school.Interpretation:Rapid antigen detection tests did not perform well compared with PCR in asymptomatic individuals. Reinforcing policies for symptom screening when entering schools and testing symptomatic individuals with RADTs on the spot may avoid subsequent substantial exposures in class. Preprint: medRxiv — doi.org/10.1101/2021.10.13.21264960

Ubiquitin is a small molecule protein consisting of 76 amino acids,widely found in eukaryotic cells. The process by which ubiquitin binding to a specific protein is called ubiquitination. Deubiquitination is the reversed process of ubiquitination. Ubiquitination stimulates downstream signal,including complex assembly,protein conformation and activity changes,proteolysis,autophagy,guilt,chromatin remodeling,and DNA repair. More than 80% of eukaryotic protein degradation is mediated by the ubiquitination system,and ubiquitin-dependent proteolysis is an extremely complex process involving many biomolecular processes. By regulating protein homeostasis,ubiquitination can also regulate a variety of biological processes including cell cycle,cell proliferation,and apoptosis,which are closely related to tumorigenesis and progression. Many abnormalities of androgen receptor (AR) including AR gene amplification,mutation,shear mutation,and AR activity enhancement are closely related to prostate cancer progression. In particular,prostate cancer progression is regulated by the ubiquitination/deubiquitination processes. This article summarizes the recent research advances in the roles of ubiquitination/deubiquitination in AR abnormalities and prostate cancer.

Nowadays, physiological amino acids profiling is based primarily on ion exchange chromatography (IEC) coupled to a post-column derivatization with ninhydrin and UV detection at two wavelengths. Unfortunately, this technique suffers various drawbacks such as long analysis time, high sample volume and specific costs related to the maintenance of a dedicated equipment. These reasons have led us to consider a technology switch to a mass spectrometry method. We tested the kit aTRAQ amino acids analysis for physiological samples (AB Sciex), offering a selective quantification of more than 40 amino acids, and have implemented the acquisition of various original markers to the initial method. The accuracy profiles established for each amino acid show that the results are very reliable. The linearity is assured between 1 and 1.000μmol/L for most analytes. Result comparison with IEC method showed good agreement. Reference ranges are similar to those defined for the IEC method and patients with inborn errors of metabolism were readily identified. The aTRAQ method offers a valid alternative to IEC method with several advantages: reduced sample volume, decreased run time and increased specificity. However, the procedure requires a thorough review of all chromatographic peaks, process that considerably lengthens the overall time of the procedure. Finally, financial and practical considerations of both techniques have to be counterbalanced before initiating any methodological transition.

Flow-through electrochemical immunoassay for targeted bacteria detection

The applicability of PTV splitless and solvent vent injection to the gas chromatographic analysis of 26 pesticides representing different chemical classes was evaluated. All parameters related to optimum injector performance in PTV splitless and PTV solvent vent (split vent) injection modes were tested. For PTV splitless injection of small sample volumes (1 μL), the inlet temperature program (initial inlet temperature, heating rate, final temperature), splitless time, and starting oven temperature were optimized. Parameters identified as optimal were then applied in PTV solvent vent injection. In the case of the PTV solvent vent technique, all injections were performed with pesticides dissolved in a binary mixture of cyclohexane-ethyl acetate (1 : 1, v/v). This solvent is used as a mobile phase in the HPGPC clean-up step involved in our multi-residue method. For the PTV solvent vent technique the following parameters were tested: maximum single injection volume, inlet temperature, vent flow, vent time, and vent period were determined for a single injection of 10 μL of sample. Thermodegradation and/or adsorption of some pesticides occurred as long as glass wool packed liner was used. To achieve lower detection limits, multiple injection concept was employed: 30 μL of standard solution were injected in three subsequent steps. Under optimized conditions even higher sample volumes could be injected. Good responses were obtained also for the compounds possessing relatively higher volatility compared to other tested analytes.