Multiplex Quantitative Real-time PCR Research Articles

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 mM) or rotenone (0.2-50 mM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.

Novel Tick-Borne Anaplasmataceae Genotypes in Tropical Birds from the Brazilian Pantanal Wetland.

Despite numerous reports of Anaplasmataceae agents in mammals worldwide, few studies have investigated their occurrence in birds. The present study aimed to investigate the occurrence and molecular identity of Anaplasmataceae agents in birds from the Pantanal wetland, Brazil. Blood samples were collected from 93 different species. After DNA extraction, samples positive for the avian β-actin gene were subjected to both a multiplex quantitative real-time (q)PCR for Anaplasma and Ehrlichia targeting the groEL gene and to a conventional PCR for Anaplasmataceae agents targeting the 16S rRNA gene. As a result, 37 (7.4%) birds were positive for Anaplasma spp. and 4 (0.8%) for Ehrlichia spp. in the qPCR assay; additionally, 13 (2.6%) were positive for Anaplasmataceae agents in the PCR targeting the 16S rRNA gene. The Ehrlichia 16S rRNA sequences detected in Arundinicola leucocephala, Ramphocelus carbo, and Elaenia albiceps were positioned closely to Ehrlichia sp. Magellanica. Ehrlichia dsb sequences detected in Agelasticus cyanopus and Basileuterus flaveolus grouped with Ehrlichia minasensis. The 16S rRNA genotypes detected in Crax fasciolata, Pitangus sulphuratus and Furnarius leucopus grouped with Candidatus Allocryptoplasma. The 23S-5S genotypes detected in C. fasciolata, Basileuterus flaveolus, and Saltator coerulescens were related to Anaplasma phagocytophilum. In conclusion, novel genotypes of Anaplasma, Ehrlichia, and Candidatus Allocryptoplasma were detected in birds from the Pantanal wetland.

Development and preliminary application of a quadruplex real-time PCR assay for differential detection of porcine circovirus 1-4 in Chengdu, China.

Porcine circovirus (PCV) typically causes severe immune suppression in pigs, leading to mixed clinical infections with various pathogens that can cause significant harm to the pig industry. PCV has four subgenotypes, with PCV4 being an emerging virus that requires investigation due to its potential for epidemic outbreaks. Therefore, there is a need to develop a method that can detect all four PCV strains simultaneously. In this study, four pairs of specific primers and TaqMan probes were designed based on the conserved sequence of the PCV1-4 ORF2 gene to establish a PCV1-4 TaqMan multiplex real-time quantitative PCR method. The novel method was compared to six commercial testing kits for its efficacy. Then, a total of 595 mixed samples of spleen and lymph node collected from 12 districts in Chengdu from July to December 2021 were tested using the novel method. The results showed that the novel PCV1-4 TaqMan multiplex real-time quantitative PCR detection method has satisfied specificity, sensitivity, and repeatability. The positive rates of PCV1, PCV2, and PCV3 in Chengdu were 2.18%, 31.60%, and 15.29%, respectively, while no positive PCV4 was detected. The mixed infection rate of PCV2 and PCV3 was 5.21%. Our novel method may be as a potential method for PCV1-4 detection. Currently, PCV2 is the main epidemic PCV subtype in Chengdu, while the potential threat of PCV4 should also be considered.

Development and validation of multiplex real-time PCR for simultaneous detection of six bacterial pathogens causing lower respiratory tract infections and antimicrobial resistance genes

BackgroundKlebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Streptococcus pneumoniae and Staphylococcus aureus are major bacterial causes of lower respiratory tract infections (LRTIs) globally, leading to substantial morbidity and mortality. The rapid increase of antimicrobial resistance (AMR) in these pathogens poses significant challenges for their effective antibiotic therapy. In low-resourced settings, patients with LRTIs are prescribed antibiotics empirically while awaiting several days for culture results. Rapid pathogen and AMR gene detection could prompt optimal antibiotic use and improve outcomes.MethodsHere, we developed multiplex quantitative real-time PCR using EvaGreen dye and melting curve analysis to rapidly identify six major pathogens and fourteen AMR genes directly from respiratory samples. The reproducibility, linearity, limit of detection (LOD) of real-time PCR assays for pathogen detection were evaluated using DNA control mixes and spiked tracheal aspirate. The performance of RT-PCR assays was subsequently compared with the gold standard, conventional culture on 50 tracheal aspirate and sputum specimens of ICU patients.ResultsThe sensitivity of RT-PCR assays was 100% for K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and 63.6% for S. aureus and the specificity ranged from 87.5% to 97.6%. The kappa correlation values of all pathogens between the two methods varied from 0.63 to 0.95. The limit of detection of target bacteria was 1600 CFU/ml. The quantitative results from the PCR assays demonstrated 100% concordance with quantitative culture of tracheal aspirates. Compared to culture, PCR assays exhibited higher sensitivity in detecting mixed infections and S. pneumoniae. There was a high level of concordance between the detection of AMR gene and AMR phenotype in single infections.ConclusionsOur multiplex quantitative RT-PCR assays are fast and simple, but sensitive and specific in detecting six bacterial pathogens of LRTIs and their antimicrobial resistance genes and should be further evaluated for clinical utility.

Molecular characterization of human bocavirus in municipal wastewaters using amplicon target sequencing

Human bocavirus (HBoV) is an emerging health concern worldwide, associated with range of clinical manifestations, including gastroenteritis and respiratory infections. Therefore, it is crucial to comprehend and minimize their prevalence in different systems. In this study, we conducted regular sampling throughout the year in two different sizes and work processes of wastewater treatment plants (WWTPs) in Tianjin, China. Our objective was to investigate the occurrence, prevalence, and endurance of HBoV in wastewater, while also evaluating the efficacy of amplicon target sequencing in directly detecting HBoV in wastewater. At two WWTPs, HBoV2 (45.51 %—45.67 %) and HBoV3 (38.30 %—40.25 %) were the most common genotypes identified, and the mean concentration range of HBoV was 2.54—7.40 log10 equivalent copies/l as determined by multiplex real-time quantitative PCR assay. A positive rate of HBoV was found in 96.6 % (29/30) samples of A-WWTP, and 96.6 % (26/27) samples of B-WWTP. The phylogenetic analysis indicated that the nucleotide similarity between the HBoV DNA sequences to the reference HBoV sequences published globally ranged from 90.14 %—100 %. A significant variation in the read abundance of HBoV2 and HBoV3 in two wastewater treatment plants (p < 0.05) was detected, specifically in the Winter and Summer seasons. The findings revealed a strong correlation between the genotypes detected in wastewater and the clinical data across various regions in China. In addition, it is worth mentioning that HBoV4 was exclusively detected in wastewater and not found in the clinical samples from patients. This study highlights the high prevalence of human bocavirus in municipal wastewater. This finding illustrates that amplicon target sequencing can amplify a wide variety of viruses, enabling the identification of newly discovered viruses.

A multiplex real-time fluorescence-based quantitative PCR assay for calf diarrhea viruses.

Calf diarrhea is a significant condition that has a strong effect on the cattle industry, resulting in huge economic losses annually. Bovine torovirus (BToV), bovine enterovirus (BEV), bovine norovirus (BNoV), bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV) are key pathogens that have been implicated in calf diarrhea. Among these viruses, there remains limited research on BToV, BEV, and BNoV, with no available vaccines or drugs for their prevention and control. Although commercial vaccines exist for BCoV, BRV, and BVDV, the prevalence of these diseases remains high. To address this issue, we developed a multiplex real-time fluorescence quantitative PCR method for detecting BToV, BEV, BNoV, BCoV, BRV, and BVDV. This method can be used to effectively monitor the prevalence of these six viruses and serve as a reference for future prevention and control strategies. In this study, we specifically designed primers and probes for the BNoV Rdrp, BEV 5'UTR, BToV M, BCoV N, BRV NSP5, and BVDV 5'UTR genes. This method was determined to be efficient, stable, and sensitive. The lowest detectable levels of plasmids for BNoV, BEV, BToV, BRV, BCoV, and BVDV were 1.91 copies/μL, 96.0 copies/μL, 12.8 copies/μL, 16.4 copies/μL, 18.2 copies/μL, and 65.3 copies/μL, respectively. Moreover, the coefficients of variation for all six detection methods were < 3%; they also exhibited a strong linear relationship (R2 ≥ 0.98), and an amplification efficiency of 90%-110%. A total of 295 fecal and anal swabs were collected from calves with diarrhea in Guangdong, China. The positive rates for BToV, BEV, BNoV, BCoV, BR, and BVDV were determined to be 0.34% (1/295), 6.10% (18/295), 0.68% (2/295), 1.36% (4/295), 10.85% (32/295), and 2.03% (6/295), respectively. Notably, BEV and BRV exhibited the highest prevalence. Additionally, this study identified the occurrence of BToV and BNoV in Guangdong for the first time. In summary, this study successfully established an effective method for detecting several important bovine viruses; ultimately, this holds strong implications for the future development of the cattle industry.

Development of a quadruple qRT-PCR assay for simultaneous identification of hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

Globally, the increasing number of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp.

Comparison of Multiplex Quantitative Real-Time PCR and Culture Methods for the Diagnosis of Bacterial Meningitis in Patients with Suspected Meningitis

Comparison of Multiplex Quantitative Real-Time PCR and Culture Methods for the Diagnosis of Bacterial Meningitis in Patients with Suspected Meningitis

Levels of TREC and KREC molecules significance determining in peripheral blood for predicting the outcome of COVID-19 disease in the acute period

The disease caused by the highly contagious SARS-CoV-2 virus – “novel coronavirus infection (COVID-19)” – had killed more than 6.5 million people at the end of December 2022. The severity of the manifestation of the infectious process varies from asymptomatic forms to rapid progression to life-threatening conditions requiring emergency measures. One of the factors, the severity of which affects the outcome of the disease, is lymphopenia, the cause of which may be a violation of lymphopoiesis. The identification of laboratory markers of a high risk of mortality in patients with COVID-19 plays an important role in improving patient care algorithms and increasing their survival. Levels of TREC and KREC molecules in peripheral blood, respectively, can serve as molecular markers of the severity of T and B lymphopenias. The aim of our work was a comparative analysis of the levels of TREC and KREC molecules in the peripheral blood of surviving and deceased patients with COVID-19. The material was whole blood samples obtained from 1745 people, including: 1028 patients diagnosed with novel coronavirus infection (COVID-19) (ICD-10 code – U07.1), of which 937 patients recovered and 91 died; 717 apparently healthy individuals (control group). The levels of TREC and KREC molecules were assessed by quantitative multiplex Real-time PCR using the TREC/KREC-AMP PS reagent kit (Federal Scientific Research Institute Pasteur, St. Petersburg). Statistically significant differences in the levels of KREC and TREC molecules between the control group and patients, both surviving and deceased, were established. A significant decrease in median concentrations of KREC molecules was shown in patients with a lethal outcome compared with survivors (p = 0.0019, 95% CI). Among the deceased patients, in 63.7% of cases, the levels of TREC or KREC molecules were reduced relative to the corresponding age norms. Of these, in 20.9% of cases, both analytes were reduced in patients. When assessing the diagnostic significance of the levels of the analytes under study for predicting the outcome of the disease, the area under the AUC curve for KREC was 0.63±0.029, which indicates the average strength of the prognostic model of the patient's death depending on the level of KREC in the blood. The constructed model is statistically significant (p = 0.002). Monitoring laboratory parameters of patients with COVID-19, including those who died, allows you to determine the prognostic factors that are most significant for assessing the outcome of the disease. Based on the assessment of the KREC level, a predictive model with high specificity reflects the risk of death in patients with COVID-19. Thus, the quantitative determination of the level of KREC molecules in the peripheral blood can be attributed to the methods of preventive personalized diagnostics aimed at improving the survival of patients.

A novel multiplex RT-qPCR assay for simultaneous detection of bovine norovirus, torovirus, and kobuvirus in fecal samples from diarrheic calves.

Calf diarrhea results in significant economic loss and is caused by a variety of pathogens, including enteric viruses. Many of these viruses, including bovine norovirus (BNoV), bovine torovirus (BToV), and bovine kobuvirus (BKoV), are recognized as the causative agents of diarrhea; however, they remain understudied as major pathogens. We developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) assay for rapid and simple detection of BNoV, BToV, and BKoV. Our method had high sensitivity and specificity, with detection limits of 1 × 102 copies/μL for BNoV, BToV, and BKoV, which is a lower detection limit than conventional RT-PCR for BNoV and BKoV and identical for BToV. We tested fecal samples from 167 diarrheic calves with our multiplex RT-qPCR method. Viral detection was superior to conventional RT-PCR methods in all samples. The diagnostic sensitivity of the multiplex RT-qPCR method (100%) is higher than that of the conventional RT-PCR methods (87%). Our assay can detect BNoV, BToV, and BKoV in calf feces rapidly and with high sensitivity and specificity.

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos

In addition to fulfilling many breeders’ curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated withbacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing.

Development of TaqMan Real-Time PCR Protocols for Simultaneous Detection and Quantification of the Bacterial Pathogen Ralstonia solanacearum and Their Specific Lytic Bacteriophages.

Ralstonia solanacearum is the causal agent of bacterial wilt, one of the most destructive diseases of solanaceous plants, affecting staple crops worldwide. The bacterium survives in water, soil, and other reservoirs, and is difficult to control. In this sense, the use of three specific lytic R. solanacearum bacteriophages was recently patented for bacterial wilt biocontrol in environmental water and in plants. To optimize their applications, the phages and the bacterium need to be accurately monitored and quantified, which is laborious and time-consuming with biological methods. In this work, primers and TaqMan probes were designed, and duplex and multiplex real-time quantitative PCR (qPCR) protocols were developed and optimized for the simultaneous quantification of R. solanacearum and their phages. The quantification range was established from 108 to 10 PFU/mL for the phages and from 108 to 102 CFU/mL for R. solanacearum. Additionally, the multiplex qPCR protocol was validated for the detection and quantification of the phages with a limit ranging from 102 targets/mL in water and plant extracts to 103 targets/g in soil, and the target bacterium with a limit ranging from 103 targets/mL in water and plant extracts to 104 targets/g in soil, using direct methods of sample preparation.

Monitoring COVID-19 spread in selected Prague's schools based on the presence of SARS-CoV-2 RNA in wastewater

The COVID-19 pandemic has demanded a broad range of techniques to better monitor its extent. Owing to its consistency, non-invasiveness, and cost effectiveness, wastewater-based epidemiology has emerged as a relevant approach to monitor the pandemic's course. In this work, we analyzed the extent of the COVID-19 pandemic in five primary schools in Prague, the Czech Republic, and how different preventive measures impact the presence of SARS-CoV-2 RNA copy numbers in wastewaters. Copy numbers were measured by reverse transcription–multiplex quantitative real-time PCR. These copy numbers were compared to the number of infected individuals in each school identified through regular clinical tests. Each school had a different monitoring regime and subsequent application of preventive measures to thwart the spread of COVID-19. The schools that constantly identified and swiftly quarantined infected individuals exhibited persistently low amounts of SARS-CoV-2 RNA copies in their wastewaters. In one school, a consistent monitoring of infected individuals, coupled with a delayed action to quarantine, allowed for the estimation of a linear model to predict the number of infected individuals based on the presence of SARS-CoV-2 RNA in the wastewater. The results show the importance of case detection and quarantining to stop the spread of the pandemic and its impact on the presence of SARS-CoV-2 RNA in wastewaters. This work also shows that wastewater-based epidemiological models can be reliably used even in small water catchments, but difficulties arise to fit models due to the nonconstant input of viral particles into the wastewater systems.

Epidemiological characteristics of human adenovirus 2, 3 and 7 genetypes in hospitalized children with respiratory infection in a hospital of pediatric in Hebei Province from 2018 to 2020

Objective: To investigate the epidemiological characteristics of human adenovirus (HADV) 2, 3 and 7 in hospitalized children with respiratory infection. Methods: A total of 25 686 children with respiratory infection hospitalized at Children's Hospital of Hebei Province from January 2018 to December 2020 were retrospectively included.Deep sputum or nasopharyngeal aspirates of those children were collected. Then thirteen common respiratory pathogens were detected by multiplex PCR. 510 HADV positive specimens were randomly selected via random number and classified for type 2, 3 and 7 using a multiplex real-time quantitative PCR. SPSS 21.0 software was used to perform all of the statistical analyses. Enumeration data were expressed by frequency and percentage. χ2 test was used for comparison between groups. Results: The HADV-positive rate was 7.99% (2 052/25 686). Children at age 3-<6 years had the highest HADV-positive rate (11.44%). The HADV-positive rate in 2019 was highest (10.64%). Among the 510 HADV-positive specimens, the proportion of type 3 was the highest (31.16%), followed by type 7 (21.37%) and type 2 (11.18%). The rate of type 2 in 2019 was significantly lower than that in 2018 and 2020 (χ2=8.954 and 16.354; P=0.003 and <0.01), while the rate of type 3 was significantly higher than that in 2018 and 2020 (χ2=5.248 and 4.811; P=0.022 and 0.028). The rate of type 2, type 3 and type 7 were lowest in winter, spring and autumn, respectively. The rate of type 2 increased significantly in autumn and the rate of type 3 and type 7 increased significantly in winter.The co-detection rate of HADV with other respiratory pathogens was 43.33%(221/510). Among, the co-detection rate of type 3 was highest (47.32%), and the co-detection rate of type 2, 3 and 7 was significantly higher than the alone detection rate (χ2=20.438, P<0.01; χ2=42.105, P<0.01; χ2=27.573, P<0.01).The proportion of severe pneumonia in children with type 7 positive (15.89%) was higher than that in children with non-type 7 positive (8.23%) (χ2=5.260, P=0.022). Conclusion: HADV is one of the important pathogens of children with respiratory infection in Children's Hospital of Hebei Province. The susceptible population of HADV is preschool children aged 3 to 6 years. HADV often co-detects with other respiratory pathogens. Type 3 and 7 is likely to be the dominant genotypes in this region, and type 7 may be one of the risk factors of severe pneumonia in children.

Determination of the mycobiome in the lower respiratory tract of horses with equine asthma

Fungal particles are important allergenic components involved in the development of equine asthma. The aim of this study was to evaluate the mycobiome composition of the lower respiratory tract in asthmatic horses using fungal culture, quantitative multiplex real-time PCR analysis (FungiMultiPlex) and Next-Generation Sequencing approach. Bronchoalveolar lavage fluid (BALF) samples obtained from 45 client-owned horses diagnosed with equine asthma were analysed by fungal culture (19 samples), FungiMultiPlex (34 samples), and Next-Generation Sequencing (14 samples). The fungal culture was positive in 11/19 (58%) cases, and FungiMultiPlex was positive in 19/34 (56%) cases. No fungal PCR product was detected by Next-Generation Sequencing analysis. Fungal culture and FungiMultiPlex methods were performed simultaneously on eight horses only. Association of results of these methods was calculated using Phi coefficient (φ= 0.333), and concordance between the methods was not confirmed (P = 0.420). The results of this study suggest that the fungal culture and quantitative multiplex real-time PCR might be considered diagnostically useful to assess the presence of fungi in BALF in a semiquantitative and quantitative manner, respectively. The Next-Generation Sequencing method seems to be less diagnostically suitable due to technical obstacles pertinent to both the low concentration of microbial agents in the rather diluted BALF samples, and, also, due to the relatively high environmental background contamination of the collected material. Based on our data, we advocate the use of the combination of quantitative multiplex real-time PCR and fungal culture in a routine clinical diagnostic setting.

Telomere length in Thai Buddhist monks and Thai males aged 40 years and above

Introduction: The daily lifestyles of Thai Buddhist monks and Thai males differ due to Buddhist practices, which potentially affect telomere length. Telomeres are DNA compounds located at the ends of chromosomes that shorten with each cell division. This study investigated the difference in telomere length between Thai Buddhist monks and Thai males aged ≥40 years. Method: This was a cross-sectional study involving 100 Thai Buddhist monks aged ≥40 years who had been ordained for more than five years and 100 Thai males aged ≥40 years. General information and health information were assessed by questionnaire. Nutritional status was determined by body composition and blood chemistry parameters. Telomere length was measured by Monochrome Multiplex RealTime Quantitative PCR and expressed as T/S ratio. Result: Mean telomere length of Thai Buddhist monks was longer than that of Thai males (1.08±0.18 vs. 1.02±0.17; p&lt;0.050). In both groups, the mean telomere length in subjects aged ≥60 years was shorter than that in subjects aged 40-59 years (p&lt;0.010). Alcohol consumption, which affected Thai males (p&lt;0.050), but meditation tend to slow down the shortening of telomeres (r=0.167; p&lt;0.050) in both groups. Conclusion: Age was the parameter that affected telomere length the most. Furthermore, various factors in the Buddhist monk group, such as a peaceful lifestyle, meditation, non-alcohol consumption, and fewer underlying diseases, could explain for the longer telomere lengths in this group.

Analysis of Relative Average Length of Telomeres in Leukocytes of Women with COVID-19.

Coronavirus disease (COVID-19) is an acute infectious disease of the respiratory tract caused by a new SARS-CoV-2 coronavirus. A global vaccination program against SARS-CoV-2 continues, and the incidence of COVID-19 worldwide is significantly decreasing. However, among millions of those who survived COVID-19, numerous groups will need assistance due to increased clinical consequences after COVID-19. Currently, there is a need to search for molecular biomarkers for monitoring the onset and progression of post-COVID syndrome. For this purpose, the relative average length of chromosome regions was studied in the groups of women of reproductive age: in the group of patients (n = 64) recovered from COVID-19 and in the control group (n = 42) of women of the same age. The analysis was carried out using a method of multiplex monochrome quantitative real-time PCR on DNA samples isolated from the peripheral blood leukocytes. According to the results of the study, it was established that the relative average length of chromosomes in the peripheral blood leukocytes was statistically significantly lower in the group of patients with COVID-19 than in the control group (p < 0.05). The results obtained allow one to state that the observed shortening of the relative average length of telomeres in the group of patients that recovered from COVID-19 can indicate that SARS-CoV-2 infection can directly cause the erosion of telomeres in the blood cells, particularly, in leukocytes. Thus, the determination of the relative average length of telomeres can be an informative prognostic marker for estimating the risk of the severity of COVID-19 disease and the development of post-COVID syndrome.

Real-life evaluation of a rapid antigen test (DPP SARS-CoV-2 Antigen) for COVID-19 diagnosis of primary healthcare patients, in the context of the Omicron-dominant wave in Brazil

ObjectivesWe aimed to investigate the real-life performance of the rapid antigen test in the context of a primary healthcare setting, including symptomatic and asymptomatic individuals that sought diagnosis during an Omicron infection wave. MethodsWe prospectively accessed the performance of the DPP SARS-CoV-2 Antigen test in the context of an Omicron-dominant real-life setting. We evaluated 347 unselected individuals (all-comers) from a public testing centre in Brazil, performing the rapid antigen test diagnosis at point-of-care with fresh samples. The combinatory result from two distinct real-time quantitative PCR (RT-qPCR) methods was employed as a reference and 13 samples with discordant PCR results were excluded. ResultsThe assessment of the rapid test in 67 PCR-positive and 265 negative samples revealed an overall sensitivity of 80.5% (CI 95% = 69.1%–89.2%), specificity of 99.2% (CI 95% = 97.3%–99.1%) and positive/negative predictive values higher than 95%. However, we observed that the sensitivity was dependent on the viral load (sensitivity in Ct < 31 = 93.7%, CI = 82.8%–98.7%; Ct > 31 = 47.4%, CI = 24.4%–71.1%). The positive samples evaluated in the study were Omicron (BA.1/BA.1.1) by whole-genome sequencing (n = 40) and multiplex RT-qPCR (n = 17). ConclusionsAltogether, the data obtained from a real-life prospective cohort supports that the rapid antigen test sensitivity for Omicron remains high and underscores the reliability of the test for COVID-19 diagnosis in settings with high disease prevalence and limited PCR testing capability.

Clinical features and outcomes of fusion gene defined adult Ph-negative B-cell precursor acute lymphoblastic leukemia patients: A single institutional report.

More clinical studies are needed to clarify the risk stratification by the integration of all fusion genes in adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A total of 320 consecutive adult Ph-negative BCP-ALL patients who had been tested classical fusions (KMT2A rearrangement and TCF3-PBX1) at diagnosis were further retrospectively screened novel fusion genes (Ph-like, ZNF384 and MEF2D fusions) by multiplex real-time quantitative PCR (RQ-PCR). Classical fusions were identified in 12.5% of patients, while 4.4%, 17.2% and 3.8% of patients were identified Ph-like, ZNF384 and MEF2D fusions, respectively. 1-course CR rate, relapse-free survival (RFS) and overall survival (OS) rates tended to show or showed statistically significant differences among fusion-defined subgroups (P = 0.084, 0.001 and 0.0093, respectively). Based on individual outcomes, patients with KMT2A rearrangement, TCF3-PBX1, Ph-like, and MEF2D fusions were classified into fusion-defined high-risk group (n = 66, 20.6%). High-risk group had significantly lower 3-year RFS and 3-year OS rates than standard-risk group (P 0.001 and = 0.0022), and was an independent adverse prognostic factor for RFS in the entire cohort (P 0.001). In conclusion, the spectrum of fusion genes in the current Chinese cohort was distinct from that in reports from western countries. Detection of fusion genes improved risk stratification in adult Ph-negative BCP-ALL patients.