PCR-based Method Research Articles

Association between theSp1-binding-site polymorphism in the collagen type I alpha 1 (COLIA1) gene and bone phenotypes: the Dubbo Osteoporosis Epidemiology Study.

Polymorphisms within the collagen 1 alpha 1 gene (COLIA1) have been shown to be associated with bone mineral density (BMD). This study aimed to test the hypothesis that COLIA1 polymorphisms are associated with bone loss and fragility fractures. The study involved 809 postmenopausal women aged 60years and above in the Dubbo Osteoporosis Epidemiology Study who had COLIA1 genotypes and at least two BMD measurements over a 30-year period. BMD at the lumbar spine (LSBMD) and femoral neck (FNBMD) was measured biennially by dual-energy X-ray absorptiometry (GE-Lunar Prodigy). Fragility fracture has been ascertained by X-ray reports between 1990 and 2020. The G-> T polymorphism at the Sp1-binding site in the COLIA1 gene (rs1800012) was determined by the PCR-based method, and coded as GG, GT, and TT. Women homozygous for the minor allele (TT) tended to have greater bone loss (-0.72%/year) than those with GT (-0.58%/year) or GG (-0.56%/year) though the difference did not achieve statistical significance (P = 0.84). Women of the TT genotype were associated with a two-fold greater risk of any fracture (adjusted hazard ratio: 2.21; 95%CI 1.42-3.46) and almost fourfold greater risk of hip fracture (3.78; 1.83-7.82) than those with either GG or GT genotype. Polymorphisms at the Sp1 site in the COLIA1 gene are associated with fracture risk, independent of bone loss.

Insights into trypanosomiasis transmission: Age, infection rates, and bloodmeal analysis of Glossina fuscipes fuscipes in N.W. Uganda.

Tsetse flies (Glossina) transmit species of Trypanosoma which cause human African trypanosomiasis (HAT) and animal African trypanosomiasis (AAT). Understanding the epidemiology of this disease and controlling the vector rationally requires analysis of the abundance, age structure, infection rates and feeding patterns of tsetse populations. We analysed a population of G. fuscipes fuscipes in the Koboko district of Uganda. Seasonal variation in the abundance of tsetse was assessed from the numbers of tsetse caught in pyramidal traps. The age structure of the population was assessed by dissecting female tsetse to estimate their ovarian categories. Classical and PCR-based methods were utilised to determine the presence of the three major pathogenic species of salivarian trypanosomes: T. vivax, T. congolense and T. brucei in a subset (n = 2369) of flies. Further, bloodmeal analysis was carried using PCR to amplify and sequence a portion of the vertebrate cytb gene. The abundance and age structure of tsetse populations were relatively stable and a slight seasonal four-fold variation in abundance appeared to be correlated with rainfall. Analyses of age structure suggests a low natural daily mortality of 1.75% (1.62-1.88). Infection rates estimated were significantly greater (1.9-9.3 times) using the PCR-based method compared to the classical dissection-based method. Positive rates for T. brucei sl, T. congolense and T. vivax were 1.6% (1.32-2.24), 2.4% (1.83-3.11and 2.0% (1.46-2.63), respectively by PCR. The majority of bloodmeals were identified as cattle (39%, 30.5-47.8) and human (37% of meals, 28.4-45.6). The seasonally stable abundance, low mortality rate and high proportion of bloodmeals from humans may explain, in part, why this district has historically been a focus of sleeping sickness. Additionally, the high rates of cattle feeding indicate insecticide treated cattle may prove to be a useful vector control strategy in the area.

Development of a triplex endpoint PCR assay for the detection of SARS-CoV-2: insights on cost-efficiency and method design.

Lower respiratory tract infections, including COVID-19, have a substantial global impact, making the development of diagnostic tests crucial. The study aimed to develop a new, accurate, fast, and cost-effective PCR-based detection method for SARS-CoV-2, applicable in limited settings and capable of detecting all current variants and potential future pathogens. The study was conducted between 2020 and 2022 at the molecular biology department of Mures County Clinical Hospital, Romania. Initially, pharyngeal and nasal secretions were collected and processed using the real-time qRT-PCR method for routine COVID-19 diagnosis. Ninety-two samples were randomly selected to develop the assay, including samples from different infection periods and negative controls. Complementary DNA (cDNA) was prepared from the selected samples, and the presence and integrity of the extracted RNA were evaluated by amplifying the GAPDH housekeeping gene. Primers for three specific viral genes (N, ORF1ab, and S) were designed, and their efficiency was evaluated using endpoint PCR and sequencing. Finally, the method was optimized and implemented as a one-step triplex PCR assay for routine diagnostic use. The molecular laboratory at the Mures County Clinical Hospital (MCCH) analyzed a total of 41,818 samples between June 2020 and December 2022. Among these samples, 26.15% tested positive for SARS-CoV-2, while 70.9% were negative and 2.95% were inconclusive or invalid. Three peaks of positive tests were observed in November 2020, April 2021, and February 2022. The study selected 92 preserved RNA samples for triplex-PCR assay development, validating the primers' specificity and confirming the quality of the nucleic acids. The comparative analysis showed the efficiency and accuracy of the endpoint reverse-transcription triplex-PCR method (RT-PCR), indicating its potential as a cost-effective alternative to real-time reverse-transcription PCR (qRT-PCR) in low-income countries with limited infrastructure for COVID-19 testing. This method has the potential to facilitate large-scale diagnosis of SARS-CoV-2 infections, allowing for rapid and appropriate therapeutic management and ongoing monitoring of patients. Additionally, the method can be easily adapted for the detection of other pathogens.

Strategy to develop and validate digital droplet PCR methods for global antimicrobial resistance wastewater surveillance.

According to World Health Organization (WHO), antimicrobial resistance (AMR) is currently one of the world's top 10 health threats, causing infections to become difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death. Accurate surveillance is a key component in the fight against AMR. Wastewater is progressively becoming a new player in AMR surveillance, with the promise of a cost-effective real-time tracking of global AMR profiles in specific regions. One of the most useful analytical methods for wastewater surveillance is currently based on real-time PCR (qPCR) and digital droplet PCR (ddPCR) technologies. As stated in the EU Wastewater Treatment Directive proposal, methodological standardization, including a workflow for method development and validation, will play a crucial role in global monitoring of AMR in wastewater. However, according to our knowledge, there are currently no qPCR and ddPCR methods for AMR surveillance available that have been validated according to international standard performance criteria. Therefore, this study proposes a workflow for the development and validation of PCR-based methods for a harmonized and global AMR surveillance, including the construction of specific sequence databases and microbial collections for an efficient method development and method specificity evaluation. Following this strategy, we have developed and validated four duplex ddPCR methods responding to international standard performance criteria, focusing on seven AMR genes (ARG's), including extended spectrum beta-lactam (blaCTX-M), carbapenem (blaKPC-2/3), tetracycline (tet(M)), erythromycin (erm(B)), vancomycin (vanA), sulfonamide (sul2), and aminoglycoside (aac(3)-IV), as well as one indicator of antibiotic (multi-) resistance and horizontal gene transfer, named the class I integron (intl1). The performance of these ddPCR methods was successfully assessed for their specificity, as no false-positive and false-negative results were observed. These ddPCR methods were also considered to be highly sensitive as showing a limit of detection below 25 copies of the targets. In addition, their applicability was confirmed using 14 wastewater samples collected from two Belgian water resource recovery facilities. The proposed study represents therefore a step forward to reinforce method harmonization in the context of the global AMR surveillance in wastewater. PRACTITIONER POINTS: In the context of wastewater surveillance, no PCR-based methods for global AMR monitoring are currently validated according to international standards. Consequently, we propose a workflow to develop and validate PCR-based methods for a harmonized and global AMR surveillance. This workflow resulted here in four duplex ddPCR methods targeting seven ARGs and one general indicator for mobilizable resistance genes. The applicability of these validated ddPCR methods was confirmed on 14 wastewater samples from two Belgian water resource recovery facilities.

Lack of Candida africana in Ugandan pregnant women: results from a pilot study using MALDI-ToF

BackgroundCandida africana is an emergent variant that has been listed as a new species or variety within the Candida albicans complex since 2001. It has a worldwide intra-albicans complex pooled prevalence of 1.67% and varies between 0 and 8% depending on geographical region. We present the results of a pilot study on its prevalence in Uganda.MethodologyWe conducted a cross-sectional study between March and June 2023. We recruited 4 pregnant women from Mulago Specialized Women and Neonatal Hospital, 102 from Kawempe National Referral Hospital, and 48 from Sebbi Hospital. Vaginal swabs were tested using microscopy, culture and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).ResultsThe prevalence of C. africana was zero. Out of the 103 isolates, the majority (81.553%) were identified as Candida albicans, followed by Nakeseomyces glabrata (13.592%) and Pichia kudriavzevii (1.942%). Cyberlindnera jadinii, Candida tropicalis, and Candida parapsilosis each accounted for 0.971% of the isolates.ConclusionThe prevalence of C. africana in Uganda is zero. However, large-scale cross-sectional studies, including studies involving the collection of vaginal samples from both urban and rural settings in Uganda and the use of both MALDI-TOF- and PCR-based laboratory methods, are needed to fully describe the public health burden of C. africana infections.

A Diagnostic Guide for Fusarium Fruit Rot of Pumpkin and Winter Squash

Fusarium fruit rot caused by Fusarium solani f. sp. cucurbitae is an important postharvest disease impacting the global production and storage of pumpkins and winter squash ( Cucurbita spp.). The pathogen infects fruit through wounds, causing water-soaked lesions that may become sunken, coalesce, and be covered by fine mycelial growth. F. solani is soilborne and can survive long term in soil and plant debris. Storage rots impact several Cucurbita spp. grown for long-term storage. F. solani f. sp. cucurbitae has two races and is the most commonly found Fusarium species infecting cucurbits. Identification includes morphologic and PCR-based methods. This guide outlines taxonomy, symptoms, isolation, and epidemiology and provides methods to identify the pathogen.

Characterization of middle ear microbiome in otitis media with effusion in Hungarian children: Alloiococcus otitidis may potentially hamper the microbial diversity

IntroductionAlthough otitis media with effusion (OME) is a common cause of hearing impairment in children, little is known about its microbiological background. We aimed to analyse the microbiome of the middle ear fluid (MEF) in OME to identify microbiological, demographic and environmental factors that may potentially influence the middle ear microbiome. In addition, we aimed to compare the results of conventional culture techniques and PCR-based methods. Methods39 samples from children with OME were investigated using conventional culture and 16S rRNA sequencing. Bioinformatic analyses were carried out to assess the microbial communities and their association with clinical data. ResultsBy conventional culture technique bacteria could be cultured in 33 % of the MEF samples; the most frequent bacterium was Haemophilus influenzae, followed by Staphylococcus species, Cutibacterium acne, and Streptococcus species. The 16S rRNA analysis showed that Alloiococcus was the most abundant bacterium, followed by Haemophilus, Streptococcus. and Sphingobium.A “High Alloiococcus group” with significantly lower and a “Low Alloiococcus group” with significantly higher alpha and beta diversity could be defined. Children born by Caesarean sections had lower beta and Shannon diversity than patients born by natural birth. Breastfeeding for at least six months showed a negative correlation with alpha diversity. Age, previous antibiotic treatment, parental smoking, duration of symptoms, existence of siblings, did not alter the significantly bacterial composition of MEF samples and there were no significant differences in regard to alpha or beta diversity. ConclusionThe conventional culture technique and 16S rRNA results were not congruent. Remarkably, Alloiococcus could not be cultured at all, even by 16S rRNA analysis the presence of Alloiococcus seems to be important. Based on our results, A. otitidis possibly impacts the diversity of middle ear microbiome in children with OME. However, further studies are required to determine the role of A. otidis in middle ear pathologies.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR and ddPCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL), B-cell chronic lymphocytic leukemia (CLL), and multiple myeloma and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. Real-time quantitative PCR (RQ-PCR) targeting circulating lymphoma cells is still the gold standard for MRD detection in indolent B-NHL and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, CLL, and FL with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy (IGH) and light chain genes. Complete and incomplete IGH and additionally IG kappa light chain rearrangements can be used as targets for MRD. Next-generation sequencing (NGS) of IG-rearrangements (IG-NGS) as new sequencing-based technology can overcome the limitation of PCR-based approaches and has a potential for higher sensitivity. Chromosomal translocations like the t(14;18)(q32;q21) translocation associated with IGH::BCL2 fusion in FL and t(11;14)(q13;q32) translocation in MCL leading to the IGH::CCND1 fusion can be used as MRD target in selected lymphoma subtypes. In patients with CLL, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 is achieved.MRD diagnostics targeting the IG loci is complex and requires extensive knowledge and experience because the junctional regions of each clonal rearranged gene have to be identified before the patient-specific PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation within the rearranged IGH gene occurring during B-cell development of germinal center and post-germinal center B-cell lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. PCR-based methods using allele-specific primers can reach a high sensitivity of up to 10-5. This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IG gene rearrangements and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results. This is available by the EuroMRD network ( https://euromrd.org ), a subgroup of ESHLO ( https://eslho.org ).

Repurposing MALDI-TOF MS for effective antibiotic resistance screening in Staphylococcus epidermidis using machine learning

The emergence of Staphylococcus epidermidis as a significant nosocomial pathogen necessitates advancements in more efficient antimicrobial resistance profiling. However, existing culture-based and PCR-based antimicrobial susceptibility testing methods are far too slow or costly. This study combines machine learning with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to develop predictive models for various antibiotics using a comprehensive dataset containing thousands of S. epidermidis isolates. Optimized machine learning models utilized feature selection and achieved high AUROC scores ranging from 0.80 to 0.95 while maintaining AUPRC scores up to 0.97. Shapley Additive exPlanations were employed to analyze relevant features and assess the significance of corresponding protein biomarkers while also verifying that predictive power was derived from the detection of proteins rather than noise. Antimicrobial resistance models were validated externally to evaluate model performance outside the original data collection site. The approaches and findings in this study demonstrate a significant advancement in rapid, cost-effective antimicrobial resistance profiling, offering a promising solution for improving treatments for nosocomial infections and being potentially applicable to other microbial pathogens in the future.

The Impact of Heavy Metal Ions on Arginase Produced from Pseudomonas aeruginosa Isolated from Sewage

Background: Researchers remain captivated by the impact of heavy metal ions, commonly found in sewage, on arginase synthesis. Objective: The study examines the presence and characteristics of Pseudomonas aeruginosa in 24 sewage samples, identifying 18 isolates (75%). It assesses DNA concentration and purity, utilizes PCR-based methods for subtype analysis and investigates the inhibitory effects of heavy metals on purified enzymes. Methods: Between November 2022 and April 2023, 52 samples were collected from various sewage sites in Baghdad, Iraq. These samples were cultured in brain heart infusion broth and on nutrient agar plates. The isolates were then subjected to laboratory procedures, including biochemical tests, analysis with the Vitek2 system and PCR to identify Pseudomonas aeruginosa. Biochemical testing specifically focused on Pseudomonas aeruginosa. DNA extraction was performed on P.aeruginosa isolates from sewage, which had already been identified through morphological and biochemical tests as P.aeruginosa. This was accomplished by screening for the presence of the 16SrRNA gene using PCR. Result: Metal ion effects on L-arginase were tested at various concentrations (100mM, 50mM, 25mM, 12.5 mM). Metal ions were obtained via the cutting method and added 15 minutes before the substrate. Enzyme activity was measured and the activity ratio (enzyme activity with and without metal ions) calculated. Activity was assessed in the absence of 100% metal ions.Arginase isolated and purified from Pseudomonas aeruginosa from sewage, heavy metals (mercury, cadmium, cobalt) has effect on arginase, mercuric chloride and cadmium chloride inhibit and decrease enzyme activity, while Cobalt chloride increases enzyme activity.

Antimicrobial Stewardship: A Game Changer in the Fight against Carbapenem-Resistant Enterobacteriaceae (CRE) among Children with Cancer

Abstract Background Carbapenem-resistant Enterobacteriaceae (CRE) is an emerging threat among pediatric cancer patients, with a high mortality rate. A previous study in our center reported that 254 patients with CRE BSI were diagnosed from (2013-2018) with mortality from CRE BSI being 30%. Previous colonization was reported in 35% of those patients. Time to start adequate antibiotics is an independent prognostic factor for patients with CRE BSI, (48%) of patients started active treatment after 48h from culture and associated with higher mortality when compared to patients who started treatment earlier. Our recommendations include antimicrobial stewardship for early detection through routine screening and timely adequate therapy that may impact the outcome for such high-risk patient groups. Methods This prospective study included all pediatric cancer patients with (CRE) bloodstream infections (BSI) at a children’s cancer hospital in Egypt (2020–2022) with adopting antimicrobial stewardship program goals by (1) applying routine screening by rectal swab for high-risk patients and starting active treatment for patients colonized by CRE. (2) Introduction of PCR-based method (Cepheid XpertCarba-R assay) is developed for detecting carbapenemase genes with the availability of results within one hour which can allow for rapid time to start active treatment in less than 48h. Results One hundred and fifty pediatric cancer patients with CRE BSI were identified; 83% had hematological malignancies, and 17% had solid tumors. Acute myeloid leukemia was the most common hematological malignancy (47%). The main clinical features for acquiring CRE-BSI were prolonged neutropenia > 7 days (62%), previous Carbapenem exposure (82%), Quinilone prophylaxis (44%), central venous catheter use (40%), previous colonization with a resistant pathogen (35%) and ICU admission within 90 days (18%). E. coli was the most common isolated pathogen (65%), followed by Klebsiella pneumoniae (31%). Documented site of infection was reported with pneumonia in (32%), typhlitis in 15% skin soft tissue infection (57%), CNS infection (18%), and urinary tract infection in (10%). Time to start active antibiotics in less than 48h was reported in 89% with clinical cure reported in 71% and microbiological clearance was reported in in less than 7 days in 70% of patients. The need for ICU admission as sepsis related to CRE was reported in 29/150 (19%). The Gentopyic profile for CRE in our study reported that class B (69%) with NDM reported in 98/150 (65%) and VIM 6/150 (4%) was the most common carbanamases followed by Oxa-48 in 89/150(59%) of patients while KPC enzyme reported only in 6/150(4%). Day 30 mortality was reported in 23/150(15%). Upon multivariate analysis, patients with Klebsiella pneumoniae BSI, NDM genotype carbapenemases, CRE with aminoglycosides resistance or associated with pneumonia, and CRE patients who developed septic shock with the need for ICU admission were predictors of poor outcome. Conclusion CRE-BSI is a major threat among pediatric cancer patients in limited-resource countries and is considered a barrier against better outcomes. Antimicrobial stewardship through routine screening for colonization detection and Rapid diagnosis by using gene expert with rapid time to start adequate active antibiotics treatment is associated with improvement of outcome and decreasing mortality. Table 1: Comparison between retrospective study and prospective study (after adopting antimicrobial stewardship goals)

Multisite community-scale monitoring of respiratory and enteric viruses in the effluent of a nursing home and in the inlet of the local wastewater treatment plant.

The aim of this study was to evaluate whether community-level monitoring of respiratory and enteric viruses in wastewater can provide a comprehensive picture of local virus circulation. Wastewater samples were collected weekly at the wastewater treatment plant (WWTP) inlet and at the outlet of a nearby nursing home (NH) in Burgundy, France, during the winter period of 2022/2023. We searched for the pepper mild mottle virus as an indicator of fecal content as well as for the main respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza, and respiratory syncytial virus] and enteric viruses (rotavirus, sapovirus, norovirus, astrovirus, and adenovirus). Samples were analyzed using real-time reverse transcription PCR-based methods. SARS-CoV-2 was the most frequently detected respiratory virus, with 66.7% of positive samples from the WWTP and 28.6% from the NH. Peaks of SARS-CoV-2 were consistent with the chronological incidence of infections recorded in the sentinel surveillance and the nearby hospital databases. The number of positive samples was lower in the NH than in WWTP for the three respiratory viruses. Enteric viruses were frequently detected, most often sapovirus and norovirus genogroup II, accounting both for 77.8% of positive samples in the WWTP and 57.1% and 37%, respectively, in the NH. The large circulation of sapovirus was unexpected in particular in the NH. Combined wastewater surveillance using simple optimized methods can be a valuable tool for monitoring viral circulation and may serve as a suitable early warning system for identifying both local outbreaks and the onset of epidemics. These results encourage the application of wastewater-based surveillance (WBS) to SARS-CoV2, norovirus, and sapovirus.IMPORTANCEWBS provides valuable information on the spread of epidemic viruses in the environment using appropriate and sensitive detection methods. By monitoring the circulation of viruses using reverse transcription PCR methods in wastewater from the inlet of a wastewater treatment plant and the outlet of a nearby retirement home (connected to the same collective sewer network), we aimed to demonstrate that implementing combined WBS at key community sites allows effective detection of the occurrence of respiratory (influenza, respiratory syncytial virus, and SARS-CoV-2) and enteric (norovirus, rotavirus, and sapovirus) virus infections within a given population. This analysis on a localized scale provided new information on the viral circulation in the two different sites. Implementing WBS to monitor the circulation or the emergence of infectious diseases is an important means of alerting the authorities and improving public health management. WBS could participate actively to the health of humans, animals, and the environment.

Gene editing of NCF1 loci is associated with homologous recombination and chromosomal rearrangements

CRISPR-based genome editing of pseudogene-associated disorders, such as p47phox-deficient chronic granulomatous disease (p47 CGD), is challenged by chromosomal rearrangements due to presence of multiple targets. We report that interactions between highly homologous sequences that are localized on the same chromosome contribute substantially to post-editing chromosomal rearrangements. We successfully employed editing approaches at the NCF1 gene and its pseudogenes, NCF1B and NCF1C, in a human cell line model of p47 CGD and in patient-derived human hematopoietic stem and progenitor cells. Upon genetic engineering, a droplet digital PCR-based method identified cells with altered copy numbers, spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context, such as the presence of homologous regions, whose instability can constitute a risk factor for chromosomal rearrangements upon genome editing.

Monitoring for PERV Following Xenotransplantation.

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs. PERV-A, PERV-B and PERV-C can be released as infectious virus particles and PERV-A and PERV-B can infect human cells in culture. PERV-C does not infect human cells, but high-titer recombinant PERV-A/C can infect them. Retroviruses are able to induce immunosuppression and/or tumors in the infected host. Numerous methods have been developed to study PERV in donor pigs. No PERV infections were observed in infection experiments as well as in preclinical and clinical xenotransplantation trials. Despite this, several strategies have been developed to prevent PERV infection of the recipient. PCR-based and immunological methods are required to screen xenotransplant recipients. Since the proviruses are integrated into the pig genome, PERV infection has to be distinguished from microchimerism, e.g., the presence of pig cells in the recipient, which is common in xenotransplantation. Sensitive PCR methods using pig short interspersed nuclear elements (SINE) sequences allow to detect pig cells easily. Virus infection can also be detected by an increase of viral genomic or mRNA in human cells. The method of choice, however, is to screen for specific antibodies against PERV using different recombinant PERV proteins, purified viruses or peptides.

First record and molecular diversity of haemosporidian parasites in Sula leucogaster (Boddaert, 1783) from the southeastern Brazilian coast.

Avian haemosporidian parasites are protozoans transmitted by vectors, belonging to the genera Plasmodium, Haemoproteus, and Leucocytozoon, capable of infecting various bird species. The prevalence of these parasites varies depending on factors such as the habitat and geographical location of the host and vector, as well as across different bird groups. Seabirds typically exhibit low infection rates due to the absence of vectors in marine environments. This study aimed to identify and characterize the molecular diversity of Haemosporida parasites in a population of Brown Boobies (Sula leucogaster) (n = 37) from the Santana Archipelago in Macaé, Rio de Janeiro state. We utilized PCR-based detection methods to confirm the presence of haemosporidian parasites and sequencing (ctyb gene) to delineate the lineages. The results uncovered a notable prevalence (48.6%) of haemosporidian parasites (Plasmodium and Haemoproteus), comprising 12 novel lineages and four previously documented lineages (establishing new host records). Our findings suggest that transmission and infection by these hemoparasites can occur both on and off the island. Overall, this study yields valuable insights into the genetic diversity of haemosporidian parasites in Brown Boobies, enriching our understanding of the ecology and epidemiology of these parasites in seabirds.

Quantification of the thermal history of insects in foods using real-time PCR-based DNA fragmentation analysis

A major challenge faced by the food industry is the contamination of food by insects. Once an insect is found in a food product, it is important to determine how it entered the food. Given that the thermal history of insects provides a clue to unraveling this mystery, in this study, we have developed a novel real-time PCR-based DNA fragmentation analysis method to quantify the thermal history of insects found in foods.We selected two species of cockroaches (Periplaneta fuliginosa and Blattela germanica) as investigative targets because cockroach contamination often causes significant economic losses in the food industry. A set of real-time PCRs was designed for each species to amplify the species-specific regions of 18S ribosomal DNA at approximate 100 and 300 bp. The degree of DNA fragmentation was evaluated using the differences between the amplification curves for the 100- and 300-bp sequences. We observed that the degree of DNA fragmentation was quantifiable when the insect was heated to >100 °C. Therefore, we determined whether cockroaches contaminate retort foods during the manufacturing or household consumption stages. This novel analytical method is expected to improve food safety and product reliability by identifying the cause of insect contamination.

Evaluation of in vitro probiotic properties and colonization of Brevibacillus Laterosporus S62-9 in the intestine of broiler chickens

Brevibacillus laterosporus has been used as a direct-fed microbial additive in chicken feed owing to its strain safety and the ability to produce broad-spectrum antimicrobial peptides. To assess the efficacy and persistence of Brevibacillus laterosporus, it is necessary to track the supplied strains throughout the gastrointestinal tract. This study aims to evaluate the in vitro probiotic properties of Brevibacillus laterosporus S62-9 and its capacity to colonize and persist in the intestines of broiler chickens. Results indicated that the spores of Brevibacillus laterosporus S62-9 exhibited higher tolerance to the digestive condition, and higher adhesion rate to mucus and intestinal cells compared to vegetative cells. The spores remained dormant during digestion in the crop and stomach but germinate in the small intestine with a germination rate exceeding 70%. Subsequently, the whole genome of S62-9 was sequenced, and specific primers were designed and validated based on comparative genome analysis. The quantitative PCR-based method had a detection limit ranging from 2.71 to 7.71 log10 CFU/mL in pure culture and from 3.25 to 9.25 log10 CFU/g in broiler cecal content. In vivo colonization assay indicated that the Brevibacillus laterosporus S62-9 can transiently colonize the broiler cecum. In summary, our results indicated that the Brevibacillus laterosporus S62-9 spores exhibited high tolerance to the digestive tract environment, germinated in the intestine, and establish colonization in the cecum of broilers. The findings provide a theoretical basis for the future application of Brevibacillus laterosporus S62-9 in broiler farming.

PCR enables rapid detection of dermatophytes in practice.

Dermatophytes cause superficial infections of skin, hair, and nails. Even though they rarely cause severe infections, they are relatively common, particularly in primary health care. Diagnosis of dermatophyte infections has relied on relatively slow culture-based methods. Nucleic acid-based detection (PCR) methods might provide results more rapidly. Here, we describe the transition from culture-based methods into PCR-based methods in Northern Finland with a catchment area of approximately 720,000 mostly Caucasian people. This study included 14,330 samples collected between 2019 and 2022. Our results showed that the PCR-based method has become the diagnostic test of choice for these infections in this area. Commercial real-time PCR assay DermaGenius 2.0 complete multiplex test detected more positive results than culture and covered the most important dermatophytes, Candida albicans, and few less common species. With PCR, the mean turn-around-time from sample request to results decreased from 19 days to 16 hours.IMPORTANCESuperficial fungal infections, dermatophytosis, are remarkably common worldwide, affecting an estimated 20%-25% of the global population. In the diagnosis of these infections, fast and accurate results by PCR shorten the time to diagnosis and help clinicians to avoid unnecessary antifungal treatments.

Harnessing non-standard nucleic acids for highly sensitive icosaplex (20-plex) detection of microbial threats.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. We benchmarked this assay using a low-cost, portable sequencing platform (Oxford Nanopore) on wastewater, soil, and human stool samples. Using parallelized multi-target TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay, there was 74% agreement on positive calls and 97% agreement on negative calls. Additionally, we show how sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish sub-species. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

Shotgun DNA sequencing for human identification: Dynamic SNP selection and likelihood ratio calculations accounting for errors

Shotgun sequencing is a DNA analysis method that potentially determines the nucleotide sequence of every DNA fragment in a sample, unlike PCR-based genotyping methods that is widely used in forensic genetics and targets predefined short tandem repeats (STRs) or predefined single nucleotide polymorphisms (SNPs). Shotgun DNA sequencing is particularly useful for highly degraded low-quality DNA samples, such as ancient samples or those from crime scenes. Here, we developed a statistical model for human identification using shotgun sequencing data and developed formulas for calculating the evidential weight as a likelihood ratio (LR). The model uses a dynamic set of binary SNP loci and takes the error rate from shotgun sequencing into consideration in a probabilistic manner. To our knowledge, the method is the first to make this possible. Results from replicated shotgun sequencing of buccal swabs (high-quality samples) and hair samples (low-quality samples) were arranged in a genotype-call confusion matrix to estimate the calling error probability by maximum likelihood and Bayesian inference. Different genotype quality filters may be applied to account for genotyping errors. An error probability of zero resulted in the commonly used LR formula for the weight of evidence. Error probabilities above zero reduced the LR contribution of matching genotypes and increased the LR in the case of a mismatch between the genotypes of the trace and the person of interest. In the latter scenario, the LR increased from zero (occurring when the error probability was zero) to low positive values, which allow for the possibility that the mismatch may be due to genotyping errors. We developed an open-source R package, wgsLR, which implements the method, including estimation of the calling error probability and calculation of LR values. The R package includes all formulas used in this paper and the functionalities to generate the formulas.