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Background/Objectives: Sarcomas are a heterogeneous group of cancers, many with high rates of recurrence and metastasis, leading to significant morbidity and mortality. Due to a lack of early diagnostic biomarkers, by the time recurrent disease can be clinically detected, it is often extensive and difficult to treat. Here, we sought to investigate methods of detecting ctDNA in sarcoma patient plasma to potentially monitor disease recurrence, progression, and response to treatment. Methods: Whole-exome sequencing of matched tumor and blood samples revealed patient-specific mutations, which were used to develop personalized assays to detect ctDNA in patient plasma. Since ctDNA is present in extremely low quantities, detection requires highly sensitive methodologies. Droplet digital PCR is highly sensitive; however, it is limited in that it can only be used to target one tumor variant at a time. Therefore, a protocol combining multiplex PCR and targeted amplicon sequencing was developed. Results: ddPCR was successfully able to detect tumor-specific mutations in plasma, confirming the presence of ctDNA in sarcoma patients. Multiplex PCR followed by amplicon sequencing was able to detect multiple tumor variants simultaneously, although it was not as sensitive as ddPCR. Additionally, ctDNA was detected in patient plasma collected at two different time points. Conclusions: This work demonstrates that although there is a lack of recurrent biomarkers, personalized assays detecting ctDNA have the potential to be used to monitor disease progression in sarcoma. [ABSTRACT FROM AUTHOR]

Canine visceral leishmaniasis (CVL) is a parasitic disease caused by the protozoan Leishmania infantum. Neurological infection occurs due to the parasite's ability to cross the blood–brain barrier. It is known that dogs can remain infected with a subclinical infection for life, potentially acting as reservoirs for L. infantum when bitten by sandflies. In this context, the objective of this study was to evaluate the occurrence of Leishmania spp. in the brains of dogs from the metropolitan region of Rio Grande do Sul, Brazil, without a history of neurological disease but residing in an endemic area for L. infantum. A total of 200 samples, from 2022 to 2023, were evaluated using conventional Polymerase Chain Reaction (PCR) with the primers Leishmini-F GGKAGGGGCGTTCTGC and Leishmini-R STATWTTACACCAACCCC, aiming to amplify a product of 120 base pairs for Leishmania spp. To identify the species, a multiplex PCR was used, differentiating L. braziliensis (127 bp), L. amazonensis (100 bp), and L. infantum (60 bp), with the molecular target being the conserved region of the kinetoplast DNA (kDNA) minicircle, specific to Leishmania spp. Of the 200 samples evaluated, 26.5% (53/200) tested positive in the conventional PCR reaction for Leishmania spp., with the PCR multiplex the only species detected was Leishmania infantum. The average age of the positive animals was 5.08 years, with 47.2% being females and 52.8% being males; among these, mixed-breed dogs were the most predominant, representing 43.4% of the total. Clinical signs varied: hepatomegaly in two dogs, pronounced neutrophilic hepatitis in one, splenomegaly in one with lymphoid hyperplasia, and glomerulonephritis and nephritis in two animals. Mild anemia and thrombocytopenia were found in eight, with pale mucous membranes in three, and diffuse alveolar edema in one case. Notable pathological findings included suspected distemper in one animal and lymphoplasmacytic meningitis in another. Histopathological findings revealed alveolar edema and acute renal failure. A third dog exhibited bilateral hydrocephalus and diffuse edema in the brain. Additional changes, such as mild inflammatory infiltrate and slight vacuolar degeneration, were observed in 11.3% of the analyzed brains. There was no clinical suspicion of leishmaniasis in any of the studied cases. Therefore, the detection of L. infantum DNA in the brains of dogs suggests that animals with subclinical infection may play a crucial role in the spread of leishmaniasis, and infection by Leishmania spp. should be considered as a differential diagnosis for neurological disease in endemic areas for the protozoan. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is an ever-increasing threat. The widespread usage of ciprofloxacin has led to the manifestation of resistance due to chromosomal mutations or the acquisition of plasmid-mediated quinolone resistance (PMQR) traits. Some particular PMQR traits, qnr genes, have been identified globally in clinical and environmental isolates. This study aimed to determine the prevalence of ciprofloxacin-resistant bacteria in aquatic environments in southern Ontario and investigate the extent of dissemination of ciprofloxacin resistance traits among the bacterial communities. We surveyed the prevalence of plasmid encoding qnr genes using a multiplex PCR assay of associated PMQR genes, qnrA, qnrB, and qnrS, on 202 isolates. Despite the absence of significant impacts on minimum inhibitory concentration levels, the presence of qnr genes correlates with heightened resistance to quinolones and nalidixic acid in some isolates. Taxonomic analysis highlights distinct differences in the composition and diversity of ciprofloxacin-sensitive (CipS) and ciprofloxacin-resistant (CipR) populations, with Proteobacteria dominating both groups. Importantly, CipR populations exhibit lower genetic diversity but higher prevalence of multiple antibiotic resistances, suggesting co-selection mechanisms. Co-occurrence analysis highlights significant associations between ciprofloxacin resistance and other antibiotic resistances, implicating complex genetic linkages. The results of our study signified the critical role of environmental monitoring in public health. [ABSTRACT FROM AUTHOR]

Lactococcosis is a common bacterial fish disease caused by Lactococcus garvieae, L. petauri and L. formosensis. Although there are different PCR‐based techniques to identify the etiological agent, none of these can differentiate these two bacteria without sequencing PCR‐amplified fragments. In the present study, we developed a multiplex PCR assay for simultaneous detection and differentiation of L. garvieae and L. petauri. The specificity of the primers was validated against the bacterial DNA of the targeted and non‐targeted bacteria. The sizes of the PCR amplicons were obtained as 204 bp for the DUF1430 domain‐containing protein gene of L. garvieae, 465 bp for the Lichenan permease IIC component gene of L. petauri, and 302 bp for the teichoic acid biosynthesis protein F gene of both L. garvieae and L. petauri. The PCR amplicons were clearly separated by agarose gel electrophoresis. The multiplex PCR assay did not produce any amplification products with the DNA of the non‐targeted bacteria. The multiplex PCR detection limits for L. garvieae and L. petauri were 5 and 4 CFU in pure culture and 50 and 40 CFU/g in spiked tissue samples, respectively. It takes less than 2 h from plate‐cultured bacteria and 3 h from tissue samples to get results. In conclusion, the developed multiplex PCR assay is a rapid, specific, accurate, and cost‐effective method for the detection and differentiation of L. garvieae and L. petauri and is suitable to be used for routine laboratory diagnosis of L. garvieae and L. petauri. [ABSTRACT FROM AUTHOR]

Piglet lethality is one of the major concerns in pig breeding programs. Deletion of a 212-kb region within the Bardet-Biedl syndrome 9 (BBS9) gene has been linked to a reduction in the number of piglets born alive per litter. The BBS9 mutant gene carrier-by-carrier mating scheme could result in mummification of piglets carrying 2 copies of the BBS9 mutant allele, which ultimately affects the reproductive performance of the sow. Our aim was to develop a simple, rapid, and cost-efficient method that could be applied in a BBS9 mutant gene carrier screening program in low- and middle-income countries within basic laboratory settings. Here, we report an optimized multiplex PCR assay that we have established successfully for detection of a 212-kb deletion within the BBS9 genomic sequence. We genotyped 420 animals from Yorkshire, Duroc, and Landrace purebred populations in Vietnam. We found that while the BBS9 mutant allele was not identified in Duroc pigs, the frequency of BBS9 carriers was 10% in both Yorkshire and Landrace populations. We subsequently validated our results using Sanger sequencing. Our multiplex PCR method could be utilized as a BBS9 screening test in pig breeding programs. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant A. baumannii pose a serious threat, limiting treatment options for severe infections induced by this emerging pathogen. This study aimed to evaluate the antimicrobial resistance profile and detect resistance mechanisms in imipenem-resistant A. baumannii from various Egyptian hospitals. Materials and Methods: A total of 59 A. baumannii isolated from various clinical specimens (blood, sputum, wound swap, urine and endotracheal tubes) collected from the microbiology lab of different Egyptian hospitals. Isolates were identified using conventional bacteriological methods. Antimicrobial susceptibility was tested using Kirby-Bauer disc diffusion method. Biofilm formation was determined spectrophotometrically using microtiter plate method. Metallo-β-lactamase (MBL) resistance was determined by combined disk test (CDT). Multiplex PCR evaluated MBL-encoding genes in imipenem-resistant isolates. Additionally, PCR was used to detect class 1 integrons in MBL-producing isolates. Results: Strong biofilm production was observed in 74% (17/23) of isolates. Class 1 integron was detected in 78.9 % (15/19) of MBL producing isolates. Out of 23 isolates 19 (32.2%) were MBL producers by CDT. Among the 19 (32.2%) CDT-positive isolates, 11 (57.9%) isolates harboured MBL encoding gene (blaNDM). Conclusion: Our findings highlight the emergence and dissemination of blaNDM in our hospitals, coupled with their biofilms and MBL production ability, represents a major public health challenge. The presence of class 1 integrons in all isolates carrying MBL-encoding gene, suggesting the critical role of Class 1 integrons in the dissemination of resistance genes. [ABSTRACT FROM AUTHOR]

Background: The resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits. Methods: To develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species. Results: Both multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test. Conclusions: The multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species. [ABSTRACT FROM AUTHOR]

Enlarged vestibular aqueduct (EVA) is a frequently occurring inner ear malformation that associates with sensorineural hearing loss (SNHL), with SLC26A4 being the responsible gene. Based on multiplex PCR enrichment and sequencing of the exonic and flanking regions of the SLC26A4 gene, we developed a panel specifically for EVA and found that up to 95% of EVA patients in our Chinese cohorts carried biallelic SLC26A4 pathogenic variants (M2). In this study, we tried to investigate the genetic etiology of 13 previously undiagnosed EVA patients with monoallelic (M1) or none (M0) SLC26A4 variant using a stepwise approach, including copy number variation (CNV) analysis of multiplex PCR enrichment and next‐generation sequencing data, single‐molecule real‐time (SMRT) sequencing of the whole SLC26A4 gene, whole exome sequencing (WES), and whole genome sequencing (WGS). CNV analysis revealed deletions in Exons 1–3, Exons 5–6, and Exons 9–10 of the SLC26A4 gene in seven patients, and SMRT sequencing identified the same heterozygous deep intronic variant (NM_000441.2:c.304+941C>T) in two patients, resulting in a final diagnosis in 9/13 patients. Notably, the variants of Exons 9–10 deletion and c.304+941C>T have not been reported previously. We further showed that the variant c.304+941C>T led to the exonization of partial AluSz6 element (126 bp) where the variant is located through sequencing of the mRNA extracted from the blood of a heterozygous variant carrier. In conclusion, our stepwise approach improved the diagnosis rate of EVA, expanded the mutational spectrum of the SLC26A4 gene, and highlighted the contribution of exonic deletions and deep intronic variants to EVA. [ABSTRACT FROM AUTHOR]

Candida onychomycosis is a common fungal infection affecting the nails, primarily caused by Candida (C.) species. Regarding the increasing trend of Candida onychomycosis and the antifungal resistant phenomenon in recent years, this study aims to evaluate the epidemiological characteristics of Candida onychomycosis, the distribution of emerging species, and the antifungal susceptibility profiles of isolates. Onychomycosis caused by yeast species was confirmed through direct examination and culture of nail scraping among all individuals suspected to have onychomycosis and referred to a medical mycology laboratory between June 2019 and March 2022. Species of yeast isolates were identified using the multiplex PCR and PCR-RFLP methods. The antifungal susceptibility of isolates to common antifungal agents and imidazole drugs was evaluated according to the M-27-A3 CLSI protocol. Among 101 yeast strains isolated from onychomycosis, Candida parapsilosis complex (50.49%) was the most common species, followed by C. albicans (20.79%) and C. tropicalis (10.89%). Rare species of yeasts such as C. guilliermondii and Saccharomyces cerevisiae were also identified by molecular methods. Results obtained from antifungal susceptibility testing showed significant differences in MIC values of isoconazole, fenticonazole, and sertaconazole among different species. Overall, a fluconazole-resistant rate of 3% was found among Candida species. Moreover, there was a statistically significant difference in MICs of fenticonazole and clotrimazole between the two most prevalent causative species, C. parapsilosis complex and C. albicans. Correct identification of the causative agents of onychomycosis and performing susceptibility testing could be helpful in choosing the most appropriate antifungal therapy. [ABSTRACT FROM AUTHOR]

Background: Candida species have emerged as a significant cause of opportunistic infections. Alongside the expression of various virulence factors, the rise of antifungal resistance among Candida species presents a considerable clinical challenge. Aim: This study aimed to identify different Candida species isolated from clinical specimens, evaluate their antifungal sensitivity patterns, identify key genes regulating virulence mechanisms using multiplex PCR and to assess any correlation between their virulence profiles and antifungal resistance patterns. Method: A total of 100 Candida spp. was isolated from 630 different clinical specimens and identified to the species level. Their antifungal susceptibility was phenotypically evaluated in accordance with CLSI guidelines using the Vitek-2 Compact System. Virulence markers, including biofilm formation capacity, protease production, melanin production, coagulase production and hemolysin production, were also phenotypically detected. The genetic determinants for biofilm formation and extracellular hydrolytic enzymes were assessed using a multiplex PCR assay. Results: The prevalence of Candida spp. was 15.9%, with C. albicans (48%) and C. glabrata (16%) being the most common. C. albicans showed the highest virulence, with strong biofilm formation, and high proteinase and melanin production. Multiplex PCR revealed Hlp in 22.0%, Hwp in 80.0%, Als in 56.0%, and Sap genes in 56.0% of isolates. Virulence genes were more common in C. albicans than in non-albicans Candida (NAC). Resistance patterns significantly correlated with virulence profiles, with notable associations between flucytosine resistance and the presence of Hlp and Hwp genes. Conclusion: The significant correlation between virulent markers such as germination, coagulase, hemolysin production and resistance patterns among different Candida isolates is crucial for predicting the severity and outcomes of Candida infections. This understanding aids in guiding tailored treatment strategies. [ABSTRACT FROM AUTHOR]

Multiplex PCR has shown to be a useful technique for identifying Listeria monocytogenes and Staphylococcus aureus in food items. It can also be used to trace toxins to enhance food safety. This study identifies Listeria monocytogenes and Staphylococcus aureus in processed beef meat products using multiplex PCR. These bacteria were found in the beef meat samples using the 16SrRNA gene of L. monocytogenes and the 23S rRNA gene of S. aureus. For this study, 40 processed meat samples—ten samples of each minced meat, beef sausage, beef burger, and beef kofta— were gathered from different supermarkets in the El-Menoufia governorate. One important byproduct produced during the culinary processing of pomegranates is the peels from the fruits. Due to their plentiful presence of broad-spectrum antibacterial agents and antioxidants, these peels can effectively prevent food spoilage. The primary objective of the study was to determine whether pomegranate peel extract had any antimicrobial effects on Listeria monocytogenes and Staphylococcus aureus in minced beef. Also, the antioxidant and antibacterial properties of a hydromethanolic extract from sweet orange (Citrus sinensis) peel were examined. The results indicated that an extract from the peel of the Citrus sinensis tree has antibacterial effects against S. aureus and Listeria monocytogenes. [ABSTRACT FROM AUTHOR]

Background: Diarrheal disease remains one of the leading causes of deaths in children below five years of age. The risk factors associated with diarrhea include poor hygiene practices such as open defecation and consumption of contaminated water and food. However, exposure of domestic animal is equally a potential risk factor for diarrhea disease in children. Methodology: We characterized animal-related exposures in a subset of households (n=73) by collecting faecal samples from 150 children with diarrhoea and 100 food animals (30 cattle, 30 chicken, 25 goats and 15 pigs). Escherichia coli was isolated from the faecal samples and biochemically confirmed using conventional microbiological techniques. The deoxyribonucleic acid (DNA) of each E. coli isolate was extracted and amplified by multiplex PCR to identify three diarrheagenic E. coli pathotypes. The amplified products were sequenced, and genetic relatedness of the isolates was determined through phylogenetic analysis. Results: We isolated and identified a total of 32 (12.8%) diarrheagenic E. coli (DEC) from the 250 faecal samples, 26 (17.3%) of which were from the 150 children with diarrhea while 6 (6.0%) were from the 100 food animals (OR=3.285, 95% CI=1.299-8.305, p=0.011). Three DEC pathotypes were confirmed by PCR in 16 DEC strains, with 9 enteroaggregative E. coli (EAEC), 2 enterotoxigenic E. coli (ETEC), 2 enteropathogenic E. coli (EPEC), 1 EAEC/ETEC, 1 EAEC/EPEC and 1 ETEC/EPEC mixed strains. The phylogenetic analysis showed that 6 DEC isolates had genetic similarity ranging between 31% to 90%. Isolates S04 originating from animal and S02 from a child with diarrhoea of the same household were closely related, with 55% similarity. Moreover, isolate S05 from animal origin and S06 of diarrheic child origin were closely related, with similarity degree as high as 82% even though they were not paired. Twenty four of the 26 (92.3%) DEC isolates from diarrhoeic children showed multidrug resistance (MDR) pattern to antibiotics but none of the 6 isolates from food animals was multi-drug resistant. Conclusion: The high degree of genetic relationship between DEC isolate S04 and S02 from animal and human origin indicated the high potency of zoonotic transmission. Further studies investigating animal husbandry practices and zoonotic transmission of DEC are needed. [ABSTRACT FROM AUTHOR]

Selecting compatible HLA‐Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA‐Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA‐Class I and HPA genotypes. Ten pairs of specific primers for HLA‐A, HLA‐B, HLA‐C, HPA‐1 through HPA‐6w, HPA‐15 and HPA‐21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA‐A, HLA‐B and HLA‐C and the TaqMan real‐time PCR method in‐house for the genotyping of HPA‐1 through HPA‐6w, HPA‐15 and HPA‐21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA‐A, HLA‐B, HLA‐C and HPA‐1 through HPA‐6w, HPA‐15 and HPA‐21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA‐A*11:451, HLA‐A*30:01:26, HLA‐B*39:201 and HLA‐B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA‐Class I and HPA simultaneously, which is high‐throughput, rapid and accurate and could be applied to build a platelet donor bank. [ABSTRACT FROM AUTHOR]

Background: Laboratory rats, as model animals, have been extensively used in the fields of life science and medicine. It is crucial to routinely monitor the genetic background of laboratory rats. The conventional approach relies on gel electrophoresis and capillary electrophoresis (CE) technologies. However, the experimental and data analysis procedures for both of these methods are time consuming and costly. Results: We established a single-nucleotide polymorphism (SNP) typing scheme using multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to address the genetic background ambiguity in laboratory rats. This methodology involved three rounds of PCR and two rounds of magnetic bead selection to improve the quality of the sequencing data. We simultaneously analysed 100 laboratory rats (including rats of 5 inbred strains and 2 in-house closed colonies), and the sequencing depth varied from an average of 108.25 to 5189.89, with sample uniformity ranging from 82.5 to 97.5%. A total of 98.9% of the amplicons were successfully genotyped (≥ 30 reads). Genetic background analysis revealed that all 38 experimental rats from the 5 inbred strains were successfully identified (without a heterozygous allele). For the 2 in-house closed colonies, the average heterozygosity (0.162 and 0.169) deviated from the typical range of 0.5–0.7, indicating a departure from the ideal heterozygosity level. Additionally, we employed multiplex PCR-CE to validate the NGS-based method, which yielded consistent results for all the rat strains. These results demonstrated that this approach significantly improves efficiency, saves time, reduces costs and ensures accuracy. Conclusion: By utilizing NGS technology, our developed method leverages SNP genotyping for genetic background identification in laboratory rats, demonstrating advantages in terms of labour efficiency and cost-effectiveness, thereby rendering it well suited for projects involving extensive sample cohorts. [ABSTRACT FROM AUTHOR]

Streptococcus agalactiae, or group B streptococcus (GBS), is a Gram-positive pathogen with an extended track record of colonization in the gastrointestinal and genitourinary tracts. GBS can induce disease in individuals across all age demographics, yet it predominantly triggers infections in neonates and the elderly. Identification of the serotype is vital for effective management of the disease as it provides critical information for clinicians on the cause of the disease. In this study, we evaluated the rapid, simple, and easy-to-adopt multiplex real-time PCR technique, NovaplexTM (NovaPCR). A total of 131 clinical isolates of different serotypes were tested using NovaPCR. Observations revealed that 129 isolates showed the same observations as LA and conventional mPCR. NovaPCR accurately identified serotypes IV and V, which were first classified as serotype Ia in the LA test and mPCR, and the difference between the traditional (LA test and mPCR) and NovaPCR methods is only 1.52%. Accurate serotype identification is helpful for monitoring the epidemics and achieving optimal clinical outcomes, and NovaPCR showed a reliable, fast, easy-to-interpret, and cost-efficient performance in GBS serotyping. [ABSTRACT FROM AUTHOR]

Recently a number of broad-range stool parasite PCR assays have been developed. However, there is ongoing disagreement regarding their diagnostic performance, as various studies have produced contradictory results. In this study, we compared the diagnostic accuracy of the Seegene Allplex GI-Parasite and Allplex GI-Helminth assays (SA) with the conventional methods used at the travel clinic of the Institute of Tropical Medicine (ITM) including microscopy, antigen testing, and molecular detection in order to provide insights into the strengths and limitations of this diagnostic tool which may be crucial to select the most appropriate diagnostic tools for the suspected pathogen. A total of 97 native stool samples from 95 patients with suspected gastrointestinal illness were analyzed, including 26 from a frozen collection and 71 prospectively collected samples. The diagnostic performance of SA was notably superior to the conventional workflow in detecting Dientamoeba fragilis (sensitivity 100% vs. 47.4%) and Blastocystis hominis (sensitivity 95% vs. 77.5%). SA had a comparable performance with the conventional workflow in detecting pathogenic protozoa (sensitivity 90% vs. 95%). In contrast, SA had a much lower diagnostic performance in detecting helminths (59.1%) compared to the conventional workflow (100%). We conclude that the Seegene Allplex GI-Parasite assay may be useful for protozoa screening in low-endemic industrialized countries. However, the Allplex GI-Helminth assay is not recommended due to its suboptimal performance compared to microscopy. [ABSTRACT FROM AUTHOR]

AbstractObjectiveMethodsResultsConclusionsAccurate and rapid identification of causative pathogens is essential to guide the clinical management of lower respiratory tract infections (LRTIs). Here we conducted a single-centre prospective study in 284 patients suspected of lower respiratory tract infections to evaluate the utility of a nucleic acid test based on highly multiplexed polymerase chain reaction (PCR) and CRISPR-Cas12a.We determined the analytical and diagnostic performance of the CRISPR assay using a combination of reference standards, including conventional microbiological tests (CMTs), metagenomic Next-Generation Sequencing (mNGS), and clinical adjudication by a panel of experts on infectious diseases and microbiology.The CRISPR assay showed a higher detection rate (63.0%) than conventional microbiological tests (38.4%) and was lower than metagenomic Next-Generation Sequencing (72.9%). In detecting polymicrobial infections, the positivity rate of the CRISPR assay (19.4%) was higher than conventional microbiological tests (3.5%) and lower than metagenomic Next-Generation Sequencing (28.9%). The overall diagnostic sensitivity of the CRISPR assay (67.8%) was higher than conventional microbiological tests (41.8%), and lower than metagenomic Next-Generation Sequencing (93.2%).Considering the low cost, ease of operation, short turnaround time, and broad range of pathogens detected in a single test, the CRISPR assay has the potential to be implemented as a screening tool for the aetiological diagnosis of lower respiratory tract infections patients, especially in cases where atypical bacteria or coinfections are suspected. [ABSTRACT FROM AUTHOR]

Background: The dairy sector faces formidable obstacles in the early detection of the bacteria causing subclinical mastitis and in the proactive treatment of those cases. The objective of the study was to access multiplex PCR assays (MPCR) and conventional methods for the concurrent identification of significant bacteria that cause sub-clinical mastitis and to compare these methods. 200 samples of pooled milk from cows and buffaloes in Rajasthan were gathered between 2020 and 2021. Methods: The primary bacterial pathogens in milk samples were identified using conventional methods such multiplex PCR, biochemical testing and culture. Result: From 200 combined samples of cow and buffalo milk, the traditional approach was able to extract 97 different strains. Staphylococcus aureus 54 (27%), Streptococcus spp. 30 (15%) and E. coli 13 (6.5%) were shown to be prevalent as single or mixed infections, respectively. Staphylococcus was the main pathogen that was discovered. concurrently, S. aureus, Streptococcus and E. coli. Direct detection of Staphylococcus 65 (32.5%), Streptococcus 37 (18.5%), E. coli and 16 (8%) by multiplex PCR was found in milk samples. The analysis revealed that because multiplex PCR assays have higher specificity and sensitivity than conventional procedures, they are more reliable. The multiplex PCR method employed in the current study was a simple and quick technique to identify the major pathogens and it has the potential to be a very helpful tool for determining the pathogens that cause environmental mastitis and evaluating the health of the herd. [ABSTRACT FROM AUTHOR]

Late blowing defects in semi-hard and hard cheeses caused by spore-forming clostridia (e.g., Clostridium tyrobutyricum, Clostridium butyricum, Clostridium sporogenes) pose a major issue for the dairy industry. With this study, we applied a multiplex PCR for the rapid and simultaneous detection of clostridia in raw milk for cheese production. Spore detection in milk usually relies on culture-dependent methods, among which the most probable number (MPN) technique is sensitive but time-consuming and nonspecific. We tested two PCR-based protocols: the one entailed direct milk analysis with results obtained within 24 h; the other included an enrichment step and gave results within 72 h. The second protocol was found to be more sensitive; it detected concentrations as low as 100 cells/L for C. sporogenes and C. butyricum and 800 cells/L for C. tyrobutyricum. Both protocols were applied to field samples (211 samples underwent protocol no. 1; 117 samples underwent protocol no. 2) collected from four dairy processing plants in Piedmont. The prevalence of C. butyricum (protocol no. 1: 9.5%; protocol no. 2: 23%) was higher than either C. sporogenes (0%; 9.4%) or C. tyrobutyricum (0%; 6.8%). Protocol no. 2 detected multiple targets in eight samples, indicating that more than one microorganism was present. Our findings underscore the importance of implementing preventive measures and early detection strategies to mitigate the risk of cheese spoilage due to clostridial contamination. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) has been spreading through Europe, Asia, and the Caribbean after its introduction in Georgia in 2007 and, due to its particularly high mortality rate, poses a continuous threat to the pig industry. The golden standard to trace back the ASFV is whole genome sequencing, but it is a cost and time-intensive methodology. A more efficient way of tracing the virus is to amplify only specific genomic regions relevant for genotyping. This is mainly accomplished by amplifying single amplicons by PCR followed by Sanger sequencing. To reduce costs and processivity time, we evaluated a multiplex PCR based on the four primer sets routinely used for ASFV genotyping (B646L, E183L, B602L, and intergenic I73R-I329L), which was followed by Nanopore ligation-based amplicon sequencing. We show that with this protocol, we can genotype ASFV DNA originating from different biological matrices and correctly classify multiple genotypes and strains using a single PCR reaction. Further optimization of this method can be accomplished by adding or swapping the primer sets used for amplification based on the needs of a specific country or region, making it a versatile tool that can speed up the processing time and lower the costs of genotyping during ASFV outbreaks. [ABSTRACT FROM AUTHOR]

Extended‑spectrum β‑lactamase (ESBL) genes are responsible for creating Multidrug‑resistant and Extensive drug resistance (XDR) patterns in Acinetobacter baumanii isolates, so limit treatment options and increase mortality and morbidity. This study aimed to development of a multiplex PCR assay for the detection of extended-spectrum beta-lactamase genes including blaCTX-M, blaSHV and blaTEM among clinical samples of Acinetobacter baumanii isolates in Tehran, Iran. In present study, 100 clinical Acinetobacter baumannii strains have been gathered from patients in Motahhari hospital in Tehran city, Iran. Antibiotic susceptibility test was conducted by Kirby–Bauer disc diffusion method. To identify ESBL-producing strains, used combined disk test and Multiplex PCR method was used for Simultaneous diagnosis of blaCTX-M, blaSHV, and blaTEM genes. Out of 100 isolates, 93% were ESBL-positive according to the phenotypic test. Most of the isolates were XDR and the highest sensitivity was for colistin. The frequency of bla CTX-M, blaSHV and blaTEM genes was 95, 1, and 2% respectively. The high percentage of antibiotic resistance and high prevalence of the blaCTX-M gene in A. baumannii isolates is a serious threat to the effectiveness of available antibiotics. This study showed Multiplex PCR can be a reliable and sensitive technique for the fast detection of ESBL genes in Acinetobacter baumannii isolates. [ABSTRACT FROM AUTHOR]

Hospitalized patients on mechanical ventilation are at high-risk of developing ventilator-associated pneumonia (VAP), making early and accurate diagnosis essential for the best possible treatment outcomes. This review examined various laboratory techniques, such as aerobic cultures, Gram's stain, and molecular techniques, to assess how well they diagnose VAP. We have done search strategies using Google Scholar, Medline Complete, and PubMed. Extensive statistical tools were utilized to examine studies and evaluate the diagnostic accuracy of laboratory results. Multiplex PCR was superior to Gram's stain and culture methods in terms of sensitivity (92%) and specificity (86%). On the other hand, Gram's stain showed the highest specificity (78.9%) and the lowest sensitivity (74.6%). The results of semiquantitative, quantitative, enrichment culture showed a lower specificity (75.97%) but a moderate sensitivity (78.5%). The most accurate diagnostic approach for VAP, according to a meta-analysis, was multiplex PCR-based testing, closely followed by culture methods. Beyond separate tests, multiplex PCR, culture, and Gram's stain combination increased sensitivity. Prompt and precise VAP diagnosis is essential for efficient treatment. With possible improvements through combination diagnostic techniques, multiplex PCR remains the most accurate diagnostic tool. However, more investigation is necessary to improve and certify VAP diagnostic instruments. [ABSTRACT FROM AUTHOR]

Fish substitution in fish products is an important issue in fish markets, as it is a widespread practice. An authentication protocol for Rohu, Thaila and Tilapia was developed by multiplex PCR. Three species-specific and one degenerate common forward primer were designed using the Cytb gene of the mitochondrial genome. These primers for Labeo rohita, Labeo catla and Oreochromis niloticus showed the fragment size of 235 bp, 186 bp and 506 bp on the agarose gel, respectively. The primers for L. rohita and L. catla were sensitive to 0.1 ng of DNA template, while for O. niloticus this value was 1 ng of DNA template. A total of 230 commercial samples (160 fried and 70 processed fish products) were screened, where 60% mislabeling in fried and 30% mislabeling in processed fish were found. This multiplex PCR protocol could give useful insights for food inspection and enforcement of regulatory food control. [ABSTRACT FROM AUTHOR]

Rice production is drastically affected by biotic stresses, namely, bacterial leaf blight (BB), blast (BL) and brown planthopper (BPH). As marker-assisted selection for uniplex gene screening in breeding population is often tedious, time-consuming and expensive, multiplex PCR is practically a reasonable choice for the testing of genes in the pyramided lines as it offers significant time- and cost-saving advantages. Therefore, a cost-effective multiplex PCR-based screening for simultaneous detection of three BB (Xa21, xa13, and Xa4), two BL (Pi1and Pi54) and two BPH resistance genes (Bph20 and Bph21) has been developed. This multiplex system can detect different combinations of BB, BL and BPH resistance. In addition to this most cost-effective method, a five-gene (Xa21, xa13, Pi54, Bph20 and Bph21) multiplex assay in a single PCR tube for three biotic stresses, BB, BL and BPH, has been developed. These standardized multiplex assays have been incorporated into our breeding programs for accelerated results. These assays have displayed no deviation and are in perfect correlation with the individual marker screenings. As one of the most efficient and cost-saving technologies, this technology will have a great impact in the seed industry. [ABSTRACT FROM AUTHOR]

Simple Summary: Gastrointestinal parasitic infections are highly prevalent among goat populations, and field samples commonly display mixed infections. Giardia duodenalis, Cryptosporidium parvum, Blastocystis spp., and Enterocytozoon bieneusi are four common zoonotic intestinal parasites with similar symptoms, including diarrhea, vomiting, and dehydration. In this study, a multiplex PCR method was developed for the differential detection of these four zoonotic protozoans. Genomic DNA extracted from 130 goat stool samples obtained from four farms in Zhanjiang city, Guangdong Province, China, was tested with both the single-target PCRs and the multiplex PCR assay developed. The positive rates of G. duodenalis, C. parvum, Blastocystis spp., and E. bieneusi were 23.08% (30/130), 24.62% (32/130), 41.54% (54/130), and 12.31% (16/130), respectively. Furthermore, the detection results showed a high prevalence of mixed infections of goat parasites, predominantly involving two parasite species. The findings of the multiplex PCR were consistent with those of single-target PCRs. This multiplex PCR method showed high specificity, sensitivity, accuracy, and cost-effectiveness, and it was suitable for the rapid large-scale screening of goat stool samples for infections caused by G. duodenalis, C. parvum, Blastocystis spp., and E. bieneusi. It thus provided a reliable and efficient tool for animal health management and disease monitoring. Giardia duodenalis, Cryptosporidium parvum, Blastocystis spp. and Enterocytozoon bieneusi are four common zoonotic parasites associated with severe diarrhea and enteric diseases. In this study, we developed a multiplex PCR assay for the simultaneous detection of these four zoonotic protozoans in goat stool samples and assessed its detection efficiency. Specific primers were designed from conserved gene sequences retrieved from GenBank, and the PCR conditions were optimized. Genomic DNA from 130 samples was subjected to both single-target PCR and multiplex PCR. The multiplex PCR assay successfully amplified specific gene fragments (G. duodenalis, 1400 bp; C. parvum, 755 bp; Blastocystis spp., 573 bp; E. bieneusi, 314 bp). The assay sensitivity was ≥102 copies of pathogenic DNA clones with high specificity confirmed by negative results for other intestinal parasites. The detection rates were 23.08% (30/130) for G. duodenalis, 24.62% (32/130) for C. parvum, 41.54% (54/130) for Blastocystis spp., and 12.31% (16/130) for E. bieneusi, matching the single-target PCR results. The sensitivity and predictive values were 100.00%. This multiplex PCR provided a rapid, sensitive, specific, and cost-effective approach for detecting these four parasites. It also provided essential technical support for the rapid detection and epidemiological investigation of G. duodenalis, C. parvum, Blastocystis spp., and E. bieneusi infections in goat fecal samples. [ABSTRACT FROM AUTHOR]

Cases of imported malaria are reported each year in several malaria non-endemic countries, including Kuwait. PCR testing is the ideal method for identification of the infecting Plasmodium spp. The present study documented the epidemiologic characteristics of molecularly confirmed cases of imported malaria in Kuwait during the first year of COVID-19 pandemic. During the period from February 2020 to February 2021, 100 travelers with suspected malaria who had come from malaria-endemic countries of Africa (n = 60) and Asia (n = 40) were examined. Malaria diagnosis was made by microscopy of blood-stained smears and confirmed by a multiplex real-time PCR assay. Samples with discordant species identification results were sequenced. A total of 27 cases (27%) [P. falciparum, 14; P. vivax, 11; P. ovale, 1; mixed P. falciparum and P. malariae, 1] were detected, of whom 12 came to Kuwait for the first time and 15 were returning after visiting their home countries. Most of the returning travelers (12 out of 15 cases, 80%) had not received malaria chemoprophylaxis. Most cases of falciparum malaria (13/15) were Africans while most of the vivax cases (9/11) were Asians. Malaria was more common among subjects entering Kuwait for the first time (OR = 4.025, CI 1.07,15.1) and illiterates (OR = 13.8, CI 1.8,101.4). In conclusion, imported malaria caused mainly by P. falciparum and P. vivax was an ongoing problem during the COVID-19 pandemic. Travel history and education level were significant predictors of malaria among suspected cases. [ABSTRACT FROM AUTHOR]

BACKGROUND: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers. RESULTS: We developed primerJinn, a tool that designs a set of multiplex primers and allows for the in silico PCR evaluation of primer sets against numerous input genomes. We used primerJinn to create a multiplex PCR for the sequencing of drug resistance-conferring gene regions from Mycobacterium tuberculosis, which were then successfully sequenced. CONCLUSIONS: primerJinn provides a user-friendly, efficient, and accurate method for designing multiplex PCR primers for targeted sequencing and performing in silico PCR. It can be used for various applications in molecular biology and bioinformatics research, including the design of assays for amplifying and sequencing drug-resistance-conferring regions in important pathogens.

Abstract Accurate identification of animals and the verification of their parentage can be used to pedigree populations and support selective breeding. The International Society for Animal Genetics recommended 16 cattle STRs for individual identification and parentage testing in cattle, but no multiplex STR typing system contains these 16 STRs. Here, we develop an efficient 17-plex multiplex typing system for cattle that contains the 16 ISAG recommend STRs and a sex-determining marker. Compared to the Bovine Parenting Typing Kit (containing 11 of the 16 ISAG recommend STRs), our new typing system not only increases the number of molecular markers, but also simplifies the PCR operation and shortens the time for the typing procedure (from 4.5 h to 1 h 37 min). Profile can be generated from a single PCR reaction using as little as 1 ng of DNA. The combined probabilities of paternity exclusion CPEduo and CPEtrio were 0.999804697 and 0.999999260, respectively. These results indicate that our 17-plex typing system is a fast, sensitive and species-specific method for the identification of individuals and their parentage for cattle. The application of this system will improve the efficiency of the identification of cattle individuals and their paternity, supporting population genetic research and the selective breeding of cattle.

Abstract Background The resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits. Methods To develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species. Results Both multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test. Conclusions The multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species. Graphical Abstract

Abstract Background Candida species have emerged as a significant cause of opportunistic infections. Alongside the expression of various virulence factors, the rise of antifungal resistance among Candida species presents a considerable clinical challenge. Aim This study aimed to identify different Candida species isolated from clinical specimens, evaluate their antifungal sensitivity patterns, identify key genes regulating virulence mechanisms using multiplex PCR and to assess any correlation between their virulence profiles and antifungal resistance patterns. Method A total of 100 Candida spp. was isolated from 630 different clinical specimens and identified to the species level. Their antifungal susceptibility was phenotypically evaluated in accordance with CLSI guidelines using the Vitek-2 Compact System. Virulence markers, including biofilm formation capacity, protease production, melanin production, coagulase production and hemolysin production, were also phenotypically detected. The genetic determinants for biofilm formation and extracellular hydrolytic enzymes were assessed using a multiplex PCR assay. Results The prevalence of Candida spp. was 15.9%, with C. albicans (48%) and C. glabrata (16%) being the most common. C. albicans showed the highest virulence, with strong biofilm formation, and high proteinase and melanin production. Multiplex PCR revealed Hlp in 22.0%, Hwp in 80.0%, Als in 56.0%, and Sap genes in 56.0% of isolates. Virulence genes were more common in C. albicans than in non-albicans Candida (NAC). Resistance patterns significantly correlated with virulence profiles, with notable associations between flucytosine resistance and the presence of Hlp and Hwp genes. Conclusion The significant correlation between virulent markers such as germination, coagulase, hemolysin production and resistance patterns among different Candida isolates is crucial for predicting the severity and outcomes of Candida infections. This understanding aids in guiding tailored treatment strategies.

Abstract Background Laboratory rats, as model animals, have been extensively used in the fields of life science and medicine. It is crucial to routinely monitor the genetic background of laboratory rats. The conventional approach relies on gel electrophoresis and capillary electrophoresis (CE) technologies. However, the experimental and data analysis procedures for both of these methods are time consuming and costly. Results We established a single-nucleotide polymorphism (SNP) typing scheme using multiplex polymerase chain reaction (PCR) and next-generation sequencing (NGS) to address the genetic background ambiguity in laboratory rats. This methodology involved three rounds of PCR and two rounds of magnetic bead selection to improve the quality of the sequencing data. We simultaneously analysed 100 laboratory rats (including rats of 5 inbred strains and 2 in-house closed colonies), and the sequencing depth varied from an average of 108.25 to 5189.89, with sample uniformity ranging from 82.5 to 97.5%. A total of 98.9% of the amplicons were successfully genotyped (≥ 30 reads). Genetic background analysis revealed that all 38 experimental rats from the 5 inbred strains were successfully identified (without a heterozygous allele). For the 2 in-house closed colonies, the average heterozygosity (0.162 and 0.169) deviated from the typical range of 0.5–0.7, indicating a departure from the ideal heterozygosity level. Additionally, we employed multiplex PCR-CE to validate the NGS-based method, which yielded consistent results for all the rat strains. These results demonstrated that this approach significantly improves efficiency, saves time, reduces costs and ensures accuracy. Conclusion By utilizing NGS technology, our developed method leverages SNP genotyping for genetic background identification in laboratory rats, demonstrating advantages in terms of labour efficiency and cost-effectiveness, thereby rendering it well suited for projects involving extensive sample cohorts.

In addition to the issue of pork contamination, processed meats frequently contain traces of rat meat. Therefore, detection and quantification of the pork and rat DNA in cases of meat and processed meat adulteration are necessary. In the current study, two gene targets of the cytochrome b for pigs and the Mt‐atp6 of Rattus norvegicus for rats were used in the absolute multiplex quantitative real‐time PCR (m‐qPCR). The sample DNA was amplified with a standard as positive control in the various concentration of 1000 pg, 100 pg, 10 pg, 0.1 pg, 0.01 pg, and 0.001 pg. There were 25 processed meat samples and 5 fresh meat samples identified in this study. Among the total of 30 samples assessed, 6 samples were successfully detected and quantified their pork and rat DNA contamination. One sample was contaminated with pork DNA with a concentration of 2.451×10‐4 pg (“Meatball 3). Five samples were contaminated with rat DNA with a concentration of 3.603×10‐11 pg (“Sempol 3”), 2.196×10‐10pg (“Meatball 6”), 4.908×10‐11 pg (“Siomay 3”), 1.489×10‐10 pg (“Grinding 2”), and 3.564×10‐10 pg (“Grinding 4”). In this study, we have discovered that the contamination of pork and rat were detected in the samples. It suggested that this method is applicable for detecting the contaminant in processed meat samples

Hospitalized patients on mechanical ventilation are at high-risk of developing ventilator-associated pneumonia (VAP), making early and accurate diagnosis essential for the best possible treatment outcomes. This review examined various laboratory techniques, such as aerobic cultures, Gram’s stain, and molecular techniques, to assess how well they diagnose VAP. We have done search strategies using Google Scholar, Medline Complete, and PubMed. Extensive statistical tools were utilized to examine studies and evaluate the diagnostic accuracy of laboratory results. Multiplex PCR was superior to Gram’s stain and culture methods in terms of sensitivity (92%) and specificity (86%). On the other hand, Gram’s stain showed the highest specificity (78.9%) and the lowest sensitivity (74.6%). The results of semi-quantitative, quantitative, enrichment culture showed a lower specificity (75.97%) but a moderate sensitivity (78.5%). The most accurate diagnostic approach for VAP, according to a meta-analysis, was multiplex PCR-based testing, closely followed by culture methods. Beyond separate tests, multiplex PCR, culture, and Gram’s stain combination increased sensitivity. Prompt and precise VAP diagnosis is essential for efficient treatment. With possible improvements through combination diagnostic techniques, multiplex PCR remains the most accurate diagnostic tool. However, more investigation is necessary to improve and certify VAP diagnostic instruments.

The giant freshwater prawn, Macrobrachium rosenbergii, is one of the most crucial crustacean species cultured in China. However, in recent years, M. rosenbergii aquaculture in China has encountered several challenges, including low productivity and growth retardation. A decline in genetic variation has been implicated in this phenomenon. This study aims to develop microsatellite multiplex PCR panels to elucidate the genetic diversity and differentiation of cultured M. rosenbergii populations in China. Based on our previous transcriptome sequencing, we developed 24 polymorphic molecular markers. The average number of alleles (Na), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC) were 4.042, 0.535, 0.605, and 0.543, respectively. Two multiplex PCR panels involving six microsatellite loci were obtained by combining different amplicon lengths from these 24 loci. The panels were used to evaluate the genetic structure of six cultured M. rosenbergii populations in China, including one population in Guangxi (GX) and another in Guangdong (GD) province, along with two populations each in Jiangsu (JSA and JSB) and Zhejiang (ZJA and ZJB) provinces. Results revealed that the polymorphism among the six populations ranged from 0.365 to 0.682 with reduced genetic diversity in ZJA and JSB populations (PIC < 0.5). Pairwise FST, Nei's genetic distance, and STRUCTURE analysis revealed significant differentiation among the six cultured populations. ZJA and GD exhibited pronounced genetic distinctiveness from the other populations and were consequently assigned to distinct groups. Populations GX and JSB (FST=0.058, DA=0.045) and populations ZJB and JSA (FST=0.009, DA=0.016) were assigned to two clusters, respectively, suggesting they share similar origins. This study provided polymorphic microsatellite markers and genetic diversity assessment of cultured M. rosenbergii populations, laying a foundation for future breeding programs. [ABSTRACT FROM AUTHOR]

Lumpy skin disease (LSD) is a viral disease that affects cattle and buffaloes in Egypt, causing considerable economic losses in the animal sector. This study aimed to investigate the recent outbreak of LSDV in cattle and buffaloes and evaluate the potential role of the hard tick Rhipicephalus annulatus in their transmission through isolation and molecular characterization by multiplex PCR (mPCR) and real-time quantitative PCR (rt-qPCR) assays. A total of 50 skin biopsies (cattle n = 30, buffaloes n = 20), 110 nasal swabs (cattle n = 76, buffaloes n = 44), and 129 blood samples (cattle n = 84, buffaloes n = 45) were collected. In addition, 145 hard ticks of different stages were collected from cattle and buffaloes of different breeds and ages in different governorates in Egypt from November 2021 to June 2022. Multiplex PCR and real-time quantitative PCR (rt-qPCR) assays based on SYBR Green and targets (P32, VP32, G protein, and viral fusion protein) were used. We identified positive results in 17 out of 30 cattle skin biopsies (56.6%), 1 out of 7 buffalo skin scabs (14.3%), and 5 out of 45 buffalo blood samples (11.11%) using mPCR and RT-qPCR methods. We successfully isolated LSDV from hard ticks and cattle infested with ticks and exhibited characteristic signs of LSD on the chorioallantois membrane (CAM) of specific pathogen-free embryonated chicken eggs (SPF-ECE). The isolates were confirmed by multiplex PCR and RT-qPCR. The cyclic threshold (Ct) with correlation-slandered curve values of rt-qPCR ranging from 10.2 to 36.5 showed the amount of LSDV-DNA in different samples. The study's findings demonstrated the widespread circulation of LSDV in both cattle and buffaloes in Egypt and provided strong evidence that hard ticks R. annulatus play a role in the transmission of LSDV in susceptible animals. [ABSTRACT FROM AUTHOR]

Introduction: Opportunistic fungi create biofilm resistant to active antifungal drugs in immunocompromised people. The present study aimed to assess the expression of CDR1 and MDR1 genes in the stages of biofilm formation by candidate species isolated from clinical samples. Material & Methods: 100 oral, vaginal, and fecal swabs were sampled from people with immune and physiological defects and normal people. The isolates were identified by laboratory tests and specific Candida chrome agar culture medium, and the presence of resistance genes was proved by molecular method. The formation of biofilm in the presence and absence of amphotericin B- in the strains was investigated using the crystal violet test and scanning electron microscope photo. The expression level of CDR1 and MDR1 genes was determined using the real-time polymerase chain reaction (PCR) technique. Results: More than 50% of isolated strains were Candida albicans, and the frequency of other strains was 8.33%. Among the 60 strains that were investigated in terms of genotype, only 48 Candida strains had both CDR1 and MDR1 genes. The statistical analysis of the results demonstrated that the amphotericin B-drug during 30 hours after biofilm formation significantly reduced the expression of resistance genes compared to the control group (P

Bacterial wilt strains in the Ralstonia solanacearum species complex (RSSC) pose serious threats to economically important crops worldwide. In 2014, Safni et al. proposed the reclassification of the RSSC into three genomospecies: R. solanacearum (Rsol), R. pseudosolanacearum (Rpseu), and R. syzygii (Rsyz). The revision requires the proper identification of strains for diagnostic and epidemiological studies. In response, we developed the inexpensive and user-friendly RSSC-Lineage Multiplex PCR, which effectively detects plant-pathogenic Ralstonia strains in general and also distinguishes between Rpseu, Rsol, Rsyz, and the high-security Select Agent “race 3 biovar 2” subgroup of Rsol, also known as the phylotype IIB-1 potato brown rot pandemic lineage. Genomes were retrieved from the NCBI GenBank database and screened for unique gene regions using OrthoMCL and other comparative genomic approaches. Specific primers were designed for each genomospecies, Ralstonia in general, and “race 3 biovar 2.” AT-rich flaps were added at the 5ʹ position of each primer to optimize the reaction thermodynamics. The specificity was tested in silico using the NCBI GenBank genome database and an in-house database. The in vitro specificity and accuracy of the tool was validated with 113 representative Ralstonia strains and 24 strains from other genera. The assay is highly specific, generating neither false positives nor false negatives. Primer set detection limits ranged from 10 to 100 pg. The assay also detected and differentiated strains from naturally and artificially inoculated plant hosts. This tool is highly specific, reliable, and economical for culture characterization, diagnostics, surveys, quarantine decisions, and epidemiological studies. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Abstract Rapid and accurate identification of Salmonella enterica serotypes Typhi and Paratyphi (A, B and C), the causal agents of enteric fever, is critical for timely treatment, case management and evaluation of health policies in low and middle-income countries where the disease still remains a serious public health problem. The present study describes the development of a multiplex assay (EFMAtyping) for simultaneous identification of pathogens causing typhoid and paratyphoid fever in a single reaction by the MeltArray approach, which could be finished within 2.5 h. Seven specific genes were chosen for differentiation of typhoidal and nontyphoidal Salmonella. All gene targets were able to be detected by the EFMAtyping assay, with expected Tm values and without cross-reactivity to other relevant Salmonella serovars. The limit of detection (LOD) for all gene targets was 50 copies per reaction. The LOD reached 102–103 CFU/ml for each pathogen in simulated clinical samples. The largest standard deviation value for mean Tm was below 0.5 °C. This newly developed EFMAtyping assay was further evaluated by testing 551 clinical Salmonella isolates, corroborated in parallel by the traditional Salmonella identification workflow, and serotype prediction was enabled by whole-genome sequencing. Compared to the traditional method, our results exhibited 100% of specificity and greater than 96% of sensitivity with a kappa correlation ranging from 0.96 to 1.00. Thus, the EFMAtyping assay provides a rapid, high throughput, and promising tool for public health laboratories to monitor typhoid and paratyphoid fever.

Abstract The impact of viral keratitis (VK) on individuals and society is notable. Early diagnosis and treatment are crucial in managing viral keratitis effectively. Timely intervention with antiviral medications and supportive care can help mitigate the severity of the infection and improve visual outcomes. We examined the prevalence of varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), adenovirus (AdV) and herpes simplex virus type 2 (HSV-2) in patients suspected for ocular infections. Patients included in the study exhibited various clinical manifestations indicative of ocular pathology, such as infectious keratitis, corneal scar, endogenous endophthalmitis, panuveitis, endothelitis, stromal edema, and other relevant conditions. Four different types of tear fluid, corneal samples epithelium, aqueous humor and vitreous humor were taken. After genome extraction, multiplex real-time PCR was used for diagnosis of viruses. 48 (29.6%) out of the total of 162 (100%) eye specimen were positive. The dominant prevalence was VZV (12.3%) and HSV-1 (11.7%) followed by AdV (4.9%) and HSV-2 (0.6%). There were 4 (8.3%) coinfections within the samples (HSV-1 and VZV). Aqueous humor samples demonstrated superior virus detection ability and our only HSV-2 positive sample was from aqueous humor. The utilization of multiplex real-time PCR assays in differential diagnosis of VK holds promise for expeditious diagnoses while also preventing unwarranted antibiotic prescriptions. Moreover, the aqueous humor appears to be a more sensitive site for detecting viral keratitis.

Abstract Background Antimicrobial stewardship (AMS) for ventilator-associated pneumonia (VAP) or ventilated hospital-acquired pneumonia (vHAP) in extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) carriers is challenging. BioFire® FilmArray® Pneumonia plus Panel (mPCR) can detect bacteria and antibiotic resistance genes, including bla CTX-M, the most common ESBL-encoding gene. Methods This monocentric, prospective study was conducted on a group of ESBL-E carriers from March 2020 to August 2022. The primary objective was to evaluate the concordance between the results of mPCR and conventional culture performed on respiratory samples of ESBL-E carriers to investigate suspected VAP/vHAP. The secondary objective was to appraise the impact of performing or not mPCR on initial antibiotic therapy adequacy in ESBL-E carriers with confirmed VAP/vHAP. Results Over the study period, 294 patients with ESBL-E carriage were admitted to the ICU, of who 168 (57%) were mechanically ventilated. (i) Diagnostic performance of mPCR was evaluated in suspected 41 episodes of VAP/vHAP: bla CTX-M gene was detected in 15/41 (37%) episodes, where 9/15 (60%) were confirmed ESBL-E-induced pneumonia. The culture and bla CTX-M were concordant in 35/41 (85%) episodes, and in all episodes where bla CTX-M was negative (n = 26), the culture never detected ESBL-E. (ii) The impact of mPCR on initial antibiotic therapy adequacy was assessed in 95 episodes of confirmed VAP/vHAP (22 episodes were tested with mPCR and 73 without); 47 (49%) episodes were ESBL-E-induced, and 24 (25%) were carbapenem-resistant bacteria-induced. The use of mPCR was significantly associated with higher prescription of adequate empirical antibiotic therapy in the multivariable logistic regression (adjusted odds ratio (aOR) (95% CI) of 7.5 (2.1–35.9), p = 0.004), propensity-weighting (aOR of 5.9 (1.6–22.1), p = 0.008), and matching-cohort models (aOR of 5.8 (1.5–22.1), p = 0.01). Conclusion mPCR bla CTX-M showed an excellent diagnostic value to rule out the diagnosis of ESBL-E related pneumonia in ESBL-E carriers with suspected VAP/vHAP. In addition, in patients with confirmed VAP/vHAP, a mPCR-based antibiotic therapy was associated with an increased prescription of adequate empirical antibiotic therapy. Performing mPCR on respiratory samples seems to be a promising tool in ESBL-E carriers with suspected vHAP/VAP. However, if mPCR is used in very low pre-test clinical probability of pneumonia, due to the high sensitivity and the rate of overdiagnosed pneumonia, the risk of overconsumption of carbapenem may prevail. Further studies are warranted.

Untreated vaginal discharge is a risk factor for complications. Correct diagnosis is crucial. The purpose of this study was to apply Femoflor-16 to study the vaginal microbiome in women with genital tract discharge. Material/Methods: A total of 45 women were included in the study. Vaginal/cervical swab - one for routine tests and Gram staining and another for PCR Femoflor-16, were tested. Clinical diagnosis of bacterial vaginosis (BV) was done by using Amsel's criteria. Microbiologically, BV was assessed by applying a Nugent score system and Femoflor-16. Results: A total of 45 women with genital tract discharge were included in the study. Based on Amsel's clinical criteria, 9 (20%) of them were diagnosed with BV. Based on Nugent's score system, 11(24,4%) were categorized as having BV. Only 40 samples were compared, and the results were tested using Femoflor-16 and Nugent score system. Of them, 33 (83%) were in agreement as a result of using both methods. Femoflor-16 detected species such as aerobic bacteria (Enterobacteriасeae, Streptococcus spp., Staphylococcus spp.), Mycoplasma spp., Ureaplasma spp. and yeast-like microorganisms (Candida spp.). Conclusions: A reliable, comprehensive, and on-time diagnosis of BV is needed. Nucleic acid amplification tests nowadays complement the "classic" laboratory methods. The real time multiplex PCR test Femoflor-16 can be effectively used in vaginal microbiota evaluation in women with discharge. It can identify a wide range of microorganisms, including bacteria that are difficult to culture, normal Lactobacilli microflora and complex of aerobic and anaerobic microorganisms, mycoplasmas and Candida spp. In addition, it is able to determine the number and ratio of microorganisms in the total bacterial mass, which further orients to the disease state and the role of the microorganism as an opportunistic or obligate pathogen.

ObjectiveTo establish a rapid， accurate， and sensitive multiplex PCR detection method for the simultaneous identification of the six common edible meats （beef， lamp， chicken， pork， goose， duck）， and to evaluate its application value in meat adulteration identification.MethodsBased on complete mitochondrial genomic sequences of six species in the GenBank database， DNA sequences （cattle： 16S rRNA; sheep： COX-1; chickens： Cytb; pig： COX-1; goose： NADH2; duck： 16S rRNA） with intra-species conservation and inter-species specificity were screened， and species-specific primers were designed to construct a multiplex PCR detection system that can simultaneously detect the meat of six common species. The species specificity， sensitivity and reproducibility of the system were studied, and the simulated mixture sample detection was performed.ResultsThis study successfully constructed a multiplex PCR detection system that can detect the meats of six common species simultaneously. The system was not effective in DNA amplification of non-target species. When the DNA template sizes were 0.062 5-2 ng/μL， the amplified products of all six species could be detected. The duck component was still detected when the mixing ratio of duck and beef was as low as 0.5%.ConclusionThis study constructs and establishes a multiplex PCR detection system with strong specificity， high sensitivity， and good reproducibility. It can accurately identify the components of animal origin in common edible meats and provide a simple and practical method for identifying adulteration of common edible meats and meat products in China.

Rice variety differentiation was based on morphological descriptors (DUS) before the advent of genomic and proteomic technology. DNA fingerprinting is becoming an important molecular marker approach because of its wide application in varietal protection, classification, and conflict settlement. In the present study, genetic diversity and variability among the 11 rice varieties of Manipur at the molecular level was studied using 42 SSR markers. The genotypic data of the SSR markers revealed four sub-populations. Cluster analysis indicated the close similarity between RC Maniphou 12 and RC Maniphou 15. To create a DNA fingerprinting database for 11 rice varieties, three separate multiplex PCR systems using different combinations of 11 polymorphic SSR markers based on amplicon size were successfully developed . These were able to distinguish the distinctness of all the eleven released rice varieties.

Ventilator-associated pneumonia (VAP) is a major health care associated infection which usually emanates from aspiration, immigration of pathogens from aerodigestive tract, adulterated appliance uses or medications. The mortality rate due to VAP is approximately 13% and the causative organisms are bacteria, viruses, and fungi. Many studies have investigated the causative organisms as Pseudomonas spp., Acinetobacter spp., Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus with varying prevalence. Intensive Care Unit (ICU) admitted patients who are ventilated, are more prone to the infections where the pathogens adhere to the mucosa of lower respiratory tract of mechanically ventilated patients and start infections. Clinical diagnosis based on Clinical Pulmonary Infection Score (CPIS) has poor specificity and microbiological findings takes 48-72 hrs, that can delay the treatment of patients. Lymphopenia on complete blood count is a predictor of mortality in VAP patients, but decreased lymphocyte count occurs in various other infections too. Multiplex PCR is a better diagnostic technique for VAP which can even diagnose atypical bacteria along with other etiological agents. Effectively employing sampling techniques is a vital step in the diagnosis of VAP, enabling the identification of pathogens responsible for lung infections. Furthermore, the emergence of novel therapeutic options approved by regulatory bodies, adds significant advancements in VAP treatment. In this review article, we have performed an in-depth study on the pathogenesis, diagnosis and therapeutic strategies involved in VAP. This study will help the researchers working in this area to design their work appropriately with the updated knowledge on VAP.

Background. Naked barley is a promising food crop. To enhance its production, active breeding is required to create productive varieties. The purpose of this study was to test the STS-marker for the Nud1 gene controlling the hulled phenotype, and use it for the production of naked barley hybrids. Materials and methods. Genotyping of 112 F2 hybrids obtained by crossing the naked black variety ‘Jet’ and the hulled white variety ‘Elf’ was carried out using wF2 and kR1, or tR2 primers in the regular PCR mode to amplify the recessive or dominant alleles of the Nud1 gene, respectively, and also in the multiplex PCR mode, which allows simultaneous amplification of both dominant and recessive alleles of the Nud1 gene. The genotyping data were compared with those on phenotypes of hybrids. Results and discussion. The possibility of using multiplex PCR with a set of primers wF2, kR1, and tR2 for identifying dominant and recessive alleles of the Nud1 gene in hybrid material has been demonstrated. However, while the observed number of hybrids homozygous for the recessive allele nud1 almost completely corresponded to their expected number, the clear predominance of homozygotes for the dominant allele Nud1 and the lack of heterozygotes compared to the expected number of hybrids of these groups indicates erroneous identification of some heterozygotes as dominant homozygotes, which must be taken into account during selection of hulled barleys by genotyping. Conclusions. The STS-marker amplified by primers wF2, kR1, and tR2, can be used to select recessive homozygotes nud1nud1 from hybrid populations, however, additional analysis is required for a more reliable identification of heterozygotes and homozygotes for the dominant allele of the Nud1 gene.

Rapid and accurate identification of Salmonella enterica serotypes Typhi and Paratyphi (A, B and C), the causal agents of enteric fever, is critical for timely treatment, case management and evaluation of health policies in low and middle-income countries where the disease still remains a serious public health problem. The present study describes the development of a multiplex assay (EFMAtyping) for simultaneous identification of pathogens causing typhoid and paratyphoid fever in a single reaction by the MeltArray approach, which could be finished within 2.5 h. Seven specific genes were chosen for differentiation of typhoidal and nontyphoidal Salmonella. All gene targets were able to be detected by the EFMAtyping assay, with expected Tm values and without cross-reactivity to other relevant Salmonella serovars. The limit of detection (LOD) for all gene targets was 50 copies per reaction. The LOD reached 102–103 CFU/ml for each pathogen in simulated clinical samples. The largest standard deviation value for mean Tm was below 0.5 °C. This newly developed EFMAtyping assay was further evaluated by testing 551 clinical Salmonella isolates, corroborated in parallel by the traditional Salmonella identification workflow, and serotype prediction was enabled by whole-genome sequencing. Compared to the traditional method, our results exhibited 100% of specificity and greater than 96% of sensitivity with a kappa correlation ranging from 0.96 to 1.00. Thus, the EFMAtyping assay provides a rapid, high throughput, and promising tool for public health laboratories to monitor typhoid and paratyphoid fever. [ABSTRACT FROM AUTHOR]

Background: The marine environment harbors high biodiversity; however, it is poorly understood. Nucleotide sequence data of all marine organisms should be accumulated before natural and/or anthropogenic environmental changes jeopardize the marine environment. In this study, we report a cost-effective and easy DNA barcoding method. This method can be readily adopted without using library preparation kits. It includes multiplex PCR of short targets, indexing PCR, and outsourcing to a sequencing service using the NovaSeq system. Methods and results: We targeted four mitochondrial genes [cytochrome c oxidase subunit I (COI), COIII, 16S rRNA (16S), and 12S rRNA (12S)] and three nuclear genes [18S rRNA (18S), 28S rRNA (28S), internal transcribed spacer 2 (ITS2)] in 95 marine invertebrate specimens, which were primarily annelids. The primers, including adapters and indices for NovaSeq sequencing, were newly designed. Two PCR runs were conducted. The 1st PCR amplified specific loci with universal primers and the 2nd added sequencing adapters and indices to the 1st PCR products. The gene sequences obtained from the FASTQ files were subjected to BLAST search and phylogenetic analyses. One run using 95 specimens yielded sequences averaging 2816 bp per specimen for a total length of six loci. Nuclear genes were more successfully assembled compared with mitochondrial genes. A weak but significantly negative correlation was observed between the average length of each locus and success rate of the assembly. Some of the sequences were almost identical to the sequences obtained from specimens collected far from Japan, indicating the presence of potentially invasive species identified for the first time. Conclusions: We obtained gene sequences efficiently using next-generation sequencing rather than Sanger sequencing. Although this method requires further optimization to increase the success rate for some loci, it is used as a first step to select specimens for further analyses by determining the specific loci of the targets. [ABSTRACT FROM AUTHOR]

The impact of viral keratitis (VK) on individuals and society is notable. Early diagnosis and treatment are crucial in managing viral keratitis effectively. Timely intervention with antiviral medications and supportive care can help mitigate the severity of the infection and improve visual outcomes. We examined the prevalence of varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), adenovirus (AdV) and herpes simplex virus type 2 (HSV-2) in patients suspected for ocular infections. Patients included in the study exhibited various clinical manifestations indicative of ocular pathology, such as infectious keratitis, corneal scar, endogenous endophthalmitis, panuveitis, endothelitis, stromal edema, and other relevant conditions. Four different types of tear fluid, corneal samples epithelium, aqueous humor and vitreous humor were taken. After genome extraction, multiplex real-time PCR was used for diagnosis of viruses. 48 (29.6%) out of the total of 162 (100%) eye specimen were positive. The dominant prevalence was VZV (12.3%) and HSV-1 (11.7%) followed by AdV (4.9%) and HSV-2 (0.6%). There were 4 (8.3%) coinfections within the samples (HSV-1 and VZV). Aqueous humor samples demonstrated superior virus detection ability and our only HSV-2 positive sample was from aqueous humor. The utilization of multiplex real-time PCR assays in differential diagnosis of VK holds promise for expeditious diagnoses while also preventing unwarranted antibiotic prescriptions. Moreover, the aqueous humor appears to be a more sensitive site for detecting viral keratitis. [ABSTRACT FROM AUTHOR]

The Pinna nobilis, a Mediterranean mollusc, has suffered population declines due to a massive mortality event associated with various factors including the parasite Haplosporidium pinnae. Some populations show resilience, possibly due to local environmental conditions. In this study, a molecular multiplex PCR method was developed using species‐specific primers targeting Internal Transcribed Spacer (ITS) regions of P. nobilis and P. rudis, allowing accurate species identification and hybrid detection. Samples from Mediterranean areas were analysed, including putative hybrids and individuals from five other bivalve species. DNA was isolated, ITS regions were amplified and sequenced, and phylogenetic analyses confirmed species differentiation and primer specificity. The multiplex‐PCR successfully identified P. nobilis, P. rudis, and their hybrids based on distinct amplicon patterns. This study highlights the value of molecular tools in species conservation, especially for monitoring and managing hybridization, supporting effective biodiversity conservation strategies. [ABSTRACT FROM AUTHOR]