Polymerase Chain Reaction Products Research Articles

Capillary flow velocity-based length identification of PCR and RPA products on paper microfluidic chips.

This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γLV) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R2 values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.

Toxoplasma Gondii In Association with TLR4 Among Regular Hemodialysis Patients

Background: Hemodialysis patients can be considered immunocompromised as they have an increased risk of infections. The activity of Toll -like receptors in defense against infections was observed particularly for Toll like receptor4 molecule. Several single-nucleotide polymorphisms (SNPs) residing in genes encoding Toll -like receptors were reported as significant genetic modifications associated with infections. Aim: To determine the association between Toll- like receptors 4 polymorphisms and the susceptibility to infection with Toxoplasma gondii in hemodialysis patients. Materials and Methods: This case- control study was conducted on 150 patients with hemodialysis and 150 apparently healthy blood donors as control group, the samples were collected in the period from march 2021 to November 2021 from Al-Karama hospital and Al-Yarmouk Teaching Hospital. All patients were investigated for the presence of Toxoplasma gondii and Abs (IgG, IgM) by using ELISA technique and conformation by molecular technique (Real time-PCR). The selected SNPs in Toll- like receptors 4 were amplified by using conventional polymerase chain reaction PCR and then sequencing there polymorphism. Results: In the current study the comparison of median age between patients and controls was with no significant difference, while the laboratory tests revealed significant difference in median level of urea, creatinin , calcium , potassium ,albumin and hemoglobin, and insignificant difference for body mass index and sodium between studied groups. The ratio of females infected with Toxoplasma gondii was higher than infected males. The seropositive result in patients for Toxoplasma gondii - IgG and IgM was 15 and 6 respectively while 19 patients were positive by PCR. However in control group there were 8 patients seropositive for IgG and positive in PCR results while the results of IgM and PCR were negative for all control group. The Polymerase chain reaction (PCR) products of Toll- like receptors 4 genes were subjected for direct sequencing by using Bio edit software. And the resultant sequences were compared with reference sequences in NCBI. The analysis of TLR-4 rs4986790 genotypes were 24(80%) AA and 6(20%) AG in patients while 6 (60%) AA and 4(40%) AG in controls with insignificant difference, while the analysis of TLR-4 rs4986791 genotypes were 24(80%) CC and 6 (20%) CT in patients while 6 (60%) CC and 4(40%) CT in controls with insignificant difference. Conclusion: There was no significant association of Toxoplasma gondii with TLR4 genotypes of rs4986790 and rs4986791 SNPs in hemodialysis patients.

Repeat-mediated recombination results in Complex DNA structure of the mitochondrial genome of Trachelospermum jasminoides

BackgroundTrachelospermum jasminoides has medicinal and ornamental value and is widely distributed in China. Although the chloroplast genome has been documented, the mitochondrial genome has not yet been studied.ResultsThe mitochondrial genome of T. jasminoides was assembled and functionally annotated using Illumina and nanopore reads. The mitochondrial genome comprises a master circular molecular structure of 605,764 bp and encodes 65 genes: 39 protein-coding genes, 23 transfer RNA (tRNA) genes and 3 ribosomal RNA genes. In addition to the single circular conformation, we found many alternative conformations of the T. jasminoides mitochondrial genome mediated by 42 repetitive sequences. Six repetitive sequences (DRS01–DRS06) were supported by nanopore long reads, polymerase chain reaction (PCR) amplifications, and Sanger sequencing of the PCR products. Eleven homologous fragments were identified by comparing the mitochondrial and chloroplast genome sequences, including three complete tRNA genes. Moreover, 531 edited RNA sites were identified in the protein-coding sequences based on RNA sequencing data, with nad4 having the highest number of sites (54).ConclusionTo our knowledge, this is the first description of the mitochondrial genome of T. jasminoides. Our results demonstrate the existence of multiple conformations. These findings lay a foundation for understanding the genetics and evolutionary dynamics of Apocynaceae.

Detection of human strongyloidiasis among patients with a high risk of complications attending selected tertiary care hospitals in Colombo, Sri Lanka using molecular and serological diagnostic tools

BackgroundStrongyloidiasis a neglected tropical disease is known to cause severe disease among immunosuppressed and has not been studied extensively in Sri Lanka. Parasitological diagnostic approaches based on faecal microscopy and culture often fail to detect low-intensity infections. This study investigates the presence of strongyloidiasis among selected immunocompromised individuals using parasitological, molecular and serological techniques.MethodsAdult patients with immunocompromising conditions admitted to three tertiary care hospitals in Sri Lanka were recruited. A faecal sample and 2 ml of venous blood were collected. The faecal samples were subjected to direct faecal smear and cultures (agar plate, charcoal and Harada-Mori) and polymerase chain reaction (PCR) using species specific primers designed for Strongyloides stercoralis. The presence of Strongyloides IgG antibodies was tested in the collected serum samples using DRG Strongyloides IgG enzyme-linked immunosorbent assay (ELISA) kits. The PCR products of the positive samples were sequenced using Sanger sequencing method.ResultsA total of 260 patients were recruited to this study, out of which 160 provided faecal samples and 122 provided blood samples. Out of the 160 faecal samples, none were positive for strongyloidiasis by direct smear, charcoal and Harada-Mori cultures. Only one sample (0.6%) was positive by agar plate culture. Out of the 123 samples subjected to PCR, 14 (11.4%), including the culture positive patient, were positive for S. stercoralis. Sequencing results of the PCR products indicated 100% similarity to S. stercoralis. Out of the 122 serum samples subjected to ELISA, 20 (16.4%), including the culture positive patient, were positive for Strongyloides IgG antibodies. However, sociodemographic, exposure factors, clinical features were not significantly associated with the presence of strongyloidiasis infection.ConclusionsStrongyloidiasis is present among the immunocompromised population in Sri Lanka, even in the absence of a significant relationship with associated factors. It is advisable to screen such patients with highly sensitive tests such as PCR for early diagnosis and treatment.Graphical

Molecular Diagnosis and Typing of Cryptosporidium spp. Species in Human Stools with Diarrhea.

This study was conducted to molecularly identify and classify Cryptosporidium spp. in fecal samples (n=150) from patients with diarrhea received at the microbiology laboratory of a private hospital in Denizli. In this study, the positivity of Cryptosporidium spp. in fecal samples was investigated using direct microscopy, Kinyoun's acid-fast staining method, and Nested polymerase chain reaction (PCR) techniques. Positive PCR products were sequenced. In the examined fecal samples of patients with diarrhea, no parasites were detected through direct microscopic examination. Using the Kinyoun acid-fast staining method, Cryptosporidium spp. was identified in 2.7% (n=4) of the samples, while Nested PCR detected it in 4.67% (n=7) of the samples. The four positive samples were sequenced using primers that amplify the 18S rRNA gene region. The sequencing results identified the isolates as C. parvum. Cryptosporidiosis is an important public health issue as it is a zoonotic disease caused by the Cryptosporidium parasite that can be transmitted from animals to humans. This study focuses on the molecular characterization of Cryptosporidium species detected in human fecal samples, which is significant for understanding which specific strains or species are involved in human infections. According to the findings, it is recommended that control measures be implemented to reduce the risk of exposure to Cryptosporidium in both humans and animals in Türkiye.

Phylogenetic and Genetic Variation Analysis of ITS1 Gene of Trypanosoma lewisi in Wild Rats Using Polymerase Chain Reaction

Murine Trypanosomiasis is a disease caused by the blood protozoan Trypanosoma lewisi in rats, with the transmission process mediated by the flea species Xenopsylla cheopis and Nosopsyllus fasciatus. Cases of trypanosomiasis have been documented due to Trypanosoma lewisi infecting rats and humans in various countries. Diagnosis of T. lewisi is typically conducted using polymerase chain reaction (PCR), which amplifies target DNA using specific primers. One such target gene for detection is the Internal Transcribed Spacer-1 (ITS1). Subsequent sequencing of PCR products enables analysis of genetic variation employing parameters such as nucleotide composition, genetic distance, and phylogenetic analysis with MEGA software. Test results based on percent identity values indicated a 98.51% homology of blood samples with the Chinese strain of T. lewisi (FJ011094.1), demonstrating genetic variation. Phylogram reconstruction revealed that samples 18, 19, and 37 of T. lewisi exhibit very close intraspecies relationships with T. lewisi from NCBI genebank with genetic distance ranging from 0.007 to 0.01. While the closest interspecies relationship was found with T. cruzi (KT305857.1) with a genetic distance of (d = 0.61).

B-208 Room Temperature Stable PCR Reagents Preparation Using Capillary-Mediated Vitrification Process

Abstract Background Polymerase chain reaction (PCR) is a sensitive, and most used molecular technique for the disease diagnosis. Reagents used for PCR is available as a one-step and ready-to-use PCR Master Mix, which contains Taq DNA polymerase, dNTPs, MgC12, PCR reaction buffer. However, to preserve bioactivity, PCR reagents must be stored and distributed as frozen. Desiccation of PCR reagents can avoid cold storage and improper handling of wet reagents and its associated errors. We discovered capillary-mediated vitrification (CMV) processes can stabilize different biomolecules and allow them to be stored at ambient temperature. Here, we hypothesized that the CMV process can preserve and improve PCR reagents thermal stability, which will reduce PCR processing time and improve the testing throughput. Methods The CMV process was performed by mixing PCR Master Mix individual or all reagents such as Taq DNA polymerase or Hot Start Taq DNA polymerase, dNTPs, 16S rRNA primers, MgC12, and PCR reaction buffer with an excipient buffer and adding the mixture to a porous scaffold. The loaded scaffolds were placed into our vitrification chamber and dried for 30 min. After drying, the CMV-stabilized sample was tested or packed in Mylar pouches and were stored at ambient and elevated temperatures for 42 days. On the day of testing, the stabilized samples were eluted with nuclease-free water and E. coli genomic DNA was added, and the 50 uL reaction was tested by conventional PCR. The PCR product was visualized under agarose gel electrophoresis. Results To assess whether the CMV process maintains PCR reagents activity and not interfering with the assay, the individual PCR reagents and as a Master mix are preserved. The CMV stabilized polymerase enzyme, primer, or dNTPs amplified DNA similar to frozen reagents and produced comparable PCR products. Notably, CMV stabilized PCR Master Mix which contains either Taq DNA polymerase or Hot Start Taq DNA polymerase enzyme, dNTPs, primers, MgC12, and reaction buffer yielded intact amplified PCR product, which is comparable to frozen reagents. Likewise, a high intense PCR product was observed in the stabilized PCR Master Mix reagents when stored at room and elevated temperatures for 42 days. Conclusions The study results indicate that the CMV process stabilized and improved the PCR Master Mix reagents activity. The study suggests that the CMV technology has the potential to enable ambient storage and shipping of PCR reagents for molecular diagnostic and other related applications.

Effect of poly(dA) stretch in a template sequence on the rate and yield of polymerase chain reaction with electroactive modified 2′-deoxyuridine-5′-triphosphates

The effective incorporation of electroactive labeled nucleotides into DNA during its enzymatic amplification provides the ground for developing assays based on direct electrochemical detection of amplification products. Yet, whether the template sequence can affect the incorporation efficiency of modified nucleotides is still poorly understood. Here we examined the effects of a poly(dA) stretch in template sequence on the rate and yield of polymerase chain reaction (PCR) in the presence of modified 2′-deoxyuridine-5′-triphosphates (dUTP) in the reaction mixture. Four dUTP derivatives carrying 4-hydroxyphenyl (tyrosine; dUTP-Y1 and dUTP-Y2), 4-nitrophenyl (dUTP-N1), or indolyl (tryptophan; dUTP-W1) aromatic groups were tested as substrates for Taq and KTN (lacking 3′-5′ exonuclease activity) polymerases at 100 % substitution of 2′-deoxythymidine-5′-triphosphate (dTTP) in the PCR mixture. The 120 base pair long synthetic DNA templates contained poly(dA)·poly(dT) tracts of various length (dAn·dTn, where n = 2–10). In all cases, full-size amplicons have been obtained. Overall, in the presence of modified dUTP, the PCR yield was higher with KTN polymerase than that with Taq polymerase. The PCR yield slightly (for dUTP-Y1 and dUTP-Y2) or significantly (for dUTP-N1 and dUTP-W1) decreased with an increase of n in the dAn·dTn tract. In most cases, in the presence of modified dUTP, the PCR rate substantially decreased with n ≥ 4 in dAn·dTn. No changes in amplicon sequences due to potential incorrect nucleotide pairing during amplification with the complete substitution of dTTP with dUTP-Y2 were revealed. Electrochemical activity of tyrosine labeled PCR products dsDNA-Y2 differing in the length of stretches of modified nucleotides was similar. The findings demonstrate that the incorporation efficiency for base-modified nucleotides can in general depend on template sequence, in particular on the presence of long poly(dA) stretches when the modified dUTP are used. That has to be taken into account when developing electrochemical or other DNA assays with a specific modified nucleotide.

Clinical Characteristics and Molecular Insights of Carbapenem-Resistant Klebsiella pneumoniae Isolates from Patients in Intensive Care Units.

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP), a significant worldwide public health threat, is common in patients in intensive care units. Methods: A retrospective study was conducted over a period of 22 months to assess the risk factors associated with infection caused by CRKP isolates. Strain identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antimicrobial sensitivity was assessed using the micro broth dilution method and Kirby-Bauer test. The genes blaKPC, blaOXA-48, blaNDM, blaVIM, and blaGES were amplified using polymerase chain reaction (PCR), followed by sequencing of the PCR products. The polymerase hypermucoviscosity phenotype was determined using the string test. Capsular serotypes (K1, K2) and presence of the virulence gene (rmpA) in positive isolates were investigated using phenotypic tests followed by PCR. Results: Length of hospitalization and use of carbapenems were associated with CRKP infection. CRKP isolates exhibited extensive drug resistance, but retained sensitivity to colistin and ceftazidime-avibactam (CZA). The main gene detected in 35 CRKP isolates was blaKPC-2. In addition, 11 strains were positive in the string test, and two of these strains carried rmpA. Conclusions: Prolonged hospitalization and carbapenem exposure increased the risk of CRKP infection in intensive care unit (ICU) patients. The prevalence of CRKP carrying the blaKPC-2 gene was high, and suspected hypervirulent carbapenem-resistant K. pneumoniae isolates were scattered.

Identification of CDH4 Gene Copy Number Alteration and Its Association with Clinical Profile of Colorectal Cancer Patient

Background: The genes cadherin 4 (CDH4), Kirsten rat sarcoma viral oncogene homolog (KRAS), V-Raf murine sarcoma viral oncogene homolog B (BRAF), and microsatellite instability (MSI) each play a role in the development of colorectal cancer (CRC). CDH4 gene is located in chromosome 20q13.33 and its amplification or gain is the earliest mutational event found in the majority of CRC and colon polyps. This study aimed to identify copy number alteration in the CDH4 gene and its association with the clinical profile of CRC patients, including gender, age, tumor location and differentiation, frequency of BRAF and KRAS mutations, and MSI status. Methods: The DNA was extracted from 50 tumors and adjacent normal tissues based on the manufacturer’s instructions. Detection of MSI status was carried out by pentaplex polymerase chain reaction (PCR) and PCR products were size separated by capillary electrophoresis (CE) using an ABI 310 Genetic Analyzer. Both KRAS and BRAF mutations were identified by PCR and sequencing while CDH4 copy measurement was measured using TaqManTM Copy Number Assay. Results: Our findings showed 22 (44%) samples with no changes in the copy number of CDH4 gene. Interestingly, 21 (42%) cases had an amplification of the CDH4 gene or CDH4 gene gain, and seven (14%) cases decreased in CDH4 gene expression or CDH4 gene loss. We found an association between changes in the CDH4 gene and gender (p=0.001). However, there was no association between changes in the CDH4 gene and age (p=0.979), tumor location (p=0.145) and differentiation (p=0.648), the frequency of BRAF (p=0.171) and KRAS mutations (p=0.184) and MSI status (p=0.923). Conclusions: Copy number alteration in CDH4 gene in CRC patients and this alteration is significantly associated with gender. Further studies with a larger number of samples are essential to confirm this result and to identify the cause of CDH4 copy number alteration and its biological significance.

Molecular Detection of Microsporum Canis Infection in Cats in Baghdad Governorate

Microsporum canis is a keratinized skin fungus that causes diseases in all animals, especially in cats. It is able to infected human causes Tinea corporis and Tinea capitis. In this study,the aim was toward detection and confirmation of Microsporum canis infection in cats in Baghdad governorate using polymerase chain reaction (PCR).The diagnosis depend on sequencing of the PCR products obtained by using a universal primer to amplification of the primer DppV which was 895 bp.A total of two hundred and eighty three hair and skin scraping samples were collected from infected cats with and without skin lesions. Preliminarily examined of samples with 10% KOH preparation, and cultured for fungal identification . For the culture; Saboruad dextrose agar was used . Culture results showed that four isolates were positive for M. canis , the best growth of these isolates was confirmed by PCR and DPPV gene then sequencing of this gene. PCR technique showed that one isolate from the total of 283 isolates were assured for the DPPV gene and yielded a band at 895 bp, this finding is regarded as distinctive for the genus Microsporum canis ,which was dumped in the GenBank nucleotide sequence database under accession number OR126262. Results sequencing of this gene in Iraq revealed compatibility with global results in USA and Belgium.

Spore PCR and qPCR Methods for Rapid Detection of Five Colletotrichum Species Responsible for Pepper Anthracnose in Korea

Pepper anthracnose, caused by <i>Colletotrichum</i> spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of <i>Colletotrichum</i> spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.

Study of the role of polymorphic variants G919A and A2039G of the follicle stimulating hormone receptor gene FSHR in the genesis of male infertility

Aim. To determine the distribution of genotypes of polymorphic variants G919A and A2039G of the gene FSHR (follicle-stimulating hormone receptor) among men with azoospermia. Methods. DNA from peripheral blood leukocytes was isolated and purification using a modified salting out method. Extracted DNA was amplified by Polymerase chain reaction (PCR). The PCR products were subsequently digested with the restriction enzyme for identify polymorphic variants of the follicle-stimulating hormone receptor gene FSHR. Electrophoresis of PCR products was performed in a 2 % agarose gel. Results. Given that idiopathic infertility is overwhelmingly caused by genetic factors, it seemed necessary to conduct a set of cytological and molecular genetic studies in a group of men with azoospermia. The genetic component was verified in 28 men with azoospermia, which is 40 % of all subjects. Molecular genetic studies were performed and the distribution of genotypes of polymorphic variants A919G and A2039G of the FSHR gene among men with azoospermia was determined. Conclusions. A slightly higher frequency of homozygous genotypes GG of polymorphic variants A919G and A2039G of the follicle stimulating hormone receptor gene FSHR was found among men with azoospermia compared to the group of fertile men.

Fall Armyworm Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) in East Nusa Tenggara Province of Indonesia: Genetic Characterization and Strain Detection

<em>Spodoptera frugiperda</em> J.E. Smith (Lepidoptera: Noctuidae) is an invasive pest of corn plants spreading throughout the world, including East Nusa Tenggara (NTT), Indonesia. Despite the wide distribution, there is a lack of information on the strain or genetic diversity of the pest in NTT. Therefore, this study aimed to determine the strain of <em>S. frugiperda</em> from several areas in NTT with a molecular method using <em>cytochrome oxidase subunit 1</em> (<em>COI</em>)<em> </em>and <em>triose phosphate isomerase</em> (<em>Tpi</em>)<em> </em>gene markers. The<em> </em>samples were collected from 3 islands: Timor, Flores, and Sumba. Amplification of the marker genes was carried out using 3 specific primers to identify the strain obtained from samples. Subsequently, polymerase chain reaction (PCR) products were sequenced and the DNA sequences were analyzed using the BioEdit and BLAST programs. Phylogeny analyses were carried out using the MEGA 11 program to verify the strain group of samples with reference isolates from other countries found in GenBank. The PCR results showed product amplicon size 811 bp for the <em>CO1A</em> marker. Based on phylogeny tree analysis using <em>COI </em>marker, <em>S. frugiperda </em>from NTT showed 2 clades, namely corn and rice strains. The characterization results showed that <em>S. frugiperda</em> in NTT comprised 63.6% corn and 36.4% rice strains. <em>COIB </em>classified <em>S. frugiperda</em> from NTT into the h4 haplotype subgroup, while <em>Tpi </em>gene marker was in the corn strain. This study provided valuable information regarding the strain of <em>S. frugiperda</em> in Indonesia to determine the appropriate control strategy.

A systematic survey of environmental DNA in Palau's lakes and waterfalls reveals an increase in Leptospira levels after flooding

ObjectiveLeptospirosis is an important bacterial zoonosis which is widespread in tropical and subtropical islands and influences human and animal health which has secondary economic effects. Although leptospirosis is endemic in Palau, an Oceanian Pacific Island country, few systematic surveys of potential risk factors for Leptospira infection, such as weather and host animals, have been conducted in the natural environment. We used environmental DNA metabarcoding to assess the distribution, species diversity, and abundance of pathogenic Leptospira in this endemic region to investigate the potential environmental risks. MethodsForty-two paired water samples, representing fine and rainy weather conditions, were collected from four representative waterfalls and lakes on Babeldaob Island, the largest island in Palau. High-throughput sequencing analysis was conducted for polymerase chain reaction products of leptospiral 16S rRNA and vertebrate animal mitochondrial 12S rRNA genes. ResultsWe revealed greater Leptospira diversity and abundance in samples collected after continuous rain, particularly in the presence of flooding, compared with samples collected under typhoon, monsoon, or fine weather conditions. From same samples, six mammalian species including cats (Felis catus), mice (Mus musculus), Yap flying fox (Pteropus yapensis), rats (Rattus spp.), and pigs (Sus scrofa) were repeatedly detected. These may be candidates of host animals of Leptospira in Palau; however, their detection was not clearly correlated with that of Leptospira. ConclusionWe repeatedly detected several species of pathogenic Leptospira from water samples of a wide region of Babeldaob Island. We confirmed that Leptospira contamination in freshwater environments increased under rainy conditions, particularly in the presence of flooding. This information could be used to improve public health control measures in this region.

High serological and molecular prevalence of Ehrlichia canis and other vector-borne pathogens in dogs from Boa Vista Island, Cape Verde

Despite the high global impacts of canine vector-borne diseases (CVBD) due to their wide distribution and zoonotic potential, the current epidemiological situation of CVBD in many tropical and subtropical regions remains unknown. This study examines the seroprevalence and molecular prevalence of Ehrlichia canis and other pathogens causing CVBDs (Leishmania infantum, Dirofilaria immitis, Babesia spp., Anaplasma spp. and Hepatozoon canis) in dogs living on the island of Boa Vista (Cape Verde Republic). Blood samples and infesting ticks were taken from 150 dogs across the island (stray, shelter, and pet dogs). Serum samples were tested using a rapid immunochromatographic test (Uranotest® Quattro) that detects antibodies against E. canis, L. infantum, Anaplasma spp. and D. immitis antigen. Levels of serum antibodies against E. canis were measured using the immunofluorescence antibody test (IFAT). In addition, tick-borne pathogens in blood samples (Anaplasma spp., Babesia spp., Hepatozoon spp., and Ehrlichia canis) were detected by microscopy observation and/or PCR plus sequencing. The seroprevalence of E. canis was extremely high at 82% (123/150), as revealed by both immunochromatography and IFAT. Most dogs returning a seropositive test result (82.92%; 102/123) had antibody titres > 1:1280 but showed no clinical signs or notable laboratory abnormalities. Of the 123 animals testing seropositive for E. canis, 67 (54.47%) also presented antibodies against Anaplasma spp., and 13 (10.56%) showed the presence of Hepatozoon spp. gamonts in the blood smear. Ehrlichia canis infection was detected in 17.1% (25/146) of dogs tested by direct sequencing of polymerase chain reaction (PCR) products. Co-infections were detected in seven of these dogs: four dogs tested PCR-positive for both E. canis and A. platys, two dogs tested positive for E. canis and Hepatozoon spp., and one dog tested positive for E. canis, A. platys and Hepatozoon spp. Rhipicephalus sanguineus sensu lato was the only tick species found infesting the canine study population. The high prevalence of tick-borne pathogens detected in dogs from Boa Vista Island highlights a need for improved control measures designed to prevent the transmission of these pathogens.Graphical

Utility of Cerebrospinal Fluid Unstimulated Interferon-Gamma (IRISA-TB) as a Same-Day Test for Tuberculous Meningitis in a Tuberculosis-Endemic, Resource-Poor Setting.

Tuberculous meningitis (TBM) mortality is high and current diagnostics perform suboptimally. We evaluated the diagnostic performance of a DNA-based assay (GeneXpert Ultra) against a new same-day immunodiagnostic assay that detects unstimulated interferon-gamma (IRISA-TB). In a stage 1 evaluation, IRISA-TB was evaluated in biobanked samples from Zambia (n = 82; tuberculosis [TB] and non-TBM), and specificity in a South African biobank (n = 291; non-TBM only). Given encouraging results, a stage 2 evaluation was performed in suspected TBM patients from Zimbabwe and Malawi (n = 668). Patients were classified as having definite, probable or possible TBM, or non-TBM based on their microbiological results, cerebrospinal fluid (CSF) chemistry, and whether they received treatment. In the stage 1 evaluation, sensitivity and specificity of IRISA-TB were 75% and 87% in the Zambian samples, and specificity was 100% in the South African samples. In the stage 2 validation, IRISA-TB sensitivity (95% confidence interval [CI]) was significantly higher than Xpert Ultra (76.2% [55.0%-89.4%] vs 25% [8.9%-53.3%]; P = .0048) when trace readouts were considered negative. Specificity (95% CI) was similar for both assays (91.4% [88.8%-93.4%] vs 86.9% [83.4%-89.8%]). When the Xpert Ultra polymerase chain reaction product was verified by sequencing, the positive predictive value of trace readouts in CSF was 27.8%. Sensitivity of IRISA-TB was higher in human immunodeficiency virus (HIV)-infected versus uninfected participants (85.8% vs 66.7%). As a same-day rule-in test, IRISA-TB had significantly better sensitivity than Xpert Ultra in a TB/HIV-endemic setting. An immunodiagnostic approach to TBM is promising, and further studies are warranted.

Isolation, identification and histopathological observation of Francisella noatunensis subsp. orientalis from Nile tilapia (Oreochromis niloticus)

In April 2023, a substantial mortality event struck the primary Nile tilapia (Oreochromis niloticus) breeding areas in Maoming City, Guangdong Province, resulting in considerable economic losses. To unravel the etiology of this disease outbreak, thorough farm field necropsies were conducted, encompassing detailed descriptions of gross clinical signs and diagnostic sample collection. Clinically, affected tilapia primarily manifested multifocal superficial skin ulcers, mild focal congestion, and hemorrhage in the jaws, eyes' irises, and other integumentary regions. Tissue specimens from diseased fish were screened for Francisella spp., using a polymerase chain reaction (PCR) assay. The PCR product targeting polymerase gene showed 99.64 % nucleotide sequence identity to the published sequence of Francisella noatunensis subsp. orientalis (F. noatunensis subsp. orientalis). Histopathological examination of selected tissues revealed acute to subacute, severe necrotizing bacterial splenitis accompanied by diffuse vasculitis, marked necrotizing nephritis, mild necrotizing hepatitis, and prominent proliferative vasculitis. Transmission electron microscopy (TEM) further demonstrated that F. noatunensis subsp. orientalis was predominantly found in macrophage cytoplasm, appearing as round, spheroidal bacteria. Koch's postulates were successfully confirmed through challenge testing. Moreover, F. noatunensis subsp. orientalis exhibited high sensitivity to Gentamicin, Vancomycin, Minocycline, Imipenem, and Ceftazidime. In conclusion, F. noatunensis subsp. orientalis has emerged as a significant pathogen affecting Nile tilapia in China, characterized by systemic multi-organ necrotizing vasculitis with the spleen and head kidney being the primary targets.

Diagnosis of avian salmonellosis using PCR kits of different manufacturers

Relevance. The problem in timely detection of this infection is that poultry contaminated with salmonellae may not show clinical signs of the disease. Polymerase chain reaction in the diagnosis of avian salmonellosis is designed for direct detection and identification of pathogenic agents, especially in cases where their detection is difficult by other diagnostic methods. Advantages of PCR tests allow to successfully use this method in veterinary laboratory practice. The aim of this work is to conduct comparative tests of diagnostic PCR kits of different manufacturers in the diagnosis of avian salmonellosis.Methods. To conduct comparative studies of pathological material for the presence of the DNA of Salmonella spp., Salmonella enteritidis and Salmonella typhimurium by molecular biological method, three commercial kits of domestic and imported production for isolation and detection of Salmonella DNA by PCR were used. Determination of the possibility of using membrane glass fiber materials, in the form of cards for taking dry samples of pathogenic material, for subsequent PCR was carried out in comparative tests with parallel studies of biomaterial delivered to the laboratory in frozen form.Results. Comparative studies of pathological material for the presence of Salmonella spp. DNA using three commercial kits for the isolation and detection of Salmonella DNA by polymerase chain reaction, imported and domestic production were carried out. It was found that when comparing the results obtained using kits of different manufacturers for the presence of Salmonella spp. DNA, comparable results were observed. The study was performed within the framework of the state assignment No. FGUG-2022-0009 of (FSC VIEV).

Genetic diversity, virulence profiles, and antimicrobial resistance of Salmonella enterica serovar Typhi isolated from typhoid fever patients in Baghdad, Iraq

Typhoid fever is an important health issue in developing countries, and the pathogenicity of Salmonella enterica serovar Typhi (S. ser. Typhi) depends on the presence of different virulence factors. Therefore, this study aimed to investigate the spread of virulence genes among S. Typhi isolates from patients with typhoid fever in Baghdad, Iraq. Sixty S. Typhi isolates were collected from several hospitals in Baghdad and identified using VITEK-II and confirmed by polymerase chain reaction (PCR) to detect the 16S rRNA gene. After testing their susceptibility to different antimicrobials (via the disk diffusion method), we found the highest resistance rates (100 %) were to ampicillin, piperacillin, cefotaxime, and ceftriaxone. The highest sensitivity rates (100 %) were to ertapenem, imipenem, meropenem, and sulfamethoxazole/trimethoprim. The presence of genes encoding for virulence in S. Typhi isolates was tested by conventional PCR. The results showed that out of 60 isolates, 59 (98.3 %), 59 (98.3 %), 58 (96.7 %), and 60 (100 %) were positive for viaB, staA, cdtB, and orfL genes, respectively. The sequencing of PCR products (viaB, staA, cdtB, and orfL genes) was carried out at the Macrogen Company (Seoul, Korea). The sequences were compared with nucleotide sequences in the BLAST GenBank database, and data obtained from the sequencing of these virulence genes were submitted to GenBank under different accession numbers. A phylogenetic analysis of the 16S rRNA gene sequence found a high similarity between local sequences and the closely related sequences of genes in GenBank. The presence of the viaB, staA, cdtB, and orfL virulence genes in nearly all of the isolates under examination suggests that they play an important role in the pathogenicity of local isolates.