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1.
RNA ; 30(6): 739-747, 2024 May 16.
Article in English | MEDLINE | ID: mdl-38471794

ABSTRACT

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.


Subject(s)
Adenosine , RNA, Transfer , RNA, Transfer/genetics , RNA, Transfer/metabolism , Humans , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/genetics , Nucleic Acid Conformation , Real-Time Polymerase Chain Reaction/methods
2.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Article in English | MEDLINE | ID: mdl-35197282

ABSTRACT

Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of Taq DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines Escherichia coli serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies KRAS mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.


Subject(s)
Flap Endonucleases/metabolism , Multiplex Polymerase Chain Reaction/methods , Taq Polymerase/metabolism , Chromosome Deletion , Chromosomes, Human, Y , DNA Primers , Escherichia coli/genetics , Fluorescent Dyes/chemistry , Humans , Limit of Detection
3.
J Infect Dis ; 2024 Sep 09.
Article in English | MEDLINE | ID: mdl-39248312

ABSTRACT

The causes of diarrhea after ten years of rotavirus vaccination in Rwanda were investigated in 496 children with and 298 without diarrhea using a real-time PCR. Rotavirus was detected in 11% of children with diarrhea (OR 2.48, P=0.002). Comparison of population attributable fractions (PAF) show that Shigella (PAF=11%) and ETEC-eltB (PAF=12%) have replaced rotavirus as the main causative agents. The PAF for rotavirus had declined from 41% pre-vaccination to 6.5%, indicating that rotavirus has become one among several similarly important causes of childhood diarrhea in Rwanda. A rotavirus genotype shift to G3P[8] points at the importance of continued genotype surveillance.

4.
J Infect Dis ; 229(Supplement_2): S156-S162, 2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38531075

ABSTRACT

BACKGROUND: In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak. METHODS: Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay. RESULTS: Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb. CONCLUSIONS: The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains.


Subject(s)
Biological Assay , Monkeypox virus , Humans , Disease Outbreaks , Ontario , Real-Time Polymerase Chain Reaction
5.
J Infect Dis ; 2024 Aug 29.
Article in English | MEDLINE | ID: mdl-39206960

ABSTRACT

BACKGROUND: The gradual replacement of the Streptococcus pyogenes M1global genotype by a newly emergent M1UK variant is a global public health threat warranting increased surveillance. M1UK differs from progenitor M1global genotype by 27 single nucleotide polymorphisms (SNPs) and is characterised by increased speA superantigen expression in vitro. METHODS: An allele-specific real-time PCR assay was developed for the rapid detection of M1UK strains. The assay was used in combination with whole-genome sequencing to determine emm (sub)type distribution for 51 invasive (n = 9) and non-invasive (n = 42) S. pyogenes clinical isolates. RESULTS: Emm1 was the most prevalent S. pyogenes emm serotype (n = 11) in this set of clinical isolates, with M1UK being the dominant emm1 genotype (4/5 invasive, 3/6 non-invasive isolates). The assay accurately detected M1UK strains. Whole genome sequencing revealed continued presence of Australian M1UK sub-lineages associated with epidemic scarlet fever-causing S. pyogenes in Asia. CONCLUSIONS: Our study establishes a suitable target for detection of the toxigenic M1UK, and confirms the maintenance of M1UK strains in Queensland, Australia. This assay can be deployed in laboratories and provides a valuable, cost-effective tool to enhance surveillance of the expanding M1UK clone.

6.
BMC Bioinformatics ; 25(1): 17, 2024 Jan 11.
Article in English | MEDLINE | ID: mdl-38212692

ABSTRACT

BACKGROUND: qPCR is a widely used technique in scientific research as a basic tool in gene expression analysis. Classically, the quantitative endpoint of qPCR is the threshold cycle (CT) that ignores differences in amplification efficiency among many other drawbacks. While other methods have been developed to analyze qPCR results, none has statistically proven to perform better than the CT method. Therefore, we aimed to develop a new qPCR analysis method that overcomes the limitations of the CT method. Our f0% [eff naught percent] method depends on a modified flexible sigmoid function to fit the amplification curve with a linear part to subtract the background noise. Then, the initial fluorescence is estimated and reported as a percentage of the predicted maximum fluorescence (f0%). RESULTS: The performance of the new f0% method was compared against the CT method along with another two outstanding methods-LinRegPCR and Cy0. The comparison regarded absolute and relative quantifications and used 20 dilution curves obtained from 7 different datasets that utilize different DNA-binding dyes. In the case of absolute quantification, f0% reduced CV%, variance, and absolute relative error by 1.66, 2.78, and 1.8 folds relative to CT; and by 1.65, 2.61, and 1.71 folds relative to LinRegPCR, respectively. While, regarding relative quantification, f0% reduced CV% by 1.76, 1.55, and 1.25 folds and variance by 3.13, 2.31, and 1.57 folds regarding CT, LinRegPCR, and Cy0, respectively. Finally, f0% reduced the absolute relative error caused by LinRegPCR by 1.83 folds. CONCLUSIONS: We recommend using the f0% method to analyze and report qPCR results based on its reported advantages. Finally, to simplify the usage of the f0% method, it was implemented in a macro-enabled Excel file with a user manual located on https://github.com/Mahmoud0Gamal/F0-perc/releases .

7.
BMC Genomics ; 25(1): 10, 2024 Jan 02.
Article in English | MEDLINE | ID: mdl-38166714

ABSTRACT

BACKGROUND: Plant U-box (PUB) E3 ubiquitin ligases have vital effects on various biological processes. Therefore, a comprehensive and systematic identification of the members of the U-box gene family in potato will help to understand the evolution and function of U-box E3 ubiquitin ligases in plants. RESULTS: This work identified altogether 74 PUBs in the potato (StPUBs) and examined their gene structures, chromosomal distributions, and conserved motifs. There were seventy-four StPUB genes on ten chromosomes with diverse densities. As revealed by phylogenetic analysis on PUBs within potato, Arabidopsis, tomato (Solanum lycopersicum), cabbage (Brassica oleracea), rice (Oryza sativa), and corn (Zea mays), were clustered into eight subclasses (C1-C8). According to synteny analysis, there were 40 orthologous StPUB genes to Arabidopsis, 58 to tomato, 28 to cabbage, 7 to rice, and 8 to corn. In addition, RNA-seq data downloaded from PGSC were utilized to reveal StPUBs' abiotic stress responses and tissue-specific expression in the doubled-monoploid potato (DM). Inaddition, we performed RNA-seq on the 'Atlantic' (drought-sensitive cultivar, DS) and the 'Qingshu NO.9' (drought-tolerant cultivar, DT) in early flowering, full-blooming, along with flower-falling stages to detect genes that might be involved in response to drought stress. Finally, quantitative real-time PCR (qPCR) was carried out to analyze three candidate genes for their expression levels within 100 mM NaCl- and 10% PEG 6000 (w/v)-treated potato plantlets for a 24-h period. Furthermore, we analyzed the drought tolerance of StPUB25 transgenic plants and found that overexpression of StPUB25 significantly increased peroxidase (POD) activity, reduced ROS (reactive oxygen species) and MDA (malondialdehyde) accumulation compared with wild-type (WT) plants, and enhancing drought tolerance of the transgenic plants. CONCLUSION: In this study, three candidate genes related to drought tolerance in potato were excavated, and the function of StPUB25 under drought stress was verified. These results should provide valuable information to understand the potato StPUB gene family and investigate the molecular mechanisms of StPUBs regulating potato drought tolerance.


Subject(s)
Arabidopsis , Solanum tuberosum , Ubiquitin-Protein Ligases/genetics , Solanum tuberosum/genetics , Solanum tuberosum/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Drought Resistance , Phylogeny , Droughts , Ubiquitins/genetics , Stress, Physiological/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism
8.
Emerg Infect Dis ; 30(7): 1344-1351, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38816345

ABSTRACT

The incidence of spotted fever group (SFG) rickettsioses in the United States has tripled since 2010. Rocky Mountain spotted fever, the most severe SFG rickettsiosis, is caused by Rickettsia rickettsii. The lack of species-specific confirmatory testing obfuscates the relative contribution of R. rickettsii and other SFG Rickettsia to this increase. We report a newly recognized rickettsial pathogen, Rickettsia sp. CA6269, as the cause of severe Rocky Mountain spotted fever-like illness in 2 case-patients residing in northern California. Multilocus sequence typing supported the recognition of this pathogen as a novel Rickettsia genotype most closely related to R. rickettsii. Cross-reactivity observed for an established molecular diagnostic test indicated that Rickettsia sp. CA6269 might be misidentified as R. rickettsii. We developed a Rickettsia sp. CA6269-specific real-time PCR to help resolve this diagnostic challenge and better characterize the spectrum of clinical disease and ecologic epidemiology of this pathogen.


Subject(s)
Multilocus Sequence Typing , Phylogeny , Rickettsia , Rocky Mountain Spotted Fever , Humans , California/epidemiology , Rocky Mountain Spotted Fever/diagnosis , Rocky Mountain Spotted Fever/microbiology , Rocky Mountain Spotted Fever/epidemiology , Rickettsia/genetics , Rickettsia/isolation & purification , Rickettsia/classification , Male , Female , Middle Aged , Spotted Fever Group Rickettsiosis/diagnosis , Spotted Fever Group Rickettsiosis/microbiology , Spotted Fever Group Rickettsiosis/epidemiology , Adult , Rickettsia rickettsii/genetics
9.
Curr Issues Mol Biol ; 46(9): 9534-9554, 2024 Aug 29.
Article in English | MEDLINE | ID: mdl-39329918

ABSTRACT

Species delimitation of Culicoides complex species can be challenging. Among species within the Culicoides variipennis complex, C. sonorensis is considered the primary vector of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in North America. Morphological identification of C. sonorensis within the C. variipennis complex is laborious, time-consuming, and requires entomology expertise. Therefore, in this study we developed and validated a multiplex real-time PCR for rapid detection and differentiation of C. sonorensis from the two other main cryptic species (C. variipennis and C. occidentalis) within the C. variipennis complex. The assay targets the EF1α gene and has a built-in internal control targeting 18 S. The specificity and the sensitivity of the multiplex real-time PCR were evaluated using morphologically identified reference and field-collected specimens. The multiplex PCR was 100% specific when nucleic acid extracted from C. variipennis, sonorensis, and occidentalis specimens was tested. When nucleic acid extracted from pools of midges was tested, the multiplex PCR was able to detect all three Culicoides species with comparable sensitivity. The multiplex assay, however, failed to detect eight morphologically identified C. sonorensis specimens collected from Alberta in 2014. The EF1α gene sequences of these specimens formed a distinct phylogenetic cluster, amongst those from C. variipennis, sonorensis, and occidentalis, suggesting that they belong to a different species. We hypothesize that those specimens might be C. albertensis, the only other species remaining in the C. variipennis complex with known geographical distribution in North America. We believe that this highly sensitive and specific multiplex real-time PCR assay could be an effective tool for rapid detection and differentiation of C. sonorensis, the known vector of BTV and EHDV, in trap collections in future vector surveillance programs.

10.
Cancer ; 130(5): 713-726, 2024 03 01.
Article in English | MEDLINE | ID: mdl-37819686

ABSTRACT

BACKGROUND: Philadelphia chromosome (Ph)-like B-acute lymphoblastic leukemia (B-ALL) is a clinically significant, high-risk genetic subtype of B-ALL cases. There are few data on the incidence, characterization, and treatment outcomes of Ph-like ALL cases from low- and middle-income countries. There is a pressing need to establish a well-organized/cost-effective approach for identifying Ph-like ALL instances. METHODS: Multiplex reverse transcriptase polymerase chain reaction, nCounter NanoString, and fluorescence in situ hybridization were used to detect and characterize Ph-like ALL cases among recurrent genetic abnormalities (RGA)neg B-ALL cases. At the end of induction therapy, flow cytometry-minimal residual disease (MRD) assay was used to quantify MRD positivity in Ph-like ALL cases. RESULTS: Of 130 newly diagnosed B-ALL cases, 25% (BCR::ABL1), 4% (ETV6::RUNX1), 5% (TCF3::PBX1), 2% (KM2TA::AFF1), and 65% RGAneg B-ALL cases were revealed by multiplex reverse transcriptase polymerase chain reaction. Among RGAneg B-ALL cases, 24% Ph-like ALL cases using nCounter NanoString were identified, with 48% CRLF2high cases with 45% CRLF2::P2RY8 and 18% CRLF2::IGH rearrangements(∼r) revealed by fluorescence in situ hybridization. In 52% of CRLF2low cases, 17% ABL1 and JAK2∼r 8% EPOR::IGH & PDGRFB∼r were identified. Ph-like ALL cases had higher total leukocyte count (p < .05), male preponderance (p < .05), and high MRD-positivity/induction failure compared with RGAneg B-ALL cases. Furthermore, in Ph-like ALL cases, 11 significant genes using quantitative polymerase chain reaction were identified and validated. CRLF2, IGJ, CEACAM6, MUC4, SPATS2L and NRXN3 genes were overexpressed and show statistical significance (p < .05) in Ph-like ALL cases. CONCLUSIONS: This study showed the high incidence of Ph-like ALL cases with kinase activating alterations and treatment outcomes from low- and middle-income region. Furthermore, a surrogate cost-effective multiplex panel of 11 overexpressed genes for the prompt detection of Ph-like ALL cases is proposed. PLAIN LANGUAGE SUMMARY: Identification of recurrent gene abnormalities (RGA)neg B-acute lymphoblastic leukemia (B-ALL) cases using multiplex-reverse transcriptase polymerase chain reaction. Identification and characterization of Philadelphia (Ph)-like ALL cases using nCounter NanoString gene expression profiling and fluorescence in situ hybridization. Furthermore, Ph-like ALL cases were characterized according to CRLF2 expression and kinase-activating genomic alterations. Minimal residual disease of Ph-like ALL cases were quantified using flow cytometry-minimal residual disease assay. A surrogate molecular approach was established to detect Ph-like ALL cases from low- and middle-income countries.


Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Male , Philadelphia Chromosome , In Situ Hybridization, Fluorescence , Neoplasm, Residual , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Acute Disease
11.
Article in English | MEDLINE | ID: mdl-39080120

ABSTRACT

INTRODUCTION: Homologous recombination (HR) is a crucial DNA-repair mechanism, and its disruption can lead to the accumulation of mutations that initiate and promote cancer formation. The key HR genes, BRCA1 and BRCA2, are particularly significant as their germline pathogenic variants are associated with a hereditary predisposition to breast and/or ovarian cancer. MATERIALS AND METHODS: The study was performed on 45 FFPE breast cancer tissues obtained from 24 and 21 patients, with and without the germline BRCA1/2 mutation, respectively. The expression of 11 genes: BRCA1, BRCA2, ATM, BARD1, FANCA, FANCB, FANCI, RAD50, RAD51D, BRIP1, and CHEK2 was assessed using Custom RT2 PCR Array (Qiagen), and results were analysed using R. RESULTS: Cancer tissues from patients with BRCA1 or BRCA2 germline mutations displayed no significant differences in the expression of the selected HR genes compared to BRCA1 or BRCA2 wild-type cancer tissues. In BRCA1mut cancer tissues, BRCA1 expression was significantly higher than in BRCA2mut and BRCA wild-type cancer tissues. CONCLUSIONS: In cancer tissues harbouring either BRCA1 or BRCA2 germline mutations, no significant differences in expression were observed at the mRNA level of any tested HR genes, except BRCA1. However, the significant differences observed in BRCA1 expression between germline BRCA1mut, germline BRCA2mut and BRCA1/2wt tissues may indicate a compensatory mechanism at the mRNA level to mitigate the loss of BRCA1 function in the cells.

12.
J Clin Microbiol ; 62(5): e0002824, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38639489

ABSTRACT

The mpox outbreak, caused by monkeypox virus (MPXV), accelerated the development of molecular diagnostics. In this study, we detail the evaluation of the Research Use Only (RUO) NeuMoDx MPXV assay by multiple European and US sites. The assay was designed and developed by Qiagen for the NeuMoDx Molecular Systems. Primers and probes were tested for specificity and inclusivity in silico. The analytical sensitivity of the assay was determined by testing dilutions of synthetic and genomic MPXV DNA. A total of 296 clinical samples were tested by three sites; the Johns Hopkins University (US), UZ Gent (Belgium, Europe), and Hospital Universitario San Cecilio (Spain, Europe). The analytical sensitivity of the assay was 50 copies/mL for both clades I and II. The assay showed 100% in silico identity for 80 clade I and 99.98% in silico identity for 5,162 clade II genomes. Clade II primers and probes showed 100% in silico specificity; however, identity of at least one of the two sets of clade I primers and probes with variola, cowpox, camelpox, and vaccinia viruses was noticed. The clinical validation showed sensitivity of 99.21% [95% confidence interval (CI): 95.66-99.98%] and specificity of 96.64% (95% CI: 91.62-99.08%) for lesion swab samples. The NeuMoDx MPXV Test shows acceptable analytical and clinical performance. The assay improves the laboratory's workflow as it consolidates nucleic acid extraction, PCR, data analysis, and interpretation and can be interfaced. The Test Strip can differentiate clades I and II, which has important laboratory safety implications. IMPORTANCE: In this manuscript, we provide detailed in silico analysis and clinical evaluation of the assay using a large cohort of clinical samples across three academic centers in Europe and the United States. Because the assay differentiates MPXV clades I and II, this manuscript is timely due to the current need to rule out the regulated clade I by diagnostic clinical laboratories. In December 2023, and due to first report of cases of sexually transmitted clade I infections in the Democratic Republic of the Congo, when generic assays that do not differentiate the clades are used, samples are considered regulated. The assay meets the need of full automation and has a marked positive impact on the laboratory workflow.


Subject(s)
Molecular Diagnostic Techniques , Monkeypox virus , Mpox (monkeypox) , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Humans , Monkeypox virus/genetics , Monkeypox virus/isolation & purification , Monkeypox virus/classification , Real-Time Polymerase Chain Reaction/methods , Mpox (monkeypox)/diagnosis , Mpox (monkeypox)/virology , Molecular Diagnostic Techniques/methods , Europe , United States , Automation, Laboratory/methods , DNA Primers/genetics , Belgium
13.
J Clin Microbiol ; 62(6): e0010424, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38661386

ABSTRACT

Leishmaniasis is a vector-borne disease caused by many Leishmania spp. which infect humans and other mammalian hosts. Leishmania infantum is the main agent of canine leishmaniasis (CanL) whose diagnosis is usually confirmed by serological and molecular tests. This study aimed to evaluate the clinical and analytical sensitivities of a lab-on-chip (LOC) real-time PCR applied on the portable Q3-Plus V2 platform (Q3 qPCR) in the detection of L. infantum. The Q3 qPCR performance was assessed by comparing the quantification cycle (Cq) values with those obtained using the qPCR run on a CFX96 Real-Time System (CFX96 qPCR). A total of 173 DNA samples (extracted from bone marrow, lymph node, blood, buffy coat, conjunctival swab, and skin) as well as 93 non-extracted samples (NES) (bone marrow, lymph node, blood, and buffy coat) collected from dogs were tested with both systems. Serial dilutions of each representative DNA and NES sample were used to assess the analytical sensitivity of the Q3 qPCR system. Overlapping Cq values were obtained with the Q3 qPCR and CFX96 qPCR, both using DNA extracted from L. infantum promastigotes (limit of detection, <1 promastigote per milliliter) and from biological samples as well as with NES. However, the Q3 qPCR system showed a higher sensitivity in detecting L. infantum in NES as compared with the CFX96 qPCR. Our data indicate that the Q3 qPCR system could be a reliable tool for detecting L. infantum DNA in biological samples, bypassing the DNA extraction step, which represents an advance in the point-of-care diagnostic of CanL.


Subject(s)
Dog Diseases , Leishmania infantum , Leishmaniasis, Visceral , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Dogs , Animals , Leishmania infantum/genetics , Leishmania infantum/isolation & purification , Real-Time Polymerase Chain Reaction/methods , Dog Diseases/diagnosis , Dog Diseases/parasitology , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/veterinary , Leishmaniasis, Visceral/parasitology , Lab-On-A-Chip Devices , Molecular Diagnostic Techniques/methods , DNA, Protozoan/genetics
14.
BMC Microbiol ; 24(1): 223, 2024 Jun 26.
Article in English | MEDLINE | ID: mdl-38926815

ABSTRACT

BACKGROUND: Enterocytozoon bieneusi is the most common species found in humans. Although E. bieneusi has been investigated in humans, genotype profile of E. bieneusi is not known in Türkiye. METHODS: In this study, we screened E. bieneusi in patients (n = 94) with different types of malignant solid tumors by Real Time PCR and then sequenced E. bieneusi positive samples. All cancer patients were undergoing chemotherapy and had diarrhea. Moreover, as control groups, we also screened E. bieneusi in patients with diarrhea (n = 50) and without diarrhea (n = 50). RESULTS: Among all patients analyzed, 33 (17%) were found to be E. bieneusi-positive. As the patients were categorized, the molecular prevalence of E. bieneusi increased to 25.5% among cancer patients with diarrhea. However, the molecular prevalence of E. bieneusi was found to be lower in patients with presenting only diarrhea (8%) and patients without diarrhea (10%). The high molecular prevalence value detected among cancer patients with diarrhea was also statistically significant compared to other patient groups (P = 0.00112 and P = 0.0269). Among the 33 Real Time PCR positive samples, 10 of them were amplified by nested PCR and among these 10 samples, 6 of them were successfully genotyped. The phylogenetic tree showed the presence of D and Type IV which were also identified in stray cats living in Izmir in our previous study. CONCLUSIONS: High molecular prevalence value indicates the importance of screening stool samples of cancer patients with diarrhea for E. bieneusi and genotyping results indicate that D and Type IV are circulating between humans and cats.


Subject(s)
Diarrhea , Enterocytozoon , Genotype , Microsporidiosis , Neoplasms , Humans , Enterocytozoon/genetics , Enterocytozoon/isolation & purification , Microsporidiosis/microbiology , Microsporidiosis/epidemiology , Microsporidiosis/veterinary , Neoplasms/complications , Neoplasms/drug therapy , Male , Female , Diarrhea/microbiology , Diarrhea/epidemiology , Middle Aged , Prevalence , Adult , Aged , Real-Time Polymerase Chain Reaction , Young Adult , Phylogeny , Sequence Analysis, DNA , Antineoplastic Agents , DNA, Fungal/genetics , Aged, 80 and over , Feces/microbiology
15.
BMC Microbiol ; 24(1): 117, 2024 Apr 04.
Article in English | MEDLINE | ID: mdl-38575882

ABSTRACT

BACKGROUND: Leishmaniasis as a neglected tropical disease (NTD) is caused by the inoculation of Leishmania parasites via the bite of phlebotomine sand flies. After an infected bite, a series of innate and adaptive immune responses occurs, among which neutrophils can be mentioned as the initiators. Among the multiple functions of these fighting cells, neutrophil extracellular traps (NETs) were studied in the presence of Leishmania major promastigotes and salivary gland homogenates (SGH) of Phlebotomus papatasi alone, and in combination to mimic natural conditions of transmission. MATERIAL & METHODS: The effect of L. major and SGH on NETs formation was studied in three different groups: neutrophils + SGH (NS), neutrophils + L. major (NL), neutrophils + L. major + SGH (NLS) along with negative and positive controls in 2, 4 and 6 h post-incubation. Different microscopic methods were used to visualize NETs comprising: fluorescence microscopy by Acridine Orange/ Ethidium Bromide staining, optical microscopy by Giemsa staining and scanning electron microscopy. In addition, the expression level of three different genes NE, MPO and MMP9 was evaluated by Real-Time PCR. RESULTS: All three microscopical methods revealed similar results, as in NS group, chromatin extrusion as a sign of NETosis, was not very evident in each three time points; but, in NL and especially NLS group, more NETosis was observed and the interaction between neutrophils and promastigotes in NL and also with saliva in NLS group, gradually increased over times. Real-time reveals that, the expression of MPO, NE and MMP9 genes increased during 2 and 4 h after exposure, and then decreased at 6 h in most groups. CONCLUSION: Hence, it was determined that the simultaneous presence of parasite and saliva in NLS group has a greater impact on the formation of NETs compared to NL and NS groups.


Subject(s)
Extracellular Traps , Leishmania major , Phlebotomus , Animals , Humans , Phlebotomus/genetics , Phlebotomus/parasitology , Matrix Metalloproteinase 9 , Neutrophils , Salivary Glands
16.
BMC Microbiol ; 24(1): 383, 2024 Oct 01.
Article in English | MEDLINE | ID: mdl-39354378

ABSTRACT

BACKGROUND: The incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains. RESULTS: Fluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman's correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models. CONCLUSIONS: The fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance.


Subject(s)
Antifungal Agents , Biofilms , Candida , Candidiasis , Drug Repositioning , Drug Resistance, Fungal , Fluconazole , Microbial Sensitivity Tests , Urinary Tract Infections , Fluconazole/pharmacology , Egypt , Humans , Drug Resistance, Fungal/genetics , Antifungal Agents/pharmacology , Candida/drug effects , Candida/genetics , Candida/isolation & purification , Candida/classification , Candidiasis/microbiology , Candidiasis/drug therapy , Candidiasis/urine , Urinary Tract Infections/microbiology , Urinary Tract Infections/drug therapy , Animals , Biofilms/drug effects , Biofilms/growth & development , Mice , Virulence/genetics , Virulence/drug effects , Female , Male , Phospholipases/genetics , Phospholipases/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism
17.
J Med Virol ; 96(3): e29564, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38506145

ABSTRACT

Cytomegalovirus (CMV) DNA in plasma is mainly unprotected and highly fragmented. The size of the amplicon largely explains the variation in CMV DNA loads quantified across PCR platforms. In this proof-of-concept study, we assessed whether the CMV DNA fragmentation profile may vary across allogeneic hematopoietic stem cell transplant recipients (allo-SCT), within the same patient over time, or is affected by letermovir (LMV) use. A total of 52 plasma specimens from 14 nonconsecutive allo-SCT recipients were included. The RealTime CMV PCR (Abbott Molecular), was used to monitor CMV DNA load in plasma, and fragmentation was assessed with a laboratory-designed PCR generating overlapping amplicons (around 90-110 bp) within the CMV UL34, UL80.5, and UL54 genes. Intrapatient, inter-patient, and LMV-associated qualitative and quantitative variations in seven amplicons were observed. These variations were seemingly unrelated to the CMV DNA loads measured by the Abbott PCR assay. CMV DNA loads quantified by UL34_4, UL54.5, and UL80.5_1 PCR assays discriminate between LMV and non-LMV patients. Our observations may have relevant implications in the management of active CMV infection in allo-SCT recipients, either treated or not with LMV, although the data need further validation.


Subject(s)
Acetates , Cytomegalovirus Infections , Hematopoietic Stem Cell Transplantation , Quinazolines , Humans , Cytomegalovirus/genetics , DNA Fragmentation , Hematopoietic Stem Cell Transplantation/adverse effects , Cytomegalovirus Infections/drug therapy , Transplant Recipients , DNA, Viral , Antiviral Agents/therapeutic use , Viral Proteins/genetics
18.
Electrophoresis ; 2024 Sep 17.
Article in English | MEDLINE | ID: mdl-39287077

ABSTRACT

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1 ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG + GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

19.
Microb Pathog ; 191: 106646, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38631414

ABSTRACT

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.


Subject(s)
Porcine epidemic diarrhea virus , Real-Time Polymerase Chain Reaction , Rotavirus , Sensitivity and Specificity , Swine Diseases , Transmissible gastroenteritis virus , Animals , Swine , Real-Time Polymerase Chain Reaction/methods , Transmissible gastroenteritis virus/genetics , Transmissible gastroenteritis virus/isolation & purification , Porcine epidemic diarrhea virus/genetics , Porcine epidemic diarrhea virus/isolation & purification , Porcine epidemic diarrhea virus/classification , Swine Diseases/virology , Swine Diseases/diagnosis , Rotavirus/genetics , Rotavirus/isolation & purification , Rotavirus/classification , Gastroenteritis, Transmissible, of Swine/diagnosis , Gastroenteritis, Transmissible, of Swine/virology , Deltacoronavirus/genetics , Deltacoronavirus/isolation & purification , Diarrhea/virology , Diarrhea/veterinary , Diarrhea/diagnosis , Coronavirus/genetics , Coronavirus/isolation & purification , Coronavirus/classification , Feces/virology , Coronavirus Infections/diagnosis , Coronavirus Infections/veterinary , Coronavirus Infections/virology
20.
Anal Biochem ; 696: 115688, 2024 Oct 16.
Article in English | MEDLINE | ID: mdl-39419197

ABSTRACT

Measuring the activity of proteases is essential for investigating both the physiological functions and commercial applications of these enzymes. In contrast to the numerous protease assays that are based on chromogenic or fluorogenic peptide substrates, there is a lack of approaches to monitor degradation of proteins in real time. Here we report a protease assay where SYPRO Orange is employed as a fluorogenic probe to follow proteolysis. The functionality of the assay was demonstrated with the two subtilases of varying thermostability, using four different protein substrates. The assay is compatible with a real-time PCR instrument which allows continuous fluorescence measurements in low-volume samples even at high temperatures. This makes the assay especially suitable for high-throughput characterization of thermostable proteases.

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