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Diagnostic Microbiology and Infectious... Jun 2024A 61-year-old male with subacute headache was found to have cryptococcal meningitis despite a negative BioFire FilmArray meningitis/encephalitis panel. This case...
A 61-year-old male with subacute headache was found to have cryptococcal meningitis despite a negative BioFire FilmArray meningitis/encephalitis panel. This case underscores the importance of liberal cryptococcal antigen testing, and that a negative FilmArray panel is inadequate in excluding cryptococcal meningitis, particularly in a HIV-negative host.
Topics: Humans; Meningitis, Cryptococcal; Male; Middle Aged; Polymerase Chain Reaction; Cryptococcus neoformans
PubMed: 38492489
DOI: 10.1016/j.diagmicrobio.2024.116251 -
Archives of Razi Institute Aug 2023is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular...
is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of and using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as . The PCR-RFLP assay demonstrated a product for with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for . The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for ; moreover, it is a suitable method for differentiating between and . This technique can detect in a short time with high precision and sensitivity.
Topics: Horses; Animals; Guinea Pigs; Burkholderia mallei; Glanders; Polymorphism, Restriction Fragment Length; Glycerol; Burkholderia pseudomallei; Polymerase Chain Reaction; Horse Diseases
PubMed: 38226390
DOI: 10.32592/ARI.2023.78.4.1305 -
Nature Communications Aug 2023DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases....
DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.
Topics: Polymerase Chain Reaction; DNA; DNA Methylation; Temperature; Oligonucleotides; CpG Islands
PubMed: 37620381
DOI: 10.1038/s41467-023-40873-y -
Biotechnology and Bioengineering Dec 2022Lambda-polymerase chain reaction (λ-PCR) is a novel and open-source method for DNA assembly and cloning projects. λ-PCR uses overlap extension to ultimately assemble...
Lambda-polymerase chain reaction (λ-PCR) is a novel and open-source method for DNA assembly and cloning projects. λ-PCR uses overlap extension to ultimately assemble linear and circular DNA fragments, but it allows the single-stranded DNA (ssDNA) primers of the PCR extension to first exist as double-stranded DNA (dsDNA). Having dsDNA at this step is advantageous for the stability of large insertion products, to avoid inhibitory secondary structures during direct synthesis, and to reduce costs. Three variations of λ-PCR were created to convert an initial dsDNA product into an ssDNA "megaprimer" to be used in overlap extension: (i) complete digestion by λ-exonuclease, (ii) asymmetric PCR, and (iii) partial digestion by λ-exonuclease. Four case studies are presented that demonstrate the use of λ-PCR in simple gene cloning, simultaneous multipart assemblies, gene cloning not achievable with commercial kits, and the use of thermodynamic simulations to guide λ-PCR assembly strategies. High DNA assembly and cloning efficiencies have been achieved with λ-PCR for a fraction of the cost and time associated with conventional methods and some commercial kits.
Topics: Polymerase Chain Reaction; DNA; Cloning, Molecular; Nucleic Acid Amplification Techniques; DNA, Single-Stranded; Exonucleases
PubMed: 36148504
DOI: 10.1002/bit.28240 -
Biosensors Apr 2024RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation.... (Review)
Review
RNA is an important information and functional molecule. It can respond to the regulation of life processes and is also a key molecule in gene expression and regulation. Therefore, RNA detection technology has been widely used in many fields, especially in disease diagnosis, medical research, genetic engineering and other fields. However, the current RT-qPCR for RNA detection is complex, costly and requires the support of professional technicians, resulting in it not having great potential for rapid application in the field. PCR-free techniques are the most attractive alternative. They are a low-cost, simple operation method and do not require the support of large instruments, providing a new concept for the development of new RNA detection methods. This article reviews current PCR-free methods, overviews reported RNA biosensors based on electrochemistry, SPR, microfluidics, nanomaterials and CRISPR, and discusses their challenges and future research prospects in RNA detection.
Topics: Biosensing Techniques; RNA; Humans; Electrochemical Techniques; Polymerase Chain Reaction; Nanostructures; Surface Plasmon Resonance; Microfluidics
PubMed: 38667193
DOI: 10.3390/bios14040200 -
Clinical Microbiology and Infection :... Apr 2020Mobile microbiology is an evolving concept that has the potential to reduce morbidity and mortality associated with infectious diseases on a global level. Molecular... (Review)
Review
BACKGROUND
Mobile microbiology is an evolving concept that has the potential to reduce morbidity and mortality associated with infectious diseases on a global level. Molecular methods used in the context of mobile microbiology ensure rapid and accurate aetiological diagnostics and allow timely initiation of clinical care. The great majority of published data regarding molecular diagnostics in mobile laboratories have focused on emerging viral infections and using laboratory-developed assays. Use of clinically validated and commercially available molecular diagnostic instruments in routine diagnostics of infectious diseases in mobile laboratories has received only limited attention in the field.
OBJECTIVES
This review summarizes the suitability of a range of portable diagnostic molecular instruments for application in mobile laboratories by taking into account the instruments' analytical concepts, technical features and environmental requirements, as well as results of major validation studies.
SOURCES
Data on technical features of selected portable instruments were mainly extracted from manufacturers' websites. Information on validation studies of various molecular assays developed for the selected instruments was extracted from peer-reviewed publications searched for through PubMed.
CONTENT
Eight portable diagnostic molecular instruments (Alere q, GeneXpert Edge, GeneXpert Omni, Genedrive, PanNAT, Revogene, cobas Liat and ID Now) that are commercially available or in the launching stage are presented and evaluated in the context of the mobile microbiology concept, with particular emphasis on technical features and environmental requirements. Both the cobas Liat and the Alere i assays have been extensively validated in a variety of studies carried out in both adult and paediatric patients from various settings (ranging from primary care to emergency care departments in tertiary centres). Most studies showed comparable performance of cobas Liat and Alere i molecular assays with the standard-of-care in vitro diagnostics molecular assays routinely performed in dedicated/centralized molecular diagnostics laboratories. In addition, acceptable performance of Alere q and Genedrive instruments has been shown in implementations studies for early infant diagnosis of children born to human immunodeficiency virus-positive mothers and detection of hepatitis C virus RNA, respectively. Additional validation studies on existing (GeneXpert Edge, PanNAT, Revogene) and emerging (GeneXpert Omni) technologies are warranted.
IMPLICATIONS
Several portable molecular diagnostic platforms reviewed are suitable for mobile microbiology applications. Further development in this field should be directed toward providing a broader range of assays per instrument, multiplexing, reducing the frequency of invalid results, and price cutting.
Topics: Humans; Laboratories; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Point-of-Care Systems; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 31574340
DOI: 10.1016/j.cmi.2019.09.017 -
Malaria Journal Jul 2020Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease...
BACKGROUND
Plasmodium knowlesi and Plasmodium vivax are the predominant Plasmodium species that cause malaria in Malaysia and play a role in asymptomatic malaria disease transmission in Malaysia. The diagnostic tools available to diagnose malaria, such as microscopy and rapid diagnostic test (RDT), are less sensitive at detecting lower parasite density. Droplet digital polymerase chain reaction (ddPCR), which has been shown to have higher sensitivity at diagnosing malaria, allows direct quantification without the need for a standard curve. The aim of this study is to develop and use a duplex ddPCR assay for the detection of P. knowlesi and P. vivax, and compare this method to nested PCR and qPCR.
METHODS
The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.
RESULTS
The duplex ddPCR assay had higher analytical sensitivity (P. vivax = 10 copies/µL and P. knowlesi = 0.01 copies/µL) compared to qPCR (P. vivax = 100 copies/µL and P. knowlesi = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity (P. vivax = 80% and P. knowlesi = 90%) and clinical specificity (P. vivax = 87.84% and P. knowlesi = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.
CONCLUSIONS
Overall, the ddPCR assay demonstrated acceptable efficiency in detection of P. knowlesi and P. vivax, and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay's clinical sensitivity and specificity.
Topics: Diagnostic Tests, Routine; Humans; Malaysia; Plasmodium knowlesi; Plasmodium vivax; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 32650774
DOI: 10.1186/s12936-020-03314-5 -
Archives of Razi Institute Jul 2021Authentication of animal cell lines in cell banks is one of the most important programs regulated during cell culture and storage. This operation provides a thorough and...
Authentication of animal cell lines in cell banks is one of the most important programs regulated during cell culture and storage. This operation provides a thorough and beneficial document which can be advantageous for the functional use of animal cell lines. Therefore, various procedures are used to prevent misidentified cells, cross-contamination to other cell lines, and mislabeling errors leading to incorrect assessment. These contaminants can result in major financial disadvantages. One of the practical methods in this field is a molecular procedure which can demonstrate more accurate results. In the present study, the BHK-21 (C5) was characterized, and it was tried to determine the identity of BHK-21 (C5) as a continuous cell line by Polymerase chain reaction (PCR) molecular procedure in Iran. The cytochrome c oxidase I (CO1) gene was selected as a prevalent DNA fragment for the authentication of the BHK-21 (C5) cell line, along with six cell lines, including Chinese hamster ovary, Lamb kidney, Razi Bovine Kidney, Medical Research Council cell strain 5, Monkey Green Kidney, and Goat Lymphocyte. After amplification, PCR products were analyzed by agarose gel electrophoresis to ensure their accuracy. The results of characterization were indicated, cell viability was estimated to be about 92%, and a uniform cell culture was obtained. The doubling time and µ ratio equivalent were obtained at 20.5 h and 0.03, respectively. Sterility tests revealed that the cell seed was free of bacterial, mycoplasma, and mycobacterial infections. The results of molecular identification revealed that the identification of this cell line was approved and can be used in studies, diagnosis, production, and quality control of biological products.
Topics: Animals; CHO Cells; Cattle; Cricetinae; Cricetulus; DNA Primers; Iran; Polymerase Chain Reaction; Sheep
PubMed: 34223718
DOI: 10.22092/ari.2020.128637.1419 -
BMC Pulmonary Medicine Feb 2023Polymerase chain reaction (PCR) assays are perceived to facilitate the diagnosis of fungal infections. However, due to lack of standardization, the value of... (Meta-Analysis)
Meta-Analysis
BACKGROUND
Polymerase chain reaction (PCR) assays are perceived to facilitate the diagnosis of fungal infections. However, due to lack of standardization, the value of bronchoalveolar lavage (BAL) fluid PCR in diagnosis of invasive pulmonary aspergillosis (IPA) remains unclear.
METHODS
We conducted a systematic meta-analysis to evaluate the accuracy of BAL fluid PCR in IPA diagnosis among high-risk patients. All studies involving patients at risk for IPA were included. The sensitivity, specificity, positive and negative likelihood ratios of BAL fluid PCR were summarized for diagnosis of proven/probable IPA, or proven IPA only. Potential heterogeneity was assessed by subgroup analyses and meta-regression.
RESULTS
Forty-one studies involving 5668 patients were analyzed. The summary sensitivity, specificity, positive and negative likelihood ratios of BAL fluid PCR for proven/probable IPA were 0.75 (95% CI = 0.67-0.81), 0.94 (95% CI = 0.90-0.96), 11.8 (95% CI = 7.7-18.1) and 0.27 (95% CI = 0.20-0.36), respectively. Whereas for proven IPA only, sensitivity and specificity were 0.91 (95% CI = 0.68-0.98) and 0.80 (95% CI = 0.74-0.85) in fourteen studies involving 2061 patients. Significant heterogeneity was present due to the underlying disease, antifungal treatment and differences in DNA extraction techniques and choice of PCR assay. Compared to patients with hematological malignancies (HM) and hematopoietic stem cell/solid organ transplantation (HSCT/SOT), sensitivity was higher in the population with disease such as chronic obstructive pulmonary disease, solid tumor, autoimmune disease with prolonged use of corticosteroids, etc. (0.88 vs. 0.68, P < 0.001), which was related to the concurrent use of antifungal prophylaxis among patients with HM and HSCT/SOT.
CONCLUSION
BAL fluid PCR is a useful diagnostic tool for IPA in immunocompromised patients and is also effective for diagnosing IPA in patients without HM and HSCT/SOT. Furthermore, standard protocols for DNA extraction and PCR assays should be focused on to improve the diagnostic accuracy. Trial registration PROSPERO, registration number CRD42021239028.
Topics: Humans; Invasive Pulmonary Aspergillosis; Antifungal Agents; Bronchoalveolar Lavage Fluid; Polymerase Chain Reaction; Sensitivity and Specificity; Hematologic Neoplasms
PubMed: 36750828
DOI: 10.1186/s12890-023-02343-5 -
The Journal of Maternal-fetal &... Dec 2023The aim of this study was to evaluate if screening Group B colonization by intrapartum polymerase chain reaction could improve intrapartum administration of antibiotic...
OBJECTIVES
The aim of this study was to evaluate if screening Group B colonization by intrapartum polymerase chain reaction could improve intrapartum administration of antibiotic prophylaxis, compared with antepartum culture screening and analyze the sensitivity and specificity of polymerase chain reaction test.
METHODS
198 pregnant women with Group B colonization antepartum culture screening were included. When they arrived at hospital for delivery, two rectovaginal swabs were collected: for culture and polymerase chain reaction method.
RESULTS
The rate of Group B colonization antepartum detected by culture was 16.7%; at delivery was 17.2% when detected by culture and 19.7% using polymerase chain reaction method. The rate of inconclusive polymerase chain reaction tests was 0.5%. Considering intrapartum culture screening as gold standard, sensitivity and specificity of polymerase chain reaction test for intrapartum Group B colonization was 97.1% and 95.7%, respectively. The global rate of discordance between antepartum and intrapartum Group B colonization was 6.6%. The rate of women not treated with intrapartum antibiotic prophylaxis in the setting of positive intrapartum culture was significantly lower using intrapartum polymerase chain reaction test (0.5%) than with antepartum culture method (3.5%, = 0.035).
CONCLUSION
The use of intrapartum antibiotic prophylaxis can be more efficient when screening Group B colonization intrapartum by polymerase chain reaction test. Polymerase chain reaction method had a good performance in our study, with high sensitivity and specificity.
Topics: Pregnancy; Female; Humans; Pregnancy Complications, Infectious; Streptococcal Infections; Polymerase Chain Reaction; Parturition; Streptococcus agalactiae; Vagina
PubMed: 37766418
DOI: 10.1080/14767058.2023.2262078