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  • Middleton Cooney posted an update 3 days, 12 hours ago

    Recent studies have highlighted the interest in noninvasive sampling procedures coupled with real-time PCR methods for the detection of Leishmania species in South America. In French Guiana, the sampling method still relied on skin biopsies. Noninvasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of cutaneous leishmaniasis. Therefore, we evaluated the performance of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR green-based real-time PCR of the Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with cutaneous leishmaniasis were included in the study at the Cayenne Hospital and its remote health centers. Each patient underwent scrapings for a smear, skin biopsies for parasite culture and PCR-restriction fragment length polymorphism (RFLP) (RNA polymerase II), and sampling with a cotton swab for SYBR green-based PCR. The most accurate diagnostic test was the SYBR green-based PCR on swab samples, showing 98% sensitivity. The mean PCR cycle threshold (CT ) was 24.4 (minimum CT , 17; maximum CT , 36) and was less then 35 in 97.6% of samples. All samples positive by SYBR green-based real-time PCR were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of cutaneous leishmaniasis in South America and especially for remote areas, since noninvasive collection tools are easier to use and require fewer precautions for transportation.Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases offers a better understanding of their pathogenesis. According to the World Health Organization, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free, and cost-effective. Nucleic acid pathogen detection methods, typically PCR, have numerous limitations, such as highly sophisticated equipment requirements, reagents, and trained personnel relying on well-established laboratories, besides being time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools that are rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit unpredicted in vitro properties of CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic acid binding event. These effectors can be attached to a diversity of reporters and utilized in tandem with isothermal amplification approaches to create sensitive identification in multiple deployable field formats. Although still in their beginning, SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious diseases, with ultrasensitive tests that do not require complicated processing. this website This review describes SHERLOCK and DETECTR technologies and assesses their properties, functions, and prospective to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.Widely employed diagnostic antibody serology for Lyme disease, known as standard two-tier testing (STTT), exhibits insufficient sensitivity in early Lyme disease, yielding many thousands of false-negative test results each year. Given this problem, we applied serum antibody repertoire analysis (SERA), or next-generation sequencing (NGS)-based serology, to discover IgG and IgM antibody epitope motifs capable of detecting Lyme disease-specific antibodies with high sensitivity and specificity. Iterative motif discovery and bioinformatic analysis of epitope repertoires from subjects with Lyme disease (n = 264) and controls (n = 391) yielded a set of 28 epitope motifs representing 20 distinct IgG antibody epitopes and a set of 38 epitope motifs representing 21 distinct IgM epitopes, which performed equivalently in a large validation cohort of STTT-positive samples. In a second validation set from subjects with clinically defined early Lyme disease (n = 119) and controls (n = 257), the SERA Lyme IgG and IgM assay exhibited significantly improved sensitivity relative to STTT (77% versus 62%; Z-test; P = 0.013) and improved specificity (99% versus 97%). Early Lyme disease subjects exhibited significantly fewer reactive epitopes (Mann-Whitney U test; P  less then  0.0001) relative to subjects with Lyme arthritis. Thus, SERA Lyme IgG and M panels provided increased accuracy in early Lyme disease in a readily expandable multiplex assay format.Vibrio vulnificus is a zoonotic pathogen that is spreading worldwide due to global warming. Lineage 3 (L3; formerly biotype 3) includes the strains of the species with the unique ability to cause fish farm-linked outbreaks of septicemia. The L3 strains emerged recently and are particularly virulent and difficult to identify. Here, we describe a newly developed PCR method based on a comparative genomic study useful for both rapid identification and epidemiological studies of this interesting emerging group. The comparative genomic analysis also revealed the presence of a genetic duplication in the L3 strains that could be related to the unique ability of this lineage to produce septicemia outbreaks.The objective of this study was to design a pangenotypic PCR-based assay for the detection and quantification of hepatitis E virus (HEV) RNA from across the entire spectrum of described genotypes belonging to the Orthohepevirus A genus. The optimal conditions and the performance of the assay were determined by testing the WHO standard strain (6219/10) and the WHO HEV panel (8578/13). Similarly, performance comparisons were made with two commercial assays (Real Star HEV RT-PCR 2.0 and ampliCube HEV 2.0 Quant) to detect HEV RNA at concentrations below 1,000 IU/ml with viral strains from the WHO and to test samples from 54 patients with acute hepatitis. The assay presented in this study was able to detect the entire spectrum of described genotypes belonging to the Orthohepevirus A genus, demonstrating better performance than both commercial kits. This procedure may represent a significant improvement in the molecular diagnosis of HEV infection.

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