Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades

Candida auris is an opportunistic pathogenic yeast that emerged worldwide through the previous decade. This fungal pathogen poses a major public well being menace as a result of frequent multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have recognized 5 distinct clades which might be linked to 5 geographic areas of origin and characterised by variations in a number of phenotypic traits akin to virulence and drug resistance.
Typing of C. auris strains and the identification of clades could be a highly effective device in molecular epidemiology and is likely to be of scientific significance by estimating outbreak and MDR potential. As C. auris has brought on world outbreaks, together with in low-income international locations, typing C. auris strains rapidly and inexpensively is extremely worthwhile. We report 5 allele-specific polymerase chain response (AS-PCR) assays for the identification of C. auris and every of the 5 described clades of C. auris based mostly on conserved mutations within the inner transcribed spacer (ITS) rDNA area and a clade-specific gene cluster. This PCR technique gives a quick, low cost, sequencing-free diagnostic device for the identification of C. aurisC. auris clades, and doubtlessly, the invention of recent clades.
To help the scientific laboratory prognosis of Pneumocystis jirovecii (PJ) pneumonia (PCP), an invasive fungal an infection primarily occurring in HIV-negative sufferers, in-house or business PJ-specific real-time quantitative PCR (qPCR) assays are todays’ dependable choices. The efficiency of those assays depends upon the kind of PJ gene (multi-copy mitochondrial versus single-copy nuclear) focused by the assay.
  • We described the event of a PJ-PCR assay concentrating on the dihydrofolate reductase (DHFR)-encoding gene. After delineating its analytical efficiency, the PJ-PCR assay was used to check bronchoalveolar lavage (BAL) fluid samples from 200 sufferers (solely seven have been HIV optimistic) with suspected PCP.
  • Of 211 BAL fluid samples, 18 (8.5%) have been optimistic and 193 (91.5%) have been unfavourable by PJ-PCR. Of 18 PJ-PCR-positive samples, 11 (61.1%) examined optimistic and 7 (38.9%) examined unfavourable with the immunofluorescence assay (IFA). All (100%) of the 193 PJ-PCR-negative samples have been IFA unfavourable.
  • Based mostly on IFA/PCR outcomes, sufferers have been, respectively, categorized as having (n = 18) and never having (n = 182) confirmed (PJ-PCR+/IFA+) or possible (PJ-PCR+/IFA-) PCP. For 182 sufferers with out PCP, various infectious or non-infectious etiologies have been recognized. Our PJ-PCR assay was no less than equal to IFA, fostering research aimed toward defining a qPCR-based commonplace for PCP prognosis sooner or later.

Publish-Mortem RT-PCR Assay for SARS-CoV-2 RNA in COVID-19 Sufferers’ Corneal Epithelium, Conjunctival and Nasopharyngeal Swabs

Extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2) illness has been described to presumably be related to ocular floor disturbances. Nonetheless, whether or not the virus may invade ocular tissues nonetheless stays elusive. Within the current research, we tried to research the autopsy presence of SARS-CoV-2 RNA in corneal epithelium gathered by sufferers with an ante-mortem confirmed prognosis of Coronavirus disease-19 (COVID-19). Cadavers with an ante-mortem confirmed prognosis of reasonable to extreme COVID-19 have been examined. Medical and demographic options have been retrieved from hospital sufferers’ notes.
For every cadaver, corneal scrapings, conjunctival swabs (CS) and nasopharyngeal swabs (NPS) have been collected to carry out real-time reverse transcriptase polymerase chain response ((RT)-PCR) for SARS-CoV-2. Fourteen consecutive cadavers with an ante-mortem confirmed prognosis of reasonable to extreme COVID-19 have been examined. The final NPS carried out ante-mortem confirmed SARS-CoV-2 an infection in 12/14 (85.7%) sufferers.
The imply death-to-swab time (DtS) was 3.15 ± 0.5 (2.10-5.1) h. The autopsy NPS and CS discovered optimistic for SARS-CoV-2 RNA have been 9/14 (64.3%) and three/28 (10.7%), respectively. Not one of the corneal epithelium scrapes examined optimistic to RT-PCR for SARS-CoV-2 RNA. These knowledge promote the SARS-CoV-2 as not capable of contaminate the autopsy corneal epithelium, whereas it will probably persist in numerous different constructions of the ocular floor (i.e., the conjunctiva). It’s cheap to imagine that such a contamination can happen ante-mortem too.


Improvement and Validation of Novel PCR Assays for the Prognosis of Bovine Stephanofilariasis and Detection of Stephanofilaria sp. Nematodes in Vector Flies


Background: Stephanofilaria spp. nematodes are related to cutaneous lesions in cattle and different livestock and mammalian wildlife species. In Australia, Haematobia irritans exigua, generally often called buffalo fly (BF) transmits a well-described however presently unnamed species of Stephanofilaria, which has been speculatively implicated within the aetiology of BF lesions. The sensitivity of present methods for detecting Stephanofilaria spp. in pores and skin lesions and vector species is low, and there’s no genomic sequence for any member of the genus Stephanofilaria presently obtainable in sequence databases.
Strategies: To develop molecular assays for the detection of the Australian Stephanofilaria sp., pores and skin biopsies have been collected from freshly slaughtered cattle with typical lesions close to the medial canthus. Grownup nematodes and microfilariae have been remoted from the biopsies utilizing a saline restoration approach. The nematodes have been morphologically recognized as Stephanofilaria sp. by scanning electron microscopy. DNA was extracted and the inner transcribed spacer 2 (ITS2) area of rDNA, and the cytochrome c oxidase subunit 1 (cox1) area of mtDNA was amplified and sequenced. Stephanofilaria sp. particular polymerase chain response (PCR) and qPCR assays (SYBR Inexperienced® and TaqMan™) have been developed and optimised from the novel ITS2 sequence obtained. The specificity of every assay was confirmed by testing towards nematode species Onchocerca gibsoni and Dirofilaria immitis, in addition to host (bovine) and BF DNA.
Outcomes: Scanning electron microscopy of the anterior and posterior ends of remoted nematodes confirmed Stephanofilaria sp. A phylogenetic evaluation of the cox1 sequence demonstrated that this species is most intently associated to Thelazia callipaeda, a parasitic nematode that may be a frequent reason for thelaziasis (or eyeworm infestation) in people, canines, and cats. Each standard and qPCR assays particularly amplified DNA from Stephanofilaria sp. Standard PCR, TaqMan, and SYBR Inexperienced assays have been proven to detect 1 ng, 1 pg, and 100 fg of Stephanofilaria DNA, respectively. Each qPCR assays detected DNA from single Stephanofilaria microfilaria.
Conclusion: Molecular diagnostic assays developed on this research confirmed excessive specificity and sensitivity for Stephanofilaria sp. DNA. The supply of an correct and delicate PCR assay for Stephanofilaria will help in figuring out its function within the pathogenesis of cattle pores and skin lesions, in addition to in understanding its epidemiological dynamics. This assay may have software to be used in epidemiological research with different species of Stephanofilaria, most significantly intently associated S. stilesi, however it will require affirmation.
Key phrases: ITS2; Stephanofilaria; buffalo fly lesion; cattle dermatitis; cox1.

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