Detection of Candida albicans DNA from blood samples using a novel electrochemical assay

The genus Candida contains a number of yeast species which are opportunistic pathogens and are associated with life-threatening infections in immunocompromised individuals. Provision of appropriate therapy relies on the rapid identification of the infecting species, and existing methods of identifying Candida species in clinical samples are time and resource intensive and are not always specific enough to differentiate between drug-susceptible and drug-resistant species. We have previously developed a system for the rapid detection of yeast pathogens in clinical samples using PCR followed by hybridization with a suite of five species-specific, electrochemically labelled DNA probes. The limit of detection of the assay was shown to be 37 fg (∼1 genome) per reaction using extracted genomic DNA. We carried out a study to test the limit of detection of one of the probes, CA PR3, using blood samples from a healthy donor that were spiked with genomic DNA or with C. albicans cells. Our results demonstrated a limit of detection of 37 fg (ml blood)−1 (∼1 genome ml−1) using extracted DNA or 10 c.f.u. (ml blood)−1 using C. albicans cells, indicating that the assay is capable of detecting C. albicans nucleic acid at levels that are encountered in clinical samples.

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Procalcitonin Detection in Veterinary Species: Investigation of Commercial ELISA Kits

Animals ◽

10.3390/ani10091511 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1511

Author(s):

Federica Battaglia ◽

Valentina Meucci ◽

Rosalba Tognetti ◽

Francesca Bonelli ◽

Micaela Sgorbini ◽

...

Keyword(s):

Veterinary Medicine ◽

Recombinant Proteins ◽

Limit Of Detection ◽

Rapid Identification ◽

Human Medicine ◽

Clinical Samples ◽

Reference Ranges ◽

Plasma Samples ◽

Elisa Kit ◽

Species Specific

In human medicine, procalcitonin (PCT), the precursor of calcitonin, is used for the rapid identification of the origin and severity of sepsis. In veterinary medicine, PCT has been studied in horses, cattle, and dogs, but the use of PCT in diagnostic and/or prognostic settings is not possible because of the lack of validated assays to obtain reference ranges. The aim of the present study was the investigation of commercially available ELISA kits for the detection of canine and equine PCT in plasma samples. Validation of the ELISA kits was performed by using species-specific recombinant proteins spiked both in plasma and buffer samples; linearity, limit of detection (LOD), recovery, and intra-assay and inter-assay variability were calculated. Moreover, clinical samples obtained from sick and healthy animals were also analyzed with the tested kits. Canine PCT was measured with a recombinant canine and a canine PCT ELISA kit. Equine PCT was measured with an equine and a human ELISA PCT kit. Our data demonstrate that the canine recombinant PCT ELISA kit can be used to measure canine PCT in plasma samples, showing an intra-assay and inter-assay coefficient of variation less than 20% and a LOD of 11 pg/mL, whereas the present results do not support the use of the canine PCT ELISA kit. The human PCT ELISA kit is suitable to detect equine PCT with a LOD of 56 ng/mL, whereas the equine PCT ELISA kit did not detect recombinant equine PCT.

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SCA Medium: A New Culture Medium for the Isolation of All Candida auris Clades

Journal of Fungi ◽

10.3390/jof7060433 ◽

2021 ◽

Vol 7 (6) ◽

pp. 433

Author(s):

Ahmad Ibrahim ◽

Lucie Peyclit ◽

Rim Abdallah ◽

Saber Khelaifia ◽

Amanda Chamieh ◽

...

Keyword(s):

Culture Medium ◽

Limit Of Detection ◽

Bacterial Species ◽

Multidrug Resistant ◽

Rapid Identification ◽

Clinical Samples ◽

Candida Auris ◽

Phenotypic Profile ◽

Stool Samples ◽

Selective Culture Medium

Candida auris is an emerging multidrug-resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation are necessary for diagnosis and containing its spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection ranging between 101 and 102 CFU/mL. The 100% specificity of SCA (specific C. auris) medium is confirmed on a set of 135 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, allowing the latter to study its phenotypic profile.

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Two Target Genes Based Multiple Cross Displacement Amplification Combined with Lateral Flow Biosensor for the Detection of Mycobacrterium Tuberculosis Complex

10.21203/rs.3.rs-123493/v1 ◽

2020 ◽

Author(s):

Junfei Huang ◽

Ziyu Xiao ◽

Xinggui Yang ◽

Xu Chen ◽

Xiaojuan Wang ◽

...

Keyword(s):

Public Health ◽

Real Time ◽

Genomic Dna ◽

Target Genes ◽

Limit Of Detection ◽

Lateral Flow ◽

Clinical Samples ◽

Prevention And Treatment ◽

Multiple Cross ◽

Positive Rate

Abstract Background: Mycobacterium tuberculosis complex (MTBC) causes tuberculosis (TB), which is a global public health problem that seriously endangers public health. Hence, development of a new and rapid method to detect MTBC is of great significance for prevention and treatment of TB. Results: In this study, a multiple cross displacement amplification combined with nanoparticle-based lateral flow biosensor (MCDA-LFB) was developed to simultaneously detect two target genes (IS6110 and mpb64) of MTBC. One suit of specific multiple cross displacement amplification (MCDA) primers, which was designed for IS6110 and mpb64 gene, respectively, was validated through using the genomic DNA extracted from reference strain H37Rv. The MCDA products were analyzed using real-time turbidity curve, colorimetric indicator (Malachite Green, MG) and LFB. The conditions of amplification temperature and time were optimized and the established MCDA-LFB method was applied to detect the sputum specimens and MTBC strains from clinical samples. The results show that two sets of MCDA primers for the IS6110 and mpb64 genes have detected MTBC validly. The MCDA reaction conditions were optimized at 67 °C for 35 min. The limit of detection of MCDA assay based on IS6110 and mpb64 genes was 100 fg of genomic DNA template per reaction. The specificity of MCDA-LFB detection was 100%, and no cross-reactions for non-MTBC strains detection. The positive rate of MCDA-LFB for the detection of MTBC strains was equal to that of semi-nested automatic real-time PCR (Xpert MTB/RIF), and had a higher positive rate than acid-fast staining (AFS) when used for the detection of sputum samples. The whole procedure of MCDA-LFB, including genomic template preparation, MCDA reaction and products analysis, was completed with 70 min.Conclusion: The simplicity, rapidity, sensitivity and reliability of the MCDA-LFB based on IS6110 and mpb64 gene of MTBC developed in this study make it potentially significant for the prevention and treatment of TB.

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Recombinase polymerase amplification (RPA) with lateral flow detection for three Anaplasma species of importance to livestock health

Scientific Reports ◽

10.1038/s41598-021-95402-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Andrea Salazar ◽

Francisco M. Ochoa-Corona ◽

Justin L. Talley ◽

Bruce H. Noden

Keyword(s):

Internal Control ◽

Limit Of Detection ◽

Lateral Flow ◽

Serum Samples ◽

Laboratory Equipment ◽

Blood Samples ◽

Gapdh Gene ◽

Granulocytic Anaplasmosis ◽

Positive Serum ◽

Species Specific

AbstractAnaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited.

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RPAcan3990: an ultrasensitive recombinase polymerase assay to detect Angiostrongylus cantonensis DNA

Journal of Clinical Microbiology ◽

10.1128/jcm.01185-21 ◽

2021 ◽

Author(s):

William J. Sears ◽

Yvonne Qvarnstrom ◽

Thomas B. Nutman

Keyword(s):

Genomic Dna ◽

Incubation Temperature ◽

Limit Of Detection ◽

Angiostrongylus Cantonensis ◽

Clinical Samples ◽

Eosinophilic Meningitis ◽

Environmental Surveillance ◽

Human Body Temperature ◽

Visual Reading ◽

Optimal Incubation

Angiostrongylus cantonensis (Ac) is one of the leading causes of eosinophilic meningitis worldwide. A field deployable molecular detection method could enhance both environmental surveillance and clinical diagnosis of this emerging pathogen. Accordingly, RPAcan3990, a recombinase polymerase assay (RPA) was developed to target a region predicted to be highly repeated in the Ac genome. The assay was then adapted to produce a visually interpretable fluorescent readout using an orange camera lens filter and a blue light. Using Ac genomic DNA, the limit of detection was found to be 1fg/μl by both fluorometer measurement and visual reading. All clinical samples known to be positive for Ac from various areas of the globe were positive by RPAcan3990. Cerebrospinal fluid samples from other etiologies of eosinophilic meningitis (i.e. Toxocara sp., Gnathostoma sp.) were negative in the RPAcan3990 assay. The optimal incubation temperature range for the reaction was between 35-40°C. The assay successfully detected 1fg/μl of Ac genomic DNA after incubation at human body temperature (in a shirt pocket). In conclusion, these data suggest RPAcan3990 is potentially a point of contact molecular assay capable of sensitively detecting Ac by producing visually interpretable results with minimal instrumentation.

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T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00510-17 ◽

2017 ◽

Vol 55 (8) ◽

pp. 2453-2461 ◽

Cited By ~ 5

Author(s):

Jessica L. Snyder ◽

Heidi Giese ◽

Cheryl Bandoski-Gralinski ◽

Jessica Townsend ◽

Beck E. Jacobson ◽

...

Keyword(s):

Magnetic Resonance ◽

Real Time ◽

Whole Blood ◽

Limit Of Detection ◽

Direct Detection ◽

Erythema Migrans ◽

Clinical Samples ◽

Blood Samples ◽

Content Type ◽

Whole Blood Samples

ABSTRACTIn early Lyme disease (LD), serologic testing is insensitive and seroreactivity may reflect active or past infection. In this study, we evaluated a novel assay for the direct detection of three species ofBorreliaspirochetes in whole blood. The T2 magnetic resonance (T2MR) assay platform was used to amplifyBorreliaDNA released from intact spirochetes and to detect amplicon. Analytical sensitivity was determined from blood spiked with known concentrations of spirochetes, and the assay's limit of detection was found to be in the single-cell-per-milliliter range: 5 cells/ml forB. afzeliiand 8 cells/ml forBorrelia burgdorferiandBorrelia garinii. Clinical samples (n= 66) from confirmed or suspected early LD patients were also analyzed.B. burgdorferiwas detected using T2MR in 2/2 (100%) of blood samples from patients with confirmed early LD, based on the presence of erythema migrans and documentation of seroconversion or a positive real-time blood PCR. T2MR detectedB. burgdorferiin blood samples from 17/54 (31%) of patients with probable LD, based on the presence of erythema migrans without documented seroconversion or of documented seroconversion in patients with a compatible clinical syndrome but without erythema migrans. Out of 21 clinical samples tested by real-time PCR, only 1 was positive and 13 were negative with agreement with T2MR. An additional 7 samples that were negative by real-time PCR were positive with T2MR. Therefore, T2MR enables a low limit of detection (LoD) forBorreliaspp. in whole blood samples and is able to detectB. burgdorferiin clinical samples.

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Characterization of the Dielectrophoretic Response of Different Candida Strains Using 3D Carbon Microelectrodes

Micromachines ◽

10.3390/mi11030255 ◽

2020 ◽

Vol 11 (3) ◽

pp. 255 ◽

Cited By ~ 3

Author(s):

Monsur Islam ◽

Devin Keck ◽

Jordon Gilmore ◽

Rodrigo Martinez-Duarte

Keyword(s):

Candida Albicans ◽

Candida Tropicalis ◽

Rapid Identification ◽

Candida Strain ◽

Life Threatening ◽

Rising Incidence ◽

Candida Infections ◽

Species Specific ◽

Anti Fungal

Bloodstream infection with Candida fungal cells remains one of the most life-threatening complications among hospitalized patients around the world. Although most of the cases are still due to Candida albicans, the rising incidence of infections caused by other Candida strains that may not respond to traditional anti-fungal treatments merits the development of a method for species-specific isolation of Candida. To this end, here we present the characterization of the dielectrophoresis (DEP) response of Candida albicans, Candida tropicalis and Candida parapsilosis. We complement such characterization with a study of the Candida cells morphology. The Candida strains exhibited subtle differences in their morphology and dimensions. All the Candida strains exhibited positive DEP in the range 10–500 kHz, although the strength of the DEP response was different for each Candida strain at different frequencies. Only Candida tropicalis showed positive DEP at 750 kHz. The current results show potential for manipulation and enrichment of a specific Candida strain at specific DEP conditions towards aiding in the rapid identification of Candida strains to enable the effective and timely treatment of Candida infections.

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SCA Medium: A New Culture Medium for the Isolation of all Candida auris Clade

10.20944/preprints202104.0658.v1 ◽

2021 ◽

Author(s):

Ahmad Ibrahim ◽

Lucie Peyclit ◽

Rim Abdallah ◽

Saber Khelaifia ◽

Amanda Chamieh ◽

...

Keyword(s):

Culture Medium ◽

Limit Of Detection ◽

Bacterial Species ◽

Multidrug Resistant ◽

Rapid Identification ◽

Clinical Samples ◽

Candida Auris ◽

Phenotypic Profile ◽

Stool Samples ◽

Selective Culture Medium

Candida auris is an emerging multidrug resistant yeast causing nosocomial infections and associated with high mortality in immunocompromised patients. Rapid identification and characterisation is necessary for its diagnosis and containing spread. In this study, we present a selective culture medium for all C. auris clades. This medium is sensitive with a limit of detection of 102 CFU/ml. The 100% specificity of SCA (Specific C. auris) medium is confirmed on a set of 134 Candida strains, 50 bacterial species and 200 human stool samples. Thus, this medium specifically selects for C. auris isolation from clinical samples, and allows studying its phenotypic profile.

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Diagnostic Performance and Clinical Feasibility of a Novel One-Step RT-qPCR for Detecting Multiple Severe Acute Respiratory Syndrome Coronavirus

10.21203/rs.3.rs-1027593/v1 ◽

2021 ◽

Author(s):

Tran Bac Le ◽

Hye Kwon Kim ◽

Min-Ju Ahn ◽

Mark Zanin ◽

Van Thi Lo ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Limit Of Detection ◽

Laboratory Diagnosis ◽

Rapid Identification ◽

Qpcr Assay ◽

Clinical Samples ◽

Reverse Transcription Pcr ◽

One Step ◽

Human Coronaviruses ◽

Culture Supernatants

Abstract The pandemic coronavirus disease 2019 (COVID-19) is characterized as an acute respiratory infection in the majority of cases and is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other coronaviruses (CoVs) can infect humans, although the majority only cause mild respiratory symptoms. As early diagnosis of SARS-CoV-2 is critical to prevent further transmission events and to improve clinical outcomes, it is important to be able to distinguish SARS-CoV-2 from other CoVs in respiratory samples. Therefore, we developed and evaluated a novel quantitative reverse transcription PCR (RT-qPCR) assay, targeting the spike (S) and membrane (M) genes, to enable the rapid identification of SARS-CoV-2 including several new circulating variants, and other emerging pan-SARS-like CoVs. Using RNA extracted from cell culture supernatants, our multiple simultaneous SARS-CoV-2 assays confirmed a limit of detection of 1 × 100 TCID50/ml and no cross-reaction with human coronaviruses or other respiratory viruses. We also validated our method using human clinical samples from COVID-19 patients and healthy individuals, including nasal swab and sputum samples. This novel one-step multiplex RT-qPCR assay can be used to improve the laboratory diagnosis of human-pathogenic coronaviruses, including SARS-CoV-2, and may be useful for the identification of other pan-SARS-like CoVs of zoonotic origin.

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Isolation of Stenotrophomonas maltophilia from Clinical Samples in Some Hospitals in Shiraz, Iran

Clinical and Experimental Investigations ◽

10.31487/j.cei.2020.03.01 ◽

2020 ◽

pp. 1-3

Author(s):

Majid Baserisalehi ◽

Samira Zarezadeh ◽

Majid Baserisalehi ◽

Saeed Shoa

Keyword(s):

Stenotrophomonas Maltophilia ◽

Enterobacter Aerogenes ◽

Late Summer ◽

Early Spring ◽

Clinical Samples ◽

Bacillus Species ◽

Microbiological Analysis ◽

Blood Samples ◽

Healthcare Facilities ◽

Phenotypic Methods

Stenotrophomonas maltophilia is an emerging pathogenic non-fermentative Gram-negative Bacillus species. It has caused many nosocomial infections and can be isolated from various hospital wards and healthcare facilities. Research has shown that most of its strains are inherently resistant to many antibiotics and have multidrug resistance. This research intended to determine its occurrence frequency at some Hospitals in shiraz, Iran. The present study was conducted in six months (from early spring to late summer 2019). Clinical samples (Blood, Urine and cerebrospinal fluid (CSF)) collected from 120 patients afflicted with various infections. The samples were transferred to the Laboratory and subjected to microbiological analysis. Identification of the isolates was carried out by phenotypic methods and Stenotrophomonas maltophilia isolates verified using molecular methods. In total, various bacteria were isolated from 84 clinical samples. The isolates were Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Staphylococcus aureus and Pseudomonas aeruginosa. Stenotrophomonas maltophilia was isolated from 17 (20.2%) positive samples and most of them were isolated from blood samples. Our finding indicated that Stenotrophomonas maltophilia isolated more from blood samples follow by CSF sample. In addition, our finding illustrated that Stenotrophomonas maltophilia can be considered as the common nosocomial agent at hospitals in Shiraz, Iran.

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