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18 pages, 3603 KiB

Open AccessArticle

A Sensitive and Specific Competitive Enzyme-Linked Immunosorbent Assay for Serodiagnosis of COVID-19 in Animals

by Susanna K. P. Lau, Zirong He, Chi-Ching Tsang, Tony T. Y. Chan, Hayes K. H. Luk, Elaine Chan, Kenneth S. M. Li, Joshua Fung, Franklin W. N. Chow, Anthony R. Tam, Tom W. H. Chung, Sally C. Y. Wong, Tak-Lun Que, Kitty S. C. Fung, David C. Lung, Alan K. L. Wu, Ivan F. N. Hung, Jade L. L. Teng, Ulrich Wernery, Suk-Wai Hui, Paolo Martelli and Patrick C. Y. Woo Show full author list Hide full author list

Microorganisms 2021, 9(5), 1019; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9051019 - 10 May 2021

Cited by 3 | Viewed by 2412

Abstract

In addition to human cases, cases of COVID-19 in captive animals and pets are increasingly reported. This raises the concern for two-way COVID-19 transmission between humans and animals. Here, we developed a SARS-CoV-2 nucleocapsid protein-based competitive enzyme-linked immunosorbent assay (cELISA) for serodiagnosis of [...] Read more.

In addition to human cases, cases of COVID-19 in captive animals and pets are increasingly reported. This raises the concern for two-way COVID-19 transmission between humans and animals. Here, we developed a SARS-CoV-2 nucleocapsid protein-based competitive enzyme-linked immunosorbent assay (cELISA) for serodiagnosis of COVID-19 which can theoretically be used in virtually all kinds of animals. We used 187 serum samples from patients with/without COVID-19, laboratory animals immunized with inactive SARS-CoV-2 virions, COVID-19-negative animals, and animals seropositive to other betacoronaviruses. A cut-off percent inhibition value of 22.345% was determined and the analytical sensitivity and specificity were found to be 1:64–1:256 and 93.9%, respectively. Evaluation on its diagnostic performance using 155 serum samples from COVID-19-negative animals and COVID-19 human patients showed a diagnostic sensitivity and specificity of 80.8% and 100%, respectively. The cELISA can be incorporated into routine blood testing of farmed/captive animals for COVID-19 surveillance. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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15 pages, 1492 KiB

Open AccessArticle

Diagnostic Efficiency of Three Fully Automated Serology Assays and Their Correlation with a Novel Surrogate Virus Neutralization Test in Symptomatic and Asymptomatic SARS-COV-2 Individuals

by Salma Younes, Hadeel Al-Jighefee, Farah Shurrab, Duaa W. Al-Sadeq, Nadin Younes, Soha R. Dargham, Nader Al-Dewik, Hamda Qotba, Mohamed Syed, Ahmed Alnuaimi, Hadi M. Yassine, Patrick Tang, Laith J. Abu-Raddad and Gheyath K. Nasrallah

Microorganisms 2021, 9(2), 245; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9020245 - 25 Jan 2021

Cited by 30 | Viewed by 4477

Abstract

To support the deployment of serology assays for population screening during the COVID-19 pandemic, we compared the performance of three fully automated SARS-CoV-2 IgG assays: Mindray CL-900i^® (target: spike [S] and nucleocapsid [N]), BioMérieux VIDAS^®3 (target: receptor-binding domain [RBD]) and [...] Read more.

To support the deployment of serology assays for population screening during the COVID-19 pandemic, we compared the performance of three fully automated SARS-CoV-2 IgG assays: Mindray CL-900i^® (target: spike [S] and nucleocapsid [N]), BioMérieux VIDAS^®3 (target: receptor-binding domain [RBD]) and Diasorin LIAISON^®XL (target: S1 and S2 subunits). A total of 111 SARS-CoV-2 RT-PCR- positive samples collected at ≥ 21 days post symptom onset, and 127 pre-pandemic control samples were included. Diagnostic performance was assessed in correlation to RT-PCR and a surrogate virus-neutralizing test (sVNT). Moreover, cross-reactivity with other viral antibodies was investigated. Compared to RT-PCR, LIAISON^®XL showed the highest overall specificity (100%), followed by VIDAS^®3 (98.4%) and CL-900i^® (95.3%). The highest sensitivity was demonstrated by CL-900i^® (90.1%), followed by VIDAS^®3 (88.3%) and LIAISON^®XL (85.6%). The sensitivity of all assays was higher in symptomatic patients (91.1–98.2%) compared to asymptomatic patients (78.4–80.4%). In correlation to sVNT, all assays showed excellent sensitivities (92.2–96.1%). In addition, VIDAS^®3 demonstrated the best correlation (r = 0.75) with the sVNT. The present study provides insights on the performance of three fully automated assays, which could help diagnostic laboratories in the choice of a particular assay according to the intended use. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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16 pages, 2285 KiB

Open AccessArticle

Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of Glaesserella (Haemophilus) parasuis

by Veronika Pilchová, Diana Seinige, Isabel Hennig-Pauka, Kathrin Büttner, Amir Abdulmawjood and Corinna Kehrenberg

Microorganisms 2021, 9(1), 41; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9010041 - 25 Dec 2020

Cited by 8 | Viewed by 2932

Abstract

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we [...] Read more.

Glaesserella parasuis is a fastidious pathogen that colonizes the respiratory tract of pigs and can lead to considerable economic losses in pig production. Therefore, a rapid detection assay for the pathogen, preferably applicable in the field, is important. In the current study, we developed a new and improved detection method using loop-mediated isothermal amplification (LAMP). This assay, which targets the infB gene, was tested on a collection of 60 field isolates of G. parasuis comprising 14 different serovars. In addition, 63 isolates from seven different closely related species of the family Pasteurellaceae, including A. indolicus, A. porcinus, and A. minor, and a species frequently found in the respiratory tract of pigs were used for exclusivity experiments. This assay showed an analytical specificity of 100% (both inclusivity and exclusivity) and an analytical sensitivity of 10 fg/µL. In further steps, 36 clinical samples were tested with the LAMP assay. An agreement of 77.1 (95% CI: 59.9, 89.6) and 91.4% (95% CI: 75.9, 98.2) to the culture-based and PCR results was achieved. The mean limit of detection for the spiked bronchoalveolar lavage fluid was 2.58 × 10² CFU/mL. A colorimetric assay with visual detection by the naked eye was tested to provide an alternative method in the field and showed the same sensitivity as the fluorescence-based LAMP assay. Overall, the optimized LAMP assay represents a fast and reliable method and is suitable for detecting G. parasuis in the laboratory environment or in the field. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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9 pages, 1934 KiB

Open AccessCommunication

Highly Sensitive and Rapid Quantitative Detection of Plasmodium falciparum Using an Image Cytometer

by Muneaki Hashimoto, Kazumichi Yokota, Kazuaki Kajimoto, Musashi Matsumoto, Atsuro Tatsumi, Yoshihiro Nakajima, Toshihiro Mita, Noboru Minakawa, Hiroaki Oka and Masatoshi Kataoka

Microorganisms 2020, 8(11), 1769; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms8111769 - 11 Nov 2020

Viewed by 2030

Abstract

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, [...] Read more.

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, we investigated whether an imaging cytometer was useful for the highly sensitive quantitative detection of parasites. Whole blood samples were prepared from uninfected individuals spiked with Plasmodium falciparum-infected erythrocytes. Thereafter, erythrocytes were purified using a push column comprising of a syringe filter unit with SiO₂-nanofiber filters. After adding the erythrocytes, stained with nuclear stain, to a six-well plate, quantitative detection of the parasites was performed using an image cytometer, CQ1. Imaging of 2.6 × 10⁶ erythrocytes was completed in 3 min, and the limit of detection indicated parasitemia of 0.00010% (≈5 parasites/μL of blood). In addition to rapid, highly sensitive, and quantitative detection, the ease of application and economic costs, image cytometry could be efficiently applied to diagnose submicroscopic parasites in infected people from endemic countries. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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11 pages, 1862 KiB

Open AccessArticle

Quantitative Detection of Plasmodium falciparum Using, LUNA-FL, A Fluorescent Cell Counter

by Muneaki Hashimoto, Kazumichi Yokota, Kazuaki Kajimoto, Musashi Matsumoto, Atsuro Tatsumi, Kenichi Yamamoto, Tomonori Hyodo, Kiichiro Matsushita, Noboru Minakawa, Toshihiro Mita, Hiroaki Oka and Masatoshi Kataoka

Microorganisms 2020, 8(9), 1356; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms8091356 - 04 Sep 2020

Cited by 1 | Viewed by 2576

Abstract

The microscopic examination of Giemsa-stained thin and/or thick blood films (Giemsa microscopy) is the standard method of malaria diagnosis. However, the results of the diagnosis significantly depend on the skills of clinical technicians. Furthermore, sample preparation and analysis are laborious and time-consuming. Therefore, [...] Read more.

The microscopic examination of Giemsa-stained thin and/or thick blood films (Giemsa microscopy) is the standard method of malaria diagnosis. However, the results of the diagnosis significantly depend on the skills of clinical technicians. Furthermore, sample preparation and analysis are laborious and time-consuming. Therefore, in this study, we investigated if a commercially available fluorescent cell counter, LUNA-FL, was useful for the detection of Plasmodium parasite and the estimation of parasitemia. Whole blood samples from uninfected persons, spiked with P. falciparum-infected erythrocytes, were analysed. Most of the leucocytes and platelets were removed from whole blood samples with SiO₂-nanofiber filters set on spin columns. The filtered samples were stained with acridine orange, and automatic detection, as well as counting of erythrocytes and parasites, were performed using LUNA-FL. Whole blood, with various levels of parasites, was analysed by Giemsa microscopy or with LUNA-FL to estimate parasitemia, and a comparative analysis was performed. The coefficient determination value of the regression line was high (R² = 0.98), indicating that accurate quantitative parasite detection could be performed using LUNA-FL. LUNA-FL has a low running cost; it is compact, fast, and easy to operate, and may therefore be useful for point-of-care testing in the endemic areas. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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13 pages, 1336 KiB

Open AccessArticle

Rapid Detection and Antibiotic Susceptibility of Uropathogenic Escherichia coli by Flow Cytometry

by Alexandra Mihaela Velican, Luminiţa Măruţescu, Crina Kamerzan, Violeta Corina Cristea, Otilia Banu, Elvira Borcan and Mariana-Carmen Chifiriuc

Microorganisms 2020, 8(8), 1233; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms8081233 - 13 Aug 2020

Cited by 6 | Viewed by 2790

Abstract

Background: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: [...] Read more.

Background: Early preliminary data on antibiotic resistance patterns available before starting the empiric therapy of urinary tract infections (UTIs) in patients with risk factors for acquiring antibiotic resistance could improve both clinical and epidemiological outcomes. The aim of the present study was two-fold: (i) to assess the antibiotic susceptibility of uropathogenic Escherichia coli isolates, exhibiting different antibiotic resistance phenotypes, directly in artificially contaminated urine samples using a flow cytometry (FC) based protocol; (ii) to optimize the protocol on urine samples deliberately contaminated with bacterial suspensions prepared from uropathogenic E. coli strains. Results: The results of the FC based antimicrobial susceptibility testing (AST) protocol were compared with the reference AST methods results (disk diffusion and broth microdilution) for establishing the sensitivity and specificity. The proposed FC protocol allowed the detection and quantification of uropathogenic E. coli strains susceptibility to nitrofurantoin, trimethoprim–sulfamethoxazole, ciprofloxacin, and ceftriaxone within 4 h after the inoculation of urine specimens. The early availability of preliminary antibiotic susceptibility results provided by direct analysis of clinical specimens could essentially contribute to a more targeted emergency therapy of UTIs in the anticipation of AST results obtained by reference methodology. Conclusions: This method will increase the therapeutic success rate and help to prevent the emergence and dissemination of drug resistant pathogens. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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14 pages, 7053 KiB

Open AccessArticle

Development and Evaluation of a Single Dye Duplex Droplet Digital PCR Assay for the Rapid Detection and Quantification of Mycobacterium tuberculosis

by Raphael Nyaruaba, Jin Xiong, Caroline Mwaliko, Nuo Wang, Belindah J. Kibii, Junping Yu and Hongping Wei

Microorganisms 2020, 8(5), 701; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms8050701 - 10 May 2020

Cited by 17 | Viewed by 8690

Abstract

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite [...] Read more.

Droplet digital PCR (ddPCR) is a third generation of PCR that was recently developed to overcome the challenges of real-time fluorescence-based quantitative PCR (qPCR) in absolute quantification of pathogens. Few studies have been done on tuberculosis (TB) detection and quantification using ddPCR despite its many advantages over qPCR. From the few studies, none explores a single dye duplex assay for the detection and quantification of TB. In this study, steps toward developing and evaluating a duplex single dye (FAM) assay for detecting two targets (IS6110 and IS1081) are clearly described using simplex and duplex experiments. To achieve this, various parameters are investigated, including annealing temperature, primer and probe concentration, sensitivity and specificity, sample concentration, and inter/intra-assay variability. From the results, primer and probe concentration, annealing temperature, and sample concentration have an effect on the position and separation of droplets in both simplex and duplex assays. The copies of target genes in a duplex assay can be estimated accurately using the threshold tool with little inter-assay (CV <1%) and intra-assay (CV <6%) variability when compared to simplex assays. The ddPCR assay specificity and sensitivity are both 100% when compared to qPCR. This work shows steps toward the detection and quantification of two targets in a single channel, enabling higher multiplexing to include more targets in future works. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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Review

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27 pages, 3255 KiB

Open AccessReview

A Review on the Methods Used for the Detection and Diagnosis of Rabbit Hemorrhagic Disease Virus (RHDV)

by Joana Abrantes and Ana M. Lopes

Microorganisms 2021, 9(5), 972; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9050972 - 30 Apr 2021

Cited by 6 | Viewed by 4215

Abstract

Since the early 1980s, the European rabbit (Oryctolagus cuniculus) has been threatened by the rabbit hemorrhagic disease (RHD). The disease is caused by a lagovirus of the family Caliciviridae, the rabbit hemorrhagic disease virus (RHDV). The need for detection, identification [...] Read more.

Since the early 1980s, the European rabbit (Oryctolagus cuniculus) has been threatened by the rabbit hemorrhagic disease (RHD). The disease is caused by a lagovirus of the family Caliciviridae, the rabbit hemorrhagic disease virus (RHDV). The need for detection, identification and further characterization of RHDV led to the development of several diagnostic tests. Owing to the lack of an appropriate cell culture system for in vitro propagation of the virus, much of the methods involved in these tests contributed to our current knowledge on RHD and RHDV and to the development of vaccines to contain the disease. Here, we provide a comprehensive review of the RHDV diagnostic tests used since the first RHD outbreak and that include molecular, histological and serological techniques, ranging from simpler tests initially used, such as the hemagglutination test, to the more recent and sophisticated high-throughput sequencing, along with an overview of their potential and their limitations. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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26 pages, 1263 KiB

Open AccessReview

Phage Amplification Assay for Detection of Mycobacterial Infection: A Review

by Monika Beinhauerova and Iva Slana

Microorganisms 2021, 9(2), 237; https://0-doi-org.brum.beds.ac.uk/10.3390/microorganisms9020237 - 23 Jan 2021

Cited by 9 | Viewed by 4082

Abstract

An important prerequisite for the effective control, timely diagnosis, and successful treatment of mycobacterial infections in both humans and animals is a rapid, specific, and sensitive detection technique. Culture is still considered the gold standard in the detection of viable mycobacteria; however, mycobacteria [...] Read more.

An important prerequisite for the effective control, timely diagnosis, and successful treatment of mycobacterial infections in both humans and animals is a rapid, specific, and sensitive detection technique. Culture is still considered the gold standard in the detection of viable mycobacteria; however, mycobacteria are extremely fastidious and slow-growing microorganisms, and therefore cultivation requires a very long incubation period to obtain results. Polymerase Chain Reaction (PCR) methods are also frequently used in the diagnosis of mycobacterial infections, providing faster and more accurate results, but are unable to distinguish between a viable and non-viable microorganism, which results in an inability to determine the success of tuberculosis patient treatment or to differentiate between an active and passive infection of animals. One suitable technique that overcomes these shortcomings mentioned is the phage amplification assay (PA). PA specifically detects viable mycobacteria present in a sample within 48 h using a lytic bacteriophage isolated from the environment. Nowadays, an alternative approach to PA, a commercial kit called Actiphage™, is also employed, providing the result within 6–8 h. In this approach, the bacteriophage is used to lyse mycobacterial cells present in the sample, and the released DNA is subsequently detected by PCR. The objective of this review is to summarize information based on the PA used for detection of mycobacteria significant in both human and veterinary medicine from various kinds of matrices. Full article

(This article belongs to the Special Issue Rapid Diagnosis of Microbial Pathogens)

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