scholarly journals High Content Screening, a reliable system for Coxiella burnetii isolation from clinical samples

2019 ◽  
Author(s):  
Rania Francis ◽  
Maxime Mioulane ◽  
Marion Le Bideau ◽  
Marie-Charlotte Mati ◽  
Pierre-Edouard Fournier ◽  
...  
Keyword(s):  
Cell Culture ◽  
Coxiella Burnetii ◽  
High Throughput ◽  
Antibiotic Susceptibility ◽  
Q Fever ◽  
Standard Procedure ◽  
Epidemiological Studies ◽  
High Content Screening ◽  
Clinical Samples ◽  
Shell Vial

AbstractQ fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although the current serological and molecular detection tools enable a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to evaluate their antibiotic susceptibility and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers as it requires biosafety-level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically-designed algorithms targeting cytopathic effects. This method was more efficient than the shell-vial assay when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% vs 78% for shell-vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% vs 5% for shell-vial). In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell-vial culture and quantitative PCR. Overall, this high-throughput HCS system paves the way to the development of improved cell culture isolation of human viruses.

scholarly journals High-Content Screening, a Reliable System for Coxiella burnetii Isolation from Clinical Samples

2020 ◽  
Vol 58 (5) ◽  
Author(s):  
Rania Francis ◽  
Maxime Mioulane ◽  
Marion Le Bideau ◽  
Marie-Charlotte Mati ◽  
Pierre-Edouard Fournier ◽  
...  
Keyword(s):  
Cell Culture ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Standard Procedure ◽  
Epidemiological Studies ◽  
High Content Screening ◽  
Clinical Samples ◽  
Content Type ◽  
Shell Vial ◽  
Level 3

ABSTRACT Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers, as it requires biosafety level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high-content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically designed algorithms targeting cytopathic effects. This method was more efficient than the shell vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% versus 78% for shell vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% versus 5% for shell vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell vial culture and quantitative PCR.

scholarly journals Bacteriostatic and Bactericidal Activities of Tigecycline against Coxiella burnetii and Comparison with Those of Six Other Antibiotics

2009 ◽  
Vol 53 (6) ◽  
pp. 2690-2692 ◽  
Author(s):  
Ioanna Spyridaki ◽  
Anna Psaroulaki ◽  
Iosif Vranakis ◽  
Yannis Tselentis ◽  
Achilleas Gikas
Keyword(s):  
Coxiella Burnetii ◽  
Q Fever ◽  
Vero Cells ◽  
In Vitro Susceptibility ◽  
Shell Vial ◽  
Bactericidal Activities ◽  
Acute Q Fever ◽  
Reference Strains ◽  
Shell Vial Assay

ABSTRACT The present article is a study of the in vitro susceptibility of eight Greek Coxiella burnetii isolates, derived from patients with acute Q fever, and two reference strains of Coxiella burnetii to tigecycline. The bacteriostatic activity of tigecycline was compared with those of six other antibiotics using a shell vial assay. The MICs of the examined antibiotics were as follows: tigecycline ranged from 0.25 to 0.5 μg/ml; doxycycline, trovafloxacin, and ofloxacin ranged from 1 to 2 μg/ml; linezolid and clarithromycin ranged from 2 to 4 μg/ml; and ciprofloxacin ranged from 4 to 8 μg/ml. Tigecycline was effective in inhibiting the infection of Vero cells by C. burnetii. No bactericidal activity was observed against C. burnetii at 4 μg/ml.

scholarly journals Development of High-Throughput Multiplex Serology to Detect Serum Antibodies against Coxiella burnetii

2021 ◽  
Vol 9 (11) ◽  
pp. 2373
Author(s):  
Rima Jeske ◽  
Larissa Dangel ◽  
Leander Sauerbrey ◽  
Dimitrios Frangoulidis ◽  
Lauren R. Teras ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
High Throughput ◽  
Large Scale ◽  
Q Fever ◽  
Reference Group ◽  
Clinical Manifestations ◽  
Cost Effective ◽  
Population Monitoring ◽  
Increased Risk ◽  
Multiplex Serology

The causative agent of Q fever, the bacterium Coxiella burnetii (C. burnetii), has gained increasing interest due to outbreak events and reports about it being a potential risk factor for the development of lymphomas. In order to conduct large-scale studies for population monitoring and to investigate possible associations more closely, accurate and cost-effective high-throughput assays are highly desired. To address this need, nine C. burnetii proteins were expressed as recombinant antigens for multiplex serology. This technique enables the quantitative high-throughput detection of antibodies to multiple antigens simultaneously in a single reaction. Based on a reference group of 76 seropositive and 91 seronegative sera, three antigens were able to detect C. burnetii infections. Com1, GroEL, and DnaK achieved specificities of 93%, 69%, and 77% and sensitivities of 64%, 72%, and 47%, respectively. Double positivity to Com1 and GroEL led to a combined specificity of 90% and a sensitivity of 71%. In a subgroup of seropositives with an increased risk for chronic Q fever, the double positivity to these markers reached a specificity of 90% and a sensitivity of 86%. Multiplex serology enables the detection of antibodies against C. burnetii and appears well-suited to investigate associations between C. burnetii infections and the clinical manifestations in large-scale studies.

Hydrogel-incorporating unit in a well: 3D cell culture for high-throughput analysis

Lab on a Chip ◽  
2018 ◽  
Vol 18 (17) ◽  
pp. 2604-2613 ◽  
Author(s):  
Yeong Jun Yu ◽  
Young Hye Kim ◽  
Kyuhwan Na ◽  
Seo Yun Min ◽  
Ok Kyung Hwang ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Throughput ◽  
Culture System ◽  
3D Cell Culture ◽  
High Content Screening ◽  
Cell Culture System ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
Biochemical Assays

A microchannel-free, 3D cell culture system has a hydrogel-incorporating unit integrated with a multi-well plate. This plate provides better reproducibility in a variety of quantitative biochemical assays and high content-screening (HCS).

scholarly journals Detection of Coxiella burnetii Using Silicon Microring Resonator in Patient Blood Plasma

Micromachines ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 427 ◽  
Author(s):  
Bonhan Koo ◽  
Choong Eun Jin ◽  
Moonsuk Bae ◽  
Yoon Ok Jang ◽  
Ji Yeun Kim ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Blood Plasma ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Microring Resonator ◽  
Clinical Samples ◽  
Plasma Samples ◽  
Label Free ◽  
Highly Sensitive ◽  
Low Sensitivity

Blood plasma from patients is a powerful resource for diagnosing infectious disease due to it having many genetic materials as well as being relatively easy to obtain. Thus, various biosensors have been investigated for diagnosing diseases in blood plasma. However, there are no optimized and validated sensors for clinical use due to the low sensitivity, complexity, and difficulties of removing the inhibitors from plasma samples. In this study, we described a silicon microring resonator sensor used to detect Coxiella burnetii from the blood plasma of Q-fever patients in a label-free, real-time manner. Q-fever is an infectious disease caused by Coxiella burnetii via direct contact or inhalation aerosols. We validated this biosensor in the blood plasma of 35 clinical samples (including 16 Q fever samples infected with Coxiella burnetii and 19 samples infected with other febrile diseases. The biosensors are capable of rapid (10 min), highly sensitive (87.5%), and specific (89.5%) detection in plasma samples compared to the use of the conventional method.

scholarly journals High Prevalence and New Genotype of Coxiella burnetii in Ticks Infesting Camels in Somalia

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 741
Author(s):  
Dimitrios Frangoulidis ◽  
Claudia Kahlhofer ◽  
Ahmed Shire Said ◽  
Abdinasir Yusuf Osman ◽  
Lidia Chitimia-Dobler ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
One Health ◽  
Tandem Repeats ◽  
Q Fever ◽  
Epidemiological Studies ◽  
Variable Number ◽  
New Genotype ◽  
High Prevalence ◽  
Variable Number Tandem Repeats ◽  
Vntr Analysis

Coxiella burnetii is the causative agent of Q fever. It can infect animals, humans, and birds, as well as ticks, and it has a worldwide geographical distribution. To better understand the epidemiology of C. burnetii in Somalia, ticks infesting camels were collected from five different regions, including Bari, Nugaal, Mudug, Sool, and Sanaag, between January and March 2018. Collected ticks were tested for C. burnetii and Coxiella-like endosymbiont DNA by using IS1111, icd, and Com1-target PCR assays. Moreover, sequencing of the 16S-rRNA was conducted. Molecular characterization and typing were done by adaA-gene analysis and plasmid-type identification. Further typing was carried out by 14-marker Multi-Locus Variable-Number Tandem Repeats (MLVA/VNTR) analysis. The investigated ticks (n = 237) were identified as Hyalomma spp. (n = 227, 95.8%), Amblyomma spp. (n = 8, 3.4%), and Ripicephalus spp. (n = 2, 0.8%), and 59.1% (140/237) of them were positive for Coxiella spp. While Sanger sequencing and plasmid-type identification revealed a C. burnetii that harbours the QpRS-plasmid, MLVA/VNTR genotyping showed a new genotype which was initially named D21. In conclusion, this is the first report of C. burnetii in ticks in Somalia. The findings denote the possibility that C. burnetii is endemic in Somalia. Further epidemiological studies investigating samples from humans, animals, and ticks within the context of “One Health” are warranted.

scholarly journals Q Fever in the Greek Island of Crete: Detection, Isolation, and Molecular Identification of Eight Strains of Coxiella burnetii from Clinical Samples

1998 ◽  
Vol 36 (7) ◽  
pp. 2063-2067 ◽  
Author(s):  
Ioanna Spyridaki ◽  
Achilleas Gikas ◽  
Diamantis Kofteridis ◽  
Anna Psaroulaki ◽  
Yannis Tselentis
Keyword(s):  
Coxiella Burnetii ◽  
Nested Pcr ◽  
Q Fever ◽  
Buffy Coat ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Antibody Technique ◽  
Presenting Symptoms ◽  
Specificity And Sensitivity ◽  
Reference Strains

Over a period of 6 years (1989 to 1995), serum samples from 3,300 patients suspected to be infected by Coxiella burnetii were assayed for the presence of antibodies against antigen phase II of the microorganism by the indirect immunofluorescence antibody technique (IFAT). One hundred fifty-two cases were recorded, and blood samples from 17 patients were cultured for the isolation of the pathogen. By a centrifugation shell vial technique, eight strains were isolated from patients suffering from acute Q fever. The microorganism was detected in the cultures by IFAT, by Gimenez staining, and by the cytopathogenic effect on Vero and human embryonic lung (HEL) cells. PCR followed by restriction fragment length polymorphism analysis was used to confirm the diagnosis and identify the Coxiella burnetii strains within the cell cultures as well as to compare them with reference strains. In order to avoid time-consuming cultures, to achieve direct detection of Coxiella burnetii in clinical samples (blood, buffy coat, etc.), and to increase the specificity and sensitivity of the detection, nested PCR was performed. The first step of DNA extraction was performed with the QIAamp blood kit 250. For the second step of the PCR assays, the conditions of temperature and times of recycling were properly modified, and the microorganism was detected within 4 h. Our study demonstrates that Q fever is an endemic disease in Crete and that the diagnosis of Coxiella burnetii infection can be rapidly achieved by the detection of the microorganism in buffy coat samples by nested PCR. Although the presenting symptoms of the disease in this study differed from those in other studies, the Cretan strains do not differ genotypically from the reference strains (Nine Mile and Q212).

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