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).

scholarly journals Validation of a Novel Commercial ELISA Test for the Detection of Antibodies against Coxiella burnetii

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1075
Author(s):  
Salvatore Ledda ◽  
Cinzia Santucciu ◽  
Valentina Chisu ◽  
Giovanna Masala
Keyword(s):  
Diagnostic Accuracy ◽  
Phase Ii ◽  
Coxiella Burnetii ◽  
Q Fever ◽  
Animal Health ◽  
Elisa Test ◽  
Clinical Diagnostics ◽  
Complex Life Cycle ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specificity And Sensitivity

Q fever is a zoonosis caused by Coxiella burnetii, a Gram-negative pathogen with a complex life cycle and a high impact on public and animal health all over the world. The symptoms are indistinguishable from those belonging to other diseases, and the disease could be symptomless. For these reasons, reliable laboratory tests are essential for an accurate diagnosis. The aim of this study was to validate a novel enzyme-linked immunosorbent assay (ELISA) test, named the Chorus Q Fever Phase II IgG and IgM Kit (DIESSE, Diagnostica Senese S.p.A), which is performed by an instrument named Chorus, a new device in medical diagnostics. This diagnostic test is employed for the detection of antibodies against C. burnetii Phase II antigens in acute disease. Our validation protocol was performed according to the Italian Accreditation Body (ACCREDIA) (Regulation UNI CEI EN ISO/IEC 17025:2018 and 17043:2010), OIE (World Organization for Animal Health), and Statement for Reporting Studies of Diagnostic Accuracy (STARD). Operator performance was evaluated along with the analytical specificity and sensitivity (ASp and ASe) and diagnostic accuracy of the kit, with parameters such as diagnostic specificity and sensitivity (DSp and DSe) and positive and negative predictive values (PPV and NPV), in addition to the repeatability. According to the evaluated parameters, the diagnostic ELISA test was shown to be suitable for validation and commercialization as a screening method in human sera and a valid support for clinical diagnostics.

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 Evaluation ofhsp65Nested PCR-Restriction Analysis (PRA) for Diagnosing Tuberculosis in a High Burden Country

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Sara Macente ◽  
Clarice Queico Fujimura Leite ◽  
Adolfo Carlos Barreto Santos ◽  
Vera Lúcia Dias Siqueira ◽  
Luzia Neri Cosmo Machado ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Restriction Analysis ◽  
Mycobacterial Infection ◽  
Acid Fast Bacillus ◽  
Clinical Samples ◽  
Polymorphism Analysis ◽  
Specific Diagnosis ◽  
Acid Fast Bacillus Smear ◽  
Selection Of

Current study evaluated thehsp65Nested PCR Restriction Fragment Length Polymorphism Analysis (hsp65Nested PCR-PRA) to detect and identifyMycobacterium tuberculosiscomplex directly in clinical samples for a rapid and specific diagnosis of tuberculosis (TB).hsp65Nested PCR-PRA was applied directly to 218 clinical samples obtained from 127 patients suspected of TB or another mycobacterial infection from July 2009 to July 2010. Thehsp65Nested PCR-PRA showed 100% sensitivity and 95.0 and 93.1% specificity in comparison with culture and microscopy (acid fast bacillus smear), respectively.hsp65Nested PCR-PRA was shown to be a fast and reliable assay for diagnosing TB, which may contribute towards a fast diagnosis that could help the selection of appropriate chemotherapeutic and early epidemiological management of the cases which are of paramount importance in a high TB burden country.

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-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 Nested PCR followed by NGS: Validation and application for HPV genotyping of Tunisian cervical samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255914
Author(s):  
Monia Ardhaoui ◽  
Emna Ennaifer ◽  
Anna Christina De Matos Salim ◽  
Flávio Marcom Gomez ◽  
Thalja Laasili ◽  
...  
Keyword(s):  
Nested Pcr ◽  
High Sensitivity ◽  
Epidemiological Studies ◽  
Multiple Infection ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Hpv Genotyping ◽  
Genotyping Method ◽  
Specificity And Sensitivity ◽  
Hpv Types

The most used methodologies for HPV genotyping in Tunisian studies are based on hybridization that are limited to a restricted number of HPV types and to a lack of specificity and sensitivity for same types. Recently, Next-Generation sequencing (NGS) technology has been efficiently used for HPV genotyping. In this work we designed and validated a sensitive genotyping method based on nested PCR followed by NGS. Eighty-six samples were tested for the validation of an HPV genotyping assay based on Nested-PCR followed by NGS. These include, 43 references plasmids and 43 positive HPV clinical cervical specimens previously evaluated with the conventional genotyping method: Reverse Line Hybridization (RLH). Results of genotyping using NGS were compared to those of RLH. The analytical sensitivity of the NGS assay was 1GE/μl per sample. The NGS allowed the detection of all HPV types presented in references plasmids. On the clinical samples, a total of 19 HPV types were detected versus 14 types using RLH. Besides the identification of more HPV types in multiple infection (6 types for NGS versus 4 for RLH), NGS allowed the identification of HPV types that were not detected by RLH. In addition, the NGS assay detected newly HPV types that were not described in Tunisia so far: HPV81, HPV43, HPV74, and HPV62. The high sensitivity and specificity of NGS for HPV genotyping in addition to the identification of new HPV types may justify the use of such technique to provide with high accuracy the profile of circulating types in epidemiological studies.

scholarly journals Direct Identification of Coxiella burnetii Plasmids in Human Sera by Nested PCR

1998 ◽  
Vol 36 (8) ◽  
pp. 2210-2213 ◽  
Author(s):  
G. Q. Zhang ◽  
A. Hotta ◽  
M. Mizutani ◽  
T. Ho ◽  
T. Yamaguchi ◽  
...  
Keyword(s):  
Coxiella Burnetii ◽  
Nested Pcr ◽  
Q Fever ◽  
Serum Samples ◽  
Specific Primers ◽  
Direct Identification ◽  
Human Sera ◽  
Nested Pcr Assays ◽  
Acute Phase Serum ◽  
Specific Sequences

Nested PCR assays were used for the direct identification ofCoxiella burnetii plasmids in human sera. A total of 81 serum samples from 81 patients with Q fever were tested by nested PCR with four sets of primers. The first set of primers was used to detect the genomic sequences. The second set of primers was used to detect the conserved sequences of the plasmids. Another two sets of primers were used to identify the QpH1 and QpRS plasmids. QpH1 and QpRS plasmid-specific sequences were identified in 40 (49.4%) and 24 (29.6%) of the serum samples, respectively. Both of the QpH1 and QpRS plasmid-specific sequences were detected in 5 (8.6%) of the serum samples but were not found in 12 (20.7%) of the serum samples. Furthermore, all of the 23 acute-phase serum samples were positive for the QpH1 plasmid and negative for the QpRS plasmid. Nested PCR with plasmid-specific primers appears to be a useful method for the direct typing of C. burnetii plasmids in human sera.

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 Validation of Nonnested and Real-Time PCR for Diagnosis of Sheep-Associated Malignant Catarrhal Fever in Clinical Samples

2007 ◽  
Vol 19 (4) ◽  
pp. 405-408 ◽  
Author(s):  
Donald L. Traul ◽  
Naomi S. Taus ◽  
J. Lindsay Oaks ◽  
Donal O' Toole ◽  
Fred R. Rurangirwa ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
High Specificity ◽  
Clinical Samples ◽  
Malignant Catarrhal Fever ◽  
Fatal Disease ◽  
Tissue Samples ◽  
Specificity And Sensitivity ◽  
Experimentally Induced

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

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