Evaluation of Toxoplasma ELITe MGB Real-Time PCR Assay for Diagnosis of Toxoplasmosis

ABSTRACT Molecular diagnosis of congenital toxoplasmosis or disseminated toxoplasmosis is based mainly on PCR. The repeated DNA element rep529 has become the main DNA target used in most PCR methods, whether laboratory developed or commercial. In this multicenter study, we evaluated the Toxoplasma ELITe MGB (Elitech) commercial kit by comparison with three reference quantitative PCR assays (RAs) used routinely in three proficient laboratories of the French National Reference Center for Toxoplasmosis network, using Toxoplasma calibrated suspensions diluted to obtain a range of concentrations from 0.1 to 10,000 parasites/ml. These suspensions were extracted with either the DNA extraction kit (EXTRAblood; Elitech) recommended by the manufacturer or the QIAamp DNA minikit (Qiagen). The Toxoplasma ELITe MGB assay was also evaluated on a panel of 128 clinical samples, including 56 amniotic fluid samples, 55 placenta samples, and various other samples, of which 95 originated from patients with proven toxoplasmosis. The ELITe MGB assay amplified low-concentration replicates (<10 parasites/ml) of calibrated suspensions less frequently than the RAs of 2/3 laboratories. Additionally, the combination of EXTRAblood and Toxoplasma ELITe MGB yielded poorer sensitivity than the combination of QIAamp DNA minikit and ELITe MGB for low parasite concentrations ( P < 0.001 for 1 parasite/ml). On clinical samples, the sensitivity and the specificity of the commercial assay were 89% and 100%, respectively. The sensitivity ranged from 79% (placenta samples) to 100% (amniotic fluid samples). Overall, this study shows that the Toxoplasma ELITe MGB assay is suitable for the diagnosis of toxoplasmosis from non-cell-rich or non-hemoglobin-rich samples and that the EXTRAblood kit is not optimal.

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Multicentric Comparative Assessment of the Bio-Evolution Toxoplasma gondii Detection Kit with Eight Laboratory-Developed PCR Assays for Molecular Diagnosis of Congenital Toxoplasmosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01913-14 ◽

2014 ◽

Vol 53 (1) ◽

pp. 29-34 ◽

Cited By ~ 13

Author(s):

Denis Filisetti ◽

Yvon Sterkers ◽

Marie-Pierre Brenier-Pinchart ◽

Sophie Cassaing ◽

Frédéric Dalle ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Amniotic Fluid ◽

Molecular Diagnosis ◽

Internal Control ◽

Congenital Toxoplasmosis ◽

Research Network ◽

Multicentric Study ◽

Content Type ◽

Pcr Assays ◽

Commercial Kits

The detection ofToxoplasma gondiiin amniotic fluid is an essential tool for the prenatal diagnosis of congenital toxoplasmosis and is currently essentially based on the use of PCR. Although some consensus is emerging, this molecular diagnosis suffers from a lack of standardization and an extreme diversity of laboratory-developed methods. Commercial kits for the detection ofT. gondiiby PCR were recently developed and offer certain advantages; however, they must be assessed in comparison with optimized reference PCR assays. The present multicentric study aimed to compare the performances of the Bio-EvolutionT. gondiidetection kit and laboratory-developed PCR assays set up in eight proficient centers in France. The study compared 157 amniotic fluid samples and found concordances of 99% and 100% using 76T. gondii-infected samples and 81 uninfected samples, respectively. Moreover, taking into account the classification of the European Research Network on Congenital Toxoplasmosis, the overall diagnostic sensitivity of all assays was identical and calculated to be 86% (54/63); specificity was 100% for all assays. Finally, the relative quantification results were in good agreement between the kit and the laboratory-developed assays. The good performances of this commercial kit are probably in part linked to the use of a number of good practices: detection in multiplicate, amplification of the repetitive DNA target rep529, and the use of an internal control for the detection of PCR inhibitors. The only drawbacks noted at the time of the study were the absence of uracil-N-glycosylase and small defects in the reliability of the production of different reagents.

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Help in the Choice of Automated or Semiautomated Immunoassays for Serological Diagnosis of Toxoplasmosis: Evaluation of Nine Immunoassays by the French National Reference Center for Toxoplasmosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01193-16 ◽

2016 ◽

Vol 54 (12) ◽

pp. 3034-3042 ◽

Cited By ~ 11

Author(s):

O. Villard ◽

B. Cimon ◽

C. L'Ollivier ◽

H. Fricker-Hidalgo ◽

N. Godineau ◽

...

Keyword(s):

Congenital Toxoplasmosis ◽

Antibody Detection ◽

Clinical Settings ◽

Serum Samples ◽

Igg Antibody ◽

Routine Testing ◽

Content Type ◽

Specificity And Sensitivity ◽

National Reference Center ◽

Serology Testing

Toxoplasmosis, a benign infection, is asymptomatic or paucisymptomatic in over 80% of cases, except in immunocompetent patients suffering from ocular toxoplasmosis or in immunocompromised patients with opportunistic or congenital toxoplasmosis. Diagnosis is based mainly on serology testing. Thus, we compared the performance of the nine most commonly used commercial automated or semiautomated immunoassays for IgG and IgMToxoplasma gondiiantibody detection, that is, the Advia Centaur, Architect, AxSYM, Elecsys, Enzygnost, Liaison, Platelia, VIDAS, and VIDIA assays. The assays were conducted on four panels of serum samples derived during routine testing from patients with an interfering disease and who exhibited a low IgG antibody level in one of two clinical settings, namely, acute or chronic toxoplasmosis. As a result, IgG sensitivities ranged from 97.1% to 100%, and IgG specificities ranged from 99.5% to 100%. For IgG quantification, strong differences in IgG titers (expressed in IU/ml) were noted depending on the assay used. IgM sensitivities ranged from 65% to 97.9%, and IgM specificities ranged from 92.6% to 100%. For defining the best serological strategies to be implemented, it appears crucial to compare the diagnostic performance of the different tests with respect to their specificity and sensitivity in detecting the presence of IgG and IgM antibodies.

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Culture-Independent Detection of Nontuberculous Mycobacteria in Clinical Respiratory Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.01410-16 ◽

2016 ◽

Vol 54 (9) ◽

pp. 2395-2398 ◽

Cited By ~ 5

Author(s):

Gianny P. Scoleri ◽

Jocelyn M. Choo ◽

Lex E. X. Leong ◽

Thomas R. Goddard ◽

Lisa Shephard ◽

...

Keyword(s):

Quantitative Pcr ◽

Nontuberculous Mycobacteria ◽

Direct Detection ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

Culture Independent ◽

Quantitative Pcr Assay ◽

Single Reaction

Culture-based detection of nontuberculousMycobacteria(NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust.

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A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

Journal of Clinical Microbiology ◽

10.1128/jcm.00505-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 3

Author(s):

Nawal El Houmami ◽

Guillaume André Durand ◽

Janek Bzdrenga ◽

Anne Darmon ◽

Philippe Minodier ◽

...

Keyword(s):

Real Time ◽

Malate Dehydrogenase ◽

Real Time Pcr ◽

Detection Sensitivity ◽

Rt Pcr ◽

Pcr Assay ◽

Content Type ◽

Clinical Specimens ◽

Specificity And Sensitivity ◽

Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

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A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

Journal of Clinical Microbiology ◽

10.1128/jcm.01986-20 ◽

2020 ◽

Vol 59 (1) ◽

pp. e01986-20

Author(s):

Ibne Karim M. Ali ◽

Shantanu Roy

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Small Subunit ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

Rdna Sequences ◽

Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

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Clinical Assessment of a Nocardia PCR-Based Assay for Diagnosis of Nocardiosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00002-18 ◽

2018 ◽

Vol 56 (6) ◽

Cited By ~ 15

Author(s):

Claire Rouzaud ◽

Véronica Rodriguez-Nava ◽

Emilie Catherinot ◽

Frédéric Méchaï ◽

Emmanuelle Bergeron ◽

...

Keyword(s):

Clinical Sample ◽

Positive Control ◽

Negative Control ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

Underlying Condition ◽

Alternative Diagnosis ◽

Specificity And Sensitivity ◽

Assay Result

ABSTRACT The diagnosis of nocardiosis, a severe opportunistic infection, is challenging. We assessed the specificity and sensitivity of a 16S rRNA Nocardia PCR-based assay performed on clinical samples. In this multicenter study (January 2014 to April 2015), patients who were admitted to three hospitals and had an underlying condition favoring nocardiosis, clinical and radiological signs consistent with nocardiosis, and a Nocardia PCR assay result for a clinical sample were included. Patients were classified as negative control (NC) (negative Nocardia culture results and proven alternative diagnosis or improvement at 6 months without anti- Nocardia treatment), positive control (PC) (positive Nocardia culture results), or probable nocardiosis (positive Nocardia PCR results, negative Nocardia culture results, and no alternative diagnosis). Sixty-eight patients were included; 47 were classified as NC, 8 as PC, and 13 as probable nocardiosis. PCR results were negative for 35/47 NC patients (74%). For the 12 NC patients with positive PCR results, the PCR assay had been performed with respiratory samples. These NC patients had chronic bronchopulmonary disease more frequently than did the NC patients with negative PCR results (8/12 patients [67%] versus 11/35 patients [31%]; P = 0.044). PCR results were positive for 7/8 PC patients (88%). There were 13 cases of probable nocardiosis, diagnosed solely using the PCR results; 9 of those patients (69%) had lung involvement (consolidation or nodule). Nocardia PCR testing had a specificity of 74% and a sensitivity of 88% for the diagnosis of nocardiosis. Nocardia PCR testing may be helpful for the diagnosis of nocardiosis in immunocompromised patients but interpretation of PCR results from respiratory samples is difficult, because the PCR assay may also detect colonization.

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Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.00115-15 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2148-2153 ◽

Cited By ~ 28

Author(s):

Xuan Qin ◽

Eileen J. Klein ◽

Emmanouil Galanakis ◽

Anita A. Thomas ◽

Jennifer R. Stapp ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Primary Cultures ◽

Clinical Samples ◽

Pcr Assay ◽

Content Type ◽

E Coli ◽

Successful Recovery

Timely accurate diagnosis of Shiga toxin-producingEscherichia coli(STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targetingstx1,stx2, andrfbEO157with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagicE. coli[EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC.E. coliO157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 usingrfbEO157, and LD-PCR results prompted successful recovery ofE. coliO157 (n= 25) and non-O157 STEC (n= 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and thatE. coliO157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

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GES-18, a New Carbapenem-Hydrolyzing GES-Type β-Lactamase from Pseudomonas aeruginosa That Contains Ile80 and Ser170 Residues

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01784-12 ◽

2012 ◽

Vol 57 (1) ◽

pp. 396-401 ◽

Cited By ~ 26

Author(s):

Carine Bebrone ◽

Pierre Bogaerts ◽

Heinrich Delbrück ◽

Sandra Bennink ◽

Michaël B. Kupper ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lower Respiratory Tract ◽

Resistance Mechanism ◽

Single Amino Acid ◽

Content Type ◽

Reference Center ◽

National Reference ◽

National Reference Center ◽

Similar Kinetic

ABSTRACTA clinical isolate ofPseudomonas aeruginosarecovered from the lower respiratory tract of an 81-year-old patient hospitalized in Belgium was sent to the national reference center to determine its resistance mechanism. PCR sequencing identified a new GES variant, GES-18, which differs from the carbapenem-hydrolyzing enzyme GES-5 by a single amino acid substitution (Val80Ile, in the numbering according to Ambler) and from GES-1 by two substitutions (Val80Ile and Gly170Ser). Detailed kinetic characterization showed that GES-18 and GES-5 hydrolyze imipenem and cefoxitin with similar kinetic parameters and that GES-18 was less susceptible than GES-1 to classical β-lactamase inhibitors such as clavulanate and tazobactam. The overall structure of GES-18 is similar to the solved structures of GES-1 and GES-2, the Val80Ile and Gly170Ser substitutions causing only subtle local rearrangements. Notably, the hydrolytic water molecule and the Glu166 residue were slightly displaced compared to their counterparts in GES-1. Our kinetic and crystallographic data for GES-18 highlight the pivotal role of the Gly170Ser substitution which distinguishes GES-5 and GES-18 from GES-1.

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Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay

Applied and Environmental Microbiology ◽

10.1128/aem.00806-17 ◽

2017 ◽

Vol 83 (18) ◽

Cited By ~ 9

Author(s):

Charles H. D. Williamson ◽

Adam J. Vazquez ◽

Karen Hill ◽

Theresa J. Smith ◽

Roxanne Nottingham ◽

...

Keyword(s):

Multiplex Pcr ◽

Gene Cluster ◽

Botulinum Neurotoxin ◽

Comparative Genomic ◽

Pcr Assay ◽

Content Type ◽

Multiplex Pcr Assay ◽

Group I ◽

Pcr Assays ◽

Group Ii

ABSTRACT Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha +] or orfX +). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha + or orfX +) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

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