scholarly journals Design and Validation of Transcription-Mediated-Amplification Nucleic Acid Amplification Tests forMycoplasma genitalium

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Brett Kirkconnell ◽  
Barbara Weinbaum ◽  
Katherine Santos ◽  
Trisha Le Nguyen ◽  
Bryan Vinluan ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Mycoplasma Genitalium ◽  
Vaginal Swab ◽  
Nucleic Acid Amplification ◽  
Clinical Validation ◽  
Analytical Sensitivity ◽  
Reference Standard ◽  
Content Type ◽  
Sensitivity Specificity

ABSTRACTMycoplasma genitaliumis a sexually transmitted bacterium linked to adverse sexual and reproductive health outcomes in women and men.M. genitaliumis difficult to culture, and in the absence of validated amplified molecular methods for diagnosis of infection, there is no reference standard available for use as a comparator for the validation of newM. genitaliumdiagnostic tests. We evaluated the analytical and clinical performance of three transcription-mediated amplification (TMA) tests forM. genitalium, each targeting unique rRNA sequences, for use as a composite comparator for clinical validation of the AptimaMycoplasma genitalium(AMG) assay, anin vitrodiagnostic (IVD) TMA test that targets 16 s rRNA ofM. genitalium. Analytical sensitivity, specificity, and strain inclusivity of all four TMA tests were determined using nine laboratory strains ofM. genitaliumand 56 nontarget bacteria, protozoa, and viruses. Analytical sensitivity of the tests forM. genitaliumranged from 0.017 to 0.040 genome equivalents/ml. None of the nontarget organisms evaluated cross-reacted with any test. A composite comparator reference standard consisting of the 3 alternate (Alt) TMA tests was used to evaluate the clinical performance of the AMG assay by testing residual vaginal swab, female urine, and male urine specimens obtained from 1,400 adult subjects from three U.S. clinical sites. Using this reference standard to establish infected specimen status, the sensitivity, specificity, and overall agreement of the AMG IVD assay were 100%, 99.9%, and 99.9%, respectively. These results demonstrate the utility of molecular composite reference standard methodology for the clinical validation of future IVD tests for this organism.

scholarly journals Alternate Nucleic Acid Targets Can Be Used To Create a Composite Standard To Evaluate Clinical Performance of Nucleic Acid Amplification Tests

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Julius Schachter ◽  
Max Chernesky
Keyword(s):  
Nucleic Acid ◽  
Clinical Performance ◽  
Mycoplasma Genitalium ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Reference Standard ◽  
New Paradigm ◽  
Content Type ◽  
Nucleic Acid Amplification Tests ◽  
Link Type

ABSTRACTEvaluating the clinical performance of a new nucleic acid amplification test (NAAT) forMycoplasma genitalium, B. Kirkconnell, B. Weinbaum, K. Santos, T. Le Nguyen, et al. (J Clin Microbiol 57:e00264-19, 2019,https://doi.org/10.1128/JCM.00264-19) created 3 alternate NAATs that detected other uniqueM. genitaliumgene targets. Lacking a reference standard, they used the consensus of results with those 3 NAATs as the comparator. This approach could be a new paradigm to evaluate new NAATs when there is no previously defined reference standard.

scholarly journals Characteristics of Mycoplasma genitalium Urogenital Infections in a Diverse Patient Sample from the United States: Results from the Aptima Mycoplasma genitalium Evaluation Study (AMES)

2020 ◽  
Vol 58 (7) ◽  
Author(s):  
Lisa E. Manhart ◽  
Charlotte A. Gaydos ◽  
Stephanie N. Taylor ◽  
Rebecca A. Lillis ◽  
Edward W. Hook ◽  
...  
Keyword(s):  
United States ◽  
Mycoplasma Genitalium ◽  
Evaluation Study ◽  
The United States ◽  
Nucleic Acid Amplification ◽  
Content Type ◽  
Urogenital Infections ◽  
Asymptomatic Individuals ◽  
Urogenital Symptoms

ABSTRACT Data from a large prospective multicenter clinical validation study of a nucleic acid amplification in vitro diagnostic test for Mycoplasma genitalium were analyzed to describe the prevalence of M. genitalium infection, risk factors, and disease associations in female and male patients seeking care in diverse geographic regions of the United States. Among 1,737 female and 1,563 male participants, the overall prevalence of M. genitalium infection was 10.3% and was significantly higher in persons ages 15 to 24 years than in persons ages 35 to 39 years (for females, 19.8% versus 4.7% [odds ratio {OR} = 5.05; 95% confidence interval {CI} = 3.01 to 8.46]; for males, 16.5% versus 9.4% [OR = 1.91; 95% CI = 1.20 to 3.02]). The risk for M. genitalium infection was higher in black than in white participants (for females, 12.0% versus 6.8% [OR = 1.88; 95% CI = 1.30 to 2.72]; for males, 12.9% versus 6.9% [OR = 2.02; 95% CI = 1.38 to 2.96]) and higher in non-Hispanic than in Hispanic participants (for females, 11.2% versus 6.0% [OR = 1.97; 95% CI = 1.25 to 3.10]; for males, 11.6% versus 6.8% [OR = 1.80; 95% CI = 1.14 to 2.85]). Participants reporting urogenital symptoms had a significantly elevated risk of M. genitalium infection compared to that for asymptomatic individuals (for females, OR = 1.53 [95% CI = 1.09 to 2.14]; for males, OR = 1.42 [95% CI = 1.02 to 1.99]). Women diagnosed with vaginitis and cervicitis had a higher prevalence of M. genitalium infection than women without those diagnoses, although this was statistically significant only for vaginitis (for vaginitis, OR = 1.88 [95% CI = 1.37 to 2.58]; for cervicitis, OR = 1.42 [95% CI = 0.61 to 2.96]). A diagnosis of urethritis in men was also significantly associated with M. genitalium infection (OR = 2.97; 95% CI = 2.14 to 4.13). Few characteristics distinguished asymptomatic from symptomatic M. genitalium infections. These results from persons seeking care in the United States suggest that M. genitalium infection should be considered in young persons presenting with urogenital symptoms.

scholarly journals Evaluation of the ResistancePlus MG FleXible Assay for Detection of Wild-Type and 23S rRNA-Mutated Mycoplasma genitalium Strains

2020 ◽  
Vol 58 (3) ◽  
Author(s):  
Suhella Tulsiani Drud ◽  
Peter Njuguna ◽  
Samantha Ebeyan ◽  
Simon Erskine ◽  
Mette Holm ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Mycoplasma Genitalium ◽  
Ease Of Use ◽  
23S Rrna ◽  
Analytical Sensitivity ◽  
Wild Type ◽  
Content Type ◽  
Clinical Sensitivity ◽  
Block Based ◽  
Sensitivity Specificity

ABSTRACT Antibiotic resistance in Mycoplasma genitalium is rising globally, and resistance-guided diagnostics can facilitate targeted and timely treatment. The ResistancePlus MG FleXible (RPMG Flex) assay for the detection of M. genitalium and macrolide resistance-mediating mutations (MRMM) was evaluated for analytical sensitivity, specificity, reproducibility, and inhibition in the presence of interfering substances by simulating M. genitalium-negative pooled urine and swab matrices with M. genitalium cultures. Furthermore, the clinical sensitivity of the assay was evaluated and compared with a reference real-time PCR assay. The analytical sensitivity of the RPMG Flex assay was 157 genomes/ml for wild-type (WT) and 387 genomes/ml for MRMM strains in both matrices. For clinical specimens, the RPMG assay had an overall sensitivity of 96.1% (95% urine: 10/10 WT, 9/10 MRMM; 96.5% swab: 25/26 WT, 26/29 MRMM) compared to 85.7% for the MgPa/MagNAPure24 assay (95% urine: 19/20; 87% swab: 48/57). Clinical specificity was 100% for urine and 98.5% for swab specimens, respectively. No inhibition due to the presence of any of the tested interfering substances was observed. The RPMG Flex assay was more sensitive than the reference MgPa assay, in particular, for swab specimens. The implementation of this assay may increase ease of use and considerably decrease hands-on time for sample preparation compared to a standard block-based assay. The RPMG Flex assay for the GeneXpert Dx system provides a much-needed platform for the simultaneous detection of MG and MRMM and may thereby facilitate resistance-guided therapy for M. genitalium infections.

scholarly journals In Vitro Activity of Lefamulin against Sexually Transmitted Bacterial Pathogens

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Susanne Paukner ◽  
Astrid Gruss ◽  
Jørgen Skov Jensen
Keyword(s):  
Chlamydia Trachomatis ◽  
Bacterial Pathogens ◽  
Mycoplasma Genitalium ◽  
Standard Of Care ◽  
Multidrug Resistant ◽  
In Vitro Activity ◽  
First Line ◽  
Content Type ◽  
Sexually Transmitted

ABSTRACT The pleuromutilin antibiotic lefamulin demonstrated in vitro activity against the most relevant bacterial pathogens causing sexually transmitted infections (STI), including Chlamydia trachomatis (MIC 50/90 , 0.02/0.04 mg/liter; n = 15), susceptible and multidrug-resistant Mycoplasma genitalium (MIC range, 0.002 to 0.063 mg/liter; n = 6), and susceptible and resistant Neisseria gonorrhoeae (MIC 50/90 , 0.12/0.5 mg/liter; n = 25). The results suggest that lefamulin could be a promising first-line antibiotic for the treatment of STI, particularly in populations with high rates of resistance to standard-of-care antibiotics.

scholarly journals Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests

2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Johanna Sandlund ◽  
Joel Estis ◽  
Phoebe Katzenbach ◽  
Niamh Nolan ◽  
Kirstie Hinson ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Patient Outcomes ◽  
Single Molecule ◽  
Clinical Performance ◽  
Neutralization Assay ◽  
Nucleic Acid Amplification ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Health Care Associated Infections ◽  
Stool Specimens

ABSTRACT Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity− and 4 NAAT−/Clarity+ samples, there were 26 CCNA− and 4 CCNA− samples, respectively. CDI relapse was more common in NAAT+/toxin+ patients than in NAAT+/toxin− and NAAT−/toxin− patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT+/toxin− than in NAAT+/toxin+ patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

scholarly journals Comparison of the Panther Fusion and BD MAX Group B Streptococcus (GBS) Assays for Detection of GBS in Prenatal Screening Specimens

2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Gregory J. Berry ◽  
Fan Zhang ◽  
Ryhana Manji ◽  
Stefan Juretschko
Keyword(s):  
Late Onset ◽  
Clinical Performance ◽  
Group B Streptococcus ◽  
Turnaround Time ◽  
Nucleic Acid Amplification ◽  
Kappa Statistics ◽  
Loading Capacity ◽  
Content Type ◽  
Return Visits ◽  
Group B

ABSTRACT Streptococcus agalactiae or group B Streptococcus (GBS) is the cause of early- and late-onset GBS disease in neonates and can present as septicemia, meningitis, and pneumonia. Our objective was to compare the performance of two FDA-approved nucleic acid amplification tests (NAATs), the Panther Fusion and BD MAX systems, for detection of GBS in vaginal-rectal screening specimens. A total of 510 vaginal-rectal prepartum specimens were tested simultaneously in both NAATs following broth enrichment. Assay agreement was calculated using kappa statistics. Overall agreement between assays was 99.0% (505/510; 95% confidence interval, 0.951 to 0.997; kappa = 0.974). Discordant results were retested with both assays and by standard culture. The assays were also compared for workflow characteristics, including time to first results (TFR), total turnaround time (TAT), number of return visits to load additional specimens, and hands-on time (HoT). Using a standard run size of 60 specimens/day, the Panther Fusion assay had a longer TFR (2.4 versus 2.0 h) but showed a shorter overall TAT for all 60 samples (3.98 versus 7.18 h) due to an increased initial sample loading capacity, and it required less labor (35.0 versus 71.3 s/sample) and fewer return visits for loading additional specimens (0 versus 2). The Panther Fusion system also had a larger sample loading capacity (120 versus 24 samples) and greater 8-h throughput (335 versus 96 samples). In summary, the Panther Fusion GBS assay has clinical performance comparable to that of the BD MAX GBS assay but provides a faster TAT, less HoT, and higher throughput.

scholarly journals A Systematic Review and Meta-analysis of the Diagnostic Accuracy of Nucleic Acid Amplification Tests for Tuberculous Meningitis

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Ali Pormohammad ◽  
Mohammad Javad Nasiri ◽  
Timothy D. McHugh ◽  
Seyed Mohammad Riahi ◽  
Nathan C. Bahr
Keyword(s):  
Systematic Review ◽  
Nucleic Acid ◽  
Diagnostic Accuracy ◽  
Likelihood Ratio ◽  
Tuberculous Meningitis ◽  
Meta Analysis ◽  
Nucleic Acid Amplification ◽  
Data Sets ◽  
Reference Standard ◽  
Sensitivity Specificity

ABSTRACTThe diagnosis of tuberculous meningitis (TBM) is difficult and poses a significant challenge to physicians worldwide. Recently, nucleic acid amplification (NAA) tests have shown promise for the diagnosis of TBM, although their performance has been variable. We undertook a systematic review and meta-analysis to evaluate the diagnostic accuracy of NAA tests with cerebrospinal fluid (CSF) samples against that of culture as the reference standard or a combined reference standard (CRS) for TBM. We searched the Embase, PubMed, Web of Science, and Cochrane Library databases for the relevant records. The QUADAS-2 tool was used to assess the quality of the studies. Diagnostic accuracy measures (i.e., sensitivity and specificity) were pooled with a random-effects model. All statistical analyses were performed with STATA (version 14 IC; Stata Corporation, College Station, TX, USA), Meta-DiSc (version 1.4 for Windows; Cochrane Colloquium, Barcelona, Spain), and RevMan (version 5.3; The Nordic Cochrane Centre, the Cochrane Collaboration, Copenhagen, Denmark) software. Sixty-three studies comprising 1,381 cases of confirmed TBM and 5,712 non-TBM controls were included in the final analysis. These 63 studies were divided into two groups comprising 71 data sets (43 in-house tests and 28 commercial tests) that used culture as the reference standard and 24 data sets (21 in-house tests and 3 commercial tests) that used a CRS. Studies which used a culture reference standard had better pooled summary estimates than studies which used CRS. The overall pooled estimates of sensitivity, specificity, positive likelihood ratio (PLR), and negative likelihood ratio (NLR) of the NAA tests against culture were 82% (95% confidence interval [CI], 75 to 87%), 99% (95% CI, 98 to 99%), 58.6 (95% CI, 35.3 to 97.3), and 0.19 (95% CI, 0.14 to 0.25), respectively. The pooled sensitivity, specificity, PLR, and NLR of NAA tests against CRS were 68% (95% CI, 41 to 87%), 98% (95% CI, 95 to 99%), 36.5 (95% CI, 15.6 to 85.3), and 0.32 (95% CI, 0.15 to 0.70), respectively. The analysis has demonstrated that the diagnostic accuracy of NAA tests is currently insufficient for them to replace culture as a lone diagnostic test. NAA tests may be used in combination with culture due to the advantage of time to result and in scenarios where culture tests are not feasible. Further work to improve NAA tests would benefit from the availability of standardized reference standards and improvements to the methodology.

scholarly journals Multicenter Diagnostic Accuracy Evaluation of the Luminex Aries Real-Time PCR Assay for Group B Streptococcus Detection in Lim Broth-Enriched Samples

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
D. R. Hernandez ◽  
D. M. Wolk ◽  
K. L. Walker ◽  
S. Young ◽  
R. Dunn ◽  
...  
Keyword(s):  
Clinical Performance ◽  
Enrichment Culture ◽  
Culture Method ◽  
Nucleic Acid Amplification ◽  
Accuracy Evaluation ◽  
Culture Methods ◽  
Assay Sensitivity ◽  
Content Type ◽  
Group B ◽  
Positive Agreement

ABSTRACT The vertical transmission of group B Streptococcus (GBS) strains causing neonatal sepsis is one of the leading reasons for neonatal mortality worldwide. The gold standard for GBS detection is enriched culture with or without the aid of chromogenic agars. Given the high risk for morbidity and mortality in this population, high assay sensitivity is required to prevent the personal and economic costs of GBS disease. Nucleic acid amplification tests (NAATs) allow for objective determination of GBS colonization with a sensitivity and a specificity higher than those of traditional culture methods. In this study, we determined the analytical and clinical performance of the Aries GBS assay compared to those of the enrichment culture method, biochemical identification, and the NAATs used at the study sites. Remnant Lim broth samples were used to perform the Aries assay and reference testing. Upon first testing using enriched culture as the reference standard, the Aries GBS assay identified GBS with a 96.1% sensitivity (95% confidence interval [CI], 91.2 to 98.7%) and a 91.4% specificity (95% CI, 88.8 to 93.6%). The test performed with 100% positive agreement (95% CI, 83.2 to 100%) compared to the results of the BD Max GBS assay and 98.0% positive agreement (95% CI, 89.2 to 99.9%) compared to the results of the Cepheid Xpert GBS LB test. Repeatability and reproducibility were maintained in intra- and interlaboratory testing, regardless of the instrument, module, or user who performed the test. The Aries GBS assay can be set up in less than 5 min and produces results in 2 h. The easy setup, with minimal hands-on time, and high assay sensitivity and specificity make this a useful testing option for GBS screening in prepartum women.

scholarly journals Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool

2015 ◽  
Vol 53 (10) ◽  
pp. 3204-3212 ◽  
Author(s):  
Linan Song ◽  
Mingwei Zhao ◽  
David C. Duffy ◽  
Joshua Hansen ◽  
Kelsey Shields ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cytotoxicity Assay ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Toxin B ◽  
Content Type ◽  
Toxigenic Culture ◽  
Immunosorbent Assays ◽  
Digital Elisa ◽  
Detection And Quantification

The currently available diagnostics forClostridium difficileinfection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenicC. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinicalC. difficileisolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.

scholarly journals Clinical Evaluation of Three Sample-to-Answer Platforms for Detection of SARS-CoV-2

2020 ◽  
Vol 58 (8) ◽  
Author(s):  
Wei Zhen ◽  
Elizabeth Smith ◽  
Ryhana Manji ◽  
Deborah Schron ◽  
Gregory J. Berry
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Drug Administration ◽  
Clinical Evaluation ◽  
Limit Of Detection ◽  
Clinical Performance ◽  
Molecular Diagnostic ◽  
Analytical Sensitivity ◽  
Reference Standard ◽  
Slight Improvement ◽  
Percent Agreement

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread across the globe. As part of the worldwide response, many molecular diagnostic platforms have been granted emergency use authorization (EUA) by the Food and Drug Administration (FDA) to identify SARS-CoV-2 positive patients. Our objective was to evaluate three sample-to-answer molecular diagnostic platforms (Cepheid Xpert Xpress SARS-CoV-2 [Xpert Xpress], Abbott ID NOW COVID-19 [ID NOW], and GenMark ePlex SARS-CoV-2 Test [ePlex]) to determine analytical sensitivity, clinical performance, and workflow for the detection of SARS-CoV-2 in nasopharyngeal swabs from 108 symptomatic patients. We found that Xpert Xpress had the lowest limit of detection (100% detection at 100 copies/ml), followed by ePlex (100% detection at 1,000 copies/ml), and ID NOW (20,000 copies/ml). Xpert Xpress also had highest positive percent agreement (PPA) compared to our reference standard (98.3%) followed by ePlex (91.4%) and ID NOW (87.7%). All three assays showed 100% negative percent agreement (NPA). In the workflow analysis, ID NOW produced the lowest time to result per specimen (∼17 min) compared to Xpert Xpress (∼46 min) and ePlex (∼1.5 h), but what ID NOW gained in rapid results, it lost in analytical and clinical performance. ePlex had the longest time to results and showed a slight improvement in PPA over ID NOW. Information about the clinical and analytical performance of these assays, as well as workflow, will be critical in making informed and timely decisions on testing platforms.

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