Will droplet digital PCR become the test of choice for detecting and quantifying ocular<i>Chlamydia trachomatis</i>infection? Maybe

Burr, Sarah E.; Bailey, Robin L.; Mabey, David; Holland, Martin J.

doi:10.1586/14737159.2014.897609

Cited by 5 publications

(4 citation statements)

References 21 publications

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“…However, it was demonstrated that the discrepant results occurred in a mathematically predictable manner related to the analyte concentration and that most PCR-based technology will share an absolute limit to the number of analyte copies per test that will be reproducibly detected. It was highlighted that in a traditional discrepant analysis the sensitivity of this ddPCR assay could have been as high as 98% [ 30 ] and we therefore believe the test was appropriate in this setting.…”

Section: Discussionmentioning

confidence: 99%

Low Prevalence of Ocular Chlamydia trachomatis Infection and Active Trachoma in the Western Division of Fiji

et al. 2016

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BackgroundTrachoma is the leading infectious cause of blindness and is caused by ocular infection with the bacterium Chlamydia trachomatis (Ct). While the majority of the global disease burden is found in sub-Saharan Africa, the Western Pacific Region has been identified as trachoma endemic. Population surveys carried out throughout Fiji have shown an abundance of both clinically active trachoma and trachomatous trichiasis in all divisions. This finding is at odds with the clinical experience of local healthcare workers who do not consider trachoma to be highly prevalent. We aimed to determine whether conjunctival infection with Ct could be detected in one administrative division of Fiji.MethodsA population-based survey of 2306 individuals was conducted using the Global Trachoma Mapping Project methodology. Population prevalence of active trachoma in children and trichiasis in adults was estimated using the World Health Organization simplified grading system. Conjunctival swabs were collected from 1009 children aged 1–9 years. DNA from swabs was tested for the presence of the Ct plasmid and human endogenous control.ResultsThe prevalence of active trachoma in 1–9 year olds was 3.4%. The age-adjusted prevalence was 2.8% (95% CI: 1.4–4.3%). The unadjusted prevalence of ocular Ct infection in 1–9 year-olds was 1.9% (19/1009), and the age-adjusted infection prevalence was 2.3% (95% CI: 0.4–2.5%). The median DNA load was 41 Ct plasmid copies per swab (min 20, first quartile 32, mean 6665, third quartile 161, max 86354). There was no association between current infection and follicular trachoma. No cases of trachomatous trichiasis were identified.DiscussionThe Western Division of Fiji has a low prevalence of clinical trachoma. Ocular Ct infections were observed, but they were predominantly low load infections and were not correlated with clinical signs. Our study data suggest that trachoma does not meet the WHO definition of a public health problem in this Division of Fiji, but the inconsistency with previous studies warrants further investigation.

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Section: Discussionmentioning

confidence: 99%

Low Prevalence of Ocular Chlamydia trachomatis Infection and Active Trachoma in the Western Division of Fiji

et al. 2016

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“…There are as yet no accepted guidelines as to what level of sensitivity or specificity is required of a serological test; thus we referred to a previously published template [18] and established three possible thresholds from the ROC curve: one maximising specificity, one with a sensitivity greater than 80% [18] and one optimising the balance between sensitivity and specificity, by maximising Youden’s J-index [41]. …”

Section: Methodsmentioning

confidence: 99%

“…Numerous nucleic acid-amplification tests (NAATs) have been developed, including the adapted use of commercial kits originally designed for diagnosing genitourinary Ct infections [12–16]. NAATs have been shown to be cost-effective in some settings [17] but concerns have been raised that the per-sample cost of NAATs can be too much for national eye health programmes in countries where trachoma remains a problem [18]. The cost of specialist devices and platforms for deploying NAATs can also be prohibitive.…”

Section: Introductionmentioning

confidence: 99%

Defining Seropositivity Thresholds for Use in Trachoma Elimination Studies

et al. 2017

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BackgroundEfforts are underway to eliminate trachoma as a public health problem by 2020. Programmatic guidelines are based on clinical signs that correlate poorly with Chlamydia trachomatis (Ct) infection in post-treatment and low-endemicity settings. Age-specific seroprevalence of anti Ct Pgp3 antibodies has been proposed as an alternative indicator of the need for intervention. To standardise the use of these tools, it is necessary to develop an analytical approach that performs reproducibly both within and between studies.MethodologyDried blood spots were collected in 2014 from children aged 1–9 years in Laos (n = 952) and Uganda (n = 2700) and from people aged 1–90 years in The Gambia (n = 1868). Anti-Pgp3 antibodies were detected by ELISA. A number of visual and statistical analytical approaches for defining serological status were compared.Principal FindingsSeroprevalence was estimated at 11.3% (Laos), 13.4% (Uganda) and 29.3% (The Gambia) by visual inspection of the inflection point. The expectation-maximisation algorithm estimated seroprevalence at 10.4% (Laos), 24.3% (Uganda) and 29.3% (The Gambia). Finite mixture model estimates were 15.6% (Laos), 17.1% (Uganda) and 26.2% (The Gambia). Receiver operating characteristic (ROC) curve analysis using a threshold calibrated against external reference specimens estimated the seroprevalence at 6.7% (Laos), 6.8% (Uganda) and 20.9% (The Gambia) when the threshold was set to optimise Youden’s J index. The ROC curve analysis was found to estimate seroprevalence at lower levels than estimates based on thresholds established using internal reference data. Thresholds defined using internal reference threshold methods did not vary substantially between population samples.ConclusionsInternally calibrated approaches to threshold specification are reproducible and consistent and thus have advantages over methods that require external calibrators. We propose that future serological analyses in trachoma use a finite mixture model or expectation-maximisation algorithm as a means of setting the threshold for ELISA data. This will facilitate standardisation and harmonisation between studies and eliminate the need to establish and maintain a global calibration standard.

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“…Its applications are diverse, ranging from clinical specialties such as oncology, for example for the genotyping and monitoring of lung cancer, an area with high research activity [4], [5], [6], organ transplantation [7], [8], [9], microbiology [10], [11], [12], virology [13], [14], [15] or non-invasive prenatal testing [16], [17], [18], [19], [20]; to environmental studies [21], [22], [23], or health and safety monitoring for food and feed products [24].…”

Section: Introductionmentioning

confidence: 99%

Three-color crystal digital PCR

Madic¹,

Zocevic²,

Senlis

et al. 2016

Biomolecular Detection and Quantification

119

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Digital PCR is an exciting new field for molecular analysis, allowing unprecedented precision in the quantification of nucleic acids, as well as the fine discrimination of rare molecular events in complex samples. We here present a novel technology for digital PCR, Crystal Digital PCR™, which relies on the use of a single chip to partition samples into 2D droplet arrays, which are then subjected to thermal cycling and finally read using a three-color fluorescence scanning device. This novel technology thus allows three-color multiplexing, which entails a different approach to data analysis. In the present publication, we present this innovative workflow, which is both fast and user-friendly, and discuss associated data analysis issue, such as fluorescence spillover compensation and data representation. Lastly, we also present proof-of-concept of this three-color detection system, using a quadriplex assay for the detection of EGFR mutations L858R, L861Q and T790M.

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Will droplet digital PCR become the test of choice for detecting and quantifying ocularChlamydia trachomatisinfection? Maybe

Cited by 5 publications

References 21 publications

Low Prevalence of Ocular Chlamydia trachomatis Infection and Active Trachoma in the Western Division of Fiji

Low Prevalence of Ocular Chlamydia trachomatis Infection and Active Trachoma in the Western Division of Fiji

Defining Seropositivity Thresholds for Use in Trachoma Elimination Studies

Three-color crystal digital PCR

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