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PCR and culture for diagnosis of Acanthamoeba keratitis
  1. Helene Yera1,2,
  2. Vichita Ok2,
  3. Fiona Lee Koy Kuet2,
  4. Naima Dahane2,
  5. Frédéric Ariey1,2,
  6. Lilia Hasseine3,
  7. Pascal Delaunay3,
  8. David Martiano4,
  9. Pierre Marty3,5,
  10. Jean Louis Bourges1,6
  1. 1 Faculty of Medicine, Université de Paris, Paris, France
  2. 2 Hôpital Cochin, Laboratory of Parasitology-Mycology,Hôpitaux Universitaires Paris Centre, Paris, France
  3. 3 Laboratory of Parasitology-Mycology, Centre Hospitalier Universitaire de Nice, Nice, France
  4. 4 Ophthalmology, Centre Hospitalier Universitaire de Nice, Nice, France
  5. 5 Inserm U 1065, C3M, Université Côte d’Azur, Nice, France
  6. 6 Hôpital Cochin, Ophtalmology, Hôpitaux Universitaires Paris Centre, Paris, France
  1. Correspondence to Helene Yera, Hôpital Cochin, Laboratory of Parasitology-Mycology, Hôpitaux Universitaires Paris Centre, 27 Rue du Faubourg Saint Jacques, Paris 75679, France; helene.yera{at}aphp.fr

Abstract

Background/Aims Acanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.

Methods 1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.

Results Estimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).

Conclusions Culture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

  • Diagnostic tests/Investigation
  • Cornea
  • Infection
  • Microbiology
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Footnotes

  • Contributors HY planned the study and coordinated the preparation of the manuscript. VO carried out the statistical analysis. HY, VO, FLKK, ND, LH, PD, DM, JLB collected, analysed and interpreted the data of the study. VO, FA, LH, PD, PM, JLB reviewed all aspects of the study. All authors approved the manuscript.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests No declared.

  • Ethics approval According to French law (no. 2004-806, 9 August 2004), since the data were collected retrospectively and patient management was not modified, this study did not require approval by an Ethical Review Board. It was conducted following the law on data protection (no. 2004-801, 6 August 2004).

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement All data relevant to the study are included in the article or uploaded as supplemental information.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

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