Parasitology
Rapid detection and simultaneous molecular profile characterization of Acanthamoeba infections,☆☆

https://doi.org/10.1016/j.diagmicrobio.2012.06.001Get rights and content

Abstract

Diagnosis of Acanthamoeba by microscopic examination, culture, and polymerase chain reactions (PCRs) has several limitations (sensitivity, specificity, lack of detection of several strains, cost of testing for discrimination among strains). We developed a new high-resolution melting real-time PCR (HRM) to detect and characterize Acanthamoeba infections. HRM performances were evaluated with strains from the American Type Culture Collection (ATCC) and with 20 corneal scrapings. The DNA extracted from specimens were amplified, detected, and characterized in 1 run using 2 original primers diluted in a solution containing an intercalating dye. Detection and molecular characterization of Acanthamoeba infections could be achieved in less than 2.5 h with a dramatic reduction in cost of reactants (postamplification procedures and radioactive or fluorescent-labeled molecular probes were unnecessary). HRM detection limits were 0.1 cyst/μL or less (including genotypes T5 and T11), and its sensitivity and specificity were higher than other molecular tests. For the tested strains from the ATCC, the HRM drafted 4 different profiles: Type I (genotypes T2 and T4), Type II (T5 and T7), Type III (T8), and Type IV (T1, T3, T6, T9, T11, T12, and T13).

Introduction

Acanthamoebae are protozoa found in the soil, aquatic environment, and air. They are generally associated with eye diseases, although general infections (mostly brain and skin) were reported also. The infestations with Acanthamoeba are related to poor hygiene, inhalation of contaminated water, immunosuppression, corneal lens use, and corneal injuries (Lorenzo-Morales et al., 2010, Marciano Cabral, 2003).

To reduce morbidity, sensitive, specific, and inexpensive tests are necessary to administer therapeutic agents at the onset of symptoms. However, the direct smear analysis of corneal scrapings (Gram, May-Grünwald-Giemsa pH 7.4 staining) produces results rapidly, but misdiagnoses almost 60% of clinical cases (Behets et al., 2006, Yera et al., 2007).

The detection of Acanthamoebae by polymerase chain reactions (PCR) dramatically enhanced diagnosis performances. The tests targeting the Acanthamoeba nuclear small subunit ribosomal 18S rRNA gene detected the equivalent of 1 protozoa per sample (Clarke and Niederkorn, 2006, Gatti et al., 2010, Marciano Cabral, 2003), but confirmation of the amplicon specificities required a series of additional procedures (agarose gel electrophoresis, hybridisation, and/or molecular sequencing), for which the opening of tubes with amplified material enhanced cross-contamination risks (Boggild et al., 2009, Goldschmidt et al., 2009a, Yera et al., 2007).

The reduction in postamplification handling was possible using a real-time technology (real-time PCR [rtPCR]), for which the amplicons produced during the rtPCR could be detected in closed tubes by means of 2 different strategies: a) fluorophore-labeled TaqMan probes or b) the intercalating fluorophore SYBR-green. Nevertheless, the SYBR-green rtPCR did not fulfill the expectations for diagnosis because traces of residual double-stranded DNA from other agents found in the DNA extracted from the samples may bind the fluorophore and trigger nonspecific positive signals. Here, detecting fluorescent signals required the addition of postamplification procedures (agarose gel electrophoresis or sequencing) (Behets et al., 2006, Boggild et al., 2009) to determine species or genotypes. The use of labeled TaqMan® probes (Qvarnstrom et al., 2006, Yera et al., 2007) instead of SYBR-green improved rtPCR specificity but did not detect all the genotypes. In addition, TaqMan rtPCRs were unable to provide group or genotype data (da Rocha-Azevedo et al., 2009, Qvarnstrom et al., 2006). We reported a broad-spectrum rtPCR performed directly from clinical or environmental samples (targeting a highly conserved mitochondrial gene) that made possible the detection of all the available American Type Culture Collection (ATCC) strains (0.1 cyst/μL of sample). Like other TaqMan rtPCRs, the broad-spectrum test that required expensive fluorophore-labeled probes was not informative on groups, species, types, or genotypes (Goldschmidt et al., 2009b).

The high-resolution melting real-time PCR technology (HRM) was developed for the fine assessment of DNA sequence variations (Monis et al., 2005, Reed et al., 2007) using improved fluorescent DNA-binding dyes. In this study, we developed a new molecular strategy based on HRM that should a) detect in 1 run the equivalent of 1 cyst of Acanthamoeba or less per sample; b) produce results without molecular probes based on the dye-melting derivative curve analysis with no need for gel electrophoresis, hybridizations, or immunoenzymatic assays; and c) draft different profiles for different species according to the guanine cytosine content and to the size of the amplicons neosynthesised during the nucleic-acid amplification process

Section snippets

Material and methods

HRM sensitivity, specificity, linearity, and detection limits were first assessed with strains of Acanthamoeba purchased from LGC Promochem (Molsheim, France; an ATCC partner) (A. comandoni [ref. 30135], A. palestinensis [ref. 50708], A. sp. [ref. 50655], A. palestinensis [ref. 30870], A. griffini [ref. 30731], A. castellanii [ref. 50373], A. lenticulata [ref. 30841], A. astronyxis [ref. 30137], A. tubiashi [ref. 30867], and A. hatchetti). Acanthamoeba were cultured and maintained according to

Results

The selected primers detected Acanthamoeba with a linear range spanning from 105 to at least 10 1 cyst/mL. The interassay reproducibility coefficient variation was less than 10% among HRM runs for each dilution. The HRM coefficient of variation for the interassay reproducibility in the complete linear range of detection (105 to 1 cyst/mL) for 5 runs was inferior to 10%.

The reactants, disposable material, and environmental controls did not produce signals that could have interfered with the

Discussion

Mixing the DNA extracted from samples with a solution containing primers from a selected sequence and 1 intercalating dye that stoichiometric binds reversibly to the neoformed double-stranded DNA during amplification makes it possible to conduct an automatic melting analysis to detect Acanthamoeba that provides 4 reproducible signatures for the different genotypes. The detection of Acanthamoeba and their molecular characterization are performed by obviating gel electrophoresis, hybridizations,

Acknowledgments

The authors thank the Centre de Ressources Biologiques du Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France, for providing the biological samples.

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    Conflict of interests: none for all authors.

    ☆☆

    Funding sources: This work was supported exclusively by intramural funds of the Laboratoire d'Ophtalmobiologie du Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, Paris, France.

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