New Strategy for Rapid Diagnosis and Characterization of Fungal Infections: The Example of Corneal Scrapings

PURPOSE: The prognosis of people infected with Fungi especially immunocompromised depends on rapid and accurate diagnosis to capitalize on time administration of specific treatments. However, cultures produce false negative results and nucleic-acid amplification techniques require complex post-amplification procedures to differentiate relevant fungal types. The objective of this work was to develop a new diagnostic strategy based on real-time polymerase-chain reaction high-resolution melting analysis (PCR-HRM) that a) detects yeasts and filamentous Fungi, b) differentiates yeasts from filamentous Fungi, and c) discriminates among relevant species of yeasts. METHODS: PCR-HRM detection limits and specificity were assessed with a) isolated strains; b) human blood samples experimentally infected with Fungi; c) blood experimentally infected with other infectious agents; d) corneal scrapings from patients with suspected fungal keratitis (culture positive and negative) and e) scrapings from patients with suspected bacterial, viral or Acanthamoeba infections. The DNAs were extracted and mixed with primers diluted in the MeltDoctor® HRM Master Mix in 2 tubes, the first for yeasts, containing the forward primer CandUn (5'CATGCCTGTTTGAGCGTC) and the reverse primer FungUn (5'TCCTCCGCTT ATTGATATGCT) and the second for filamentous Fungi, containing the forward primer FilamUn (5'TGCCTGTCCGAGCGTCAT) and FungUn. Molecular probes were not necessary. The yields of DNA extraction and the PCR inhibitors were systematically monitored. RESULTS: PCR-HRM detected 0.1 Colony Forming Units (CFU)/µl of yeasts and filamentous Fungi, differentiated filamentous Fungi from yeasts and discriminated among relevant species of yeasts. PCR-HRM performances were higher than haemoculture and sensitivity and specificity was 100% for culture positive samples, detecting and characterizing Fungi in 7 out 10 culture negative suspected fungal keratitis. CONCLUSIONS: PCR-HRM appears as a new, sensitive, specific and inexpensive test that detects Fungi and differentiates filamentous Fungi from yeasts. It allows direct fungal detection from clinical samples and experimentally infected blood in less than 2.30 h after DNA extraction

PCR-HRM profiles obtained with yeasts and filamentous <i>Fungi</i> using the primers FilamUn + FungUn.

<p>In red: <i>Aspergillus sp</i>.; black: <i>Fusarium solani</i>; violet, blue and green: <i>Candida sp</i>.</p

PCR-HRM profiles obtained with yeasts and filamentous <i>Fungi</i> using the primers CandUn + FungUn.

<p>In red: <i>Candida tropicalis</i>; green: <i>C. parapsilopsis</i>; violet: <i>C. albicans</i>; black: <i>C. glabrata</i>; yellow: <i>C. krusei</i>; blue: Filamentous <i>Fungi</i> (<i>Aspergillus fumigatus</i> and <i>Fusarium solani</i>.).</p

Comparison of culture and high-resolution melting analysis (PCR-HRM) performances on experimentally infected blood.

[a]<p>Haemoculture bottles were inoculated with 10 ml; S: saline; B: blood; #: results from 2 independent experiments; POS: Positive; NEG: Negative after 240 hours<b>;</b> ##: reference strains and negative controls were extracted and tested for each run<i>;</i>*: melting-curve profiles consistently superimposed on those obtained with <i>Fusarium solani</i>.</p

High-resolution melting analysis (PCR-HRM) detection limits (fungal spore suspensions titrated by plating) and discrimination among fungal species using the primers CandUn + FungUn and FilamUn + FungUn.

<p>I: yeast; II and III: Filamentous <i>Fungi</i>;</p>*<p>CandUn sequence: 5' CATGCCTGTTTGAGCGTC;</p><p>°FilamUn sequence: 5' TGCCTGTCCGAGCGTCAT;</p>**<p>FungUn sequence: 5' TCCTCCGCTTATTGATATGCT.</p

Comparison of direct microscopic examination, culture and high-resolution melting analysis performances (PCR-HRM) on corneal scrapings obtained from patients with keratitis.

<p>HRM: high-resolution melting analysis; *: after Giemsa (pH: 7.4) and Grocott staining; POS: Positive; NEG: Negative<b>;</b> FF: Filamentous Fungi<b>;</b> GNR: Gram-negative rods; GPR: Gram-positive rods; GPC: Gram-positive <i>cocci</i>; LY: lymphocytes; AEC: Altered epithelial cells; HSV1: <i>Herpes-simplex</i> virus type 1; HSV2: <i>Herpes-simplex</i> virus type 2; AC: <i>Acanthamoeba</i>; **: DNA was extracted from 6 different individuals and tested separately; ##: controls were extracted and tested for each run; ^a: The leukocytes from 4 different donors were extracted and tested separately. Air and working surface samples were obtained placing open tubes for 15 minutes under the laminar flow or in the laboratory working table. Surface samples were collected with cotton devices humidified with saline by gentle swabbing the working surface. DNA extraction procedures were conducted in duplicate in each series of experiments for the DNA extraction reactants.</p