Measurement of capillary refill time with a handheld prototype device: a comparative validation study in healthy volunteers.

Validity and reproducibility of clinical capillary refill time (CRT) measurement depend on many factors in daily routine practice. We conducted a prospective validation study of an automatized handheld prototype device providing standardized CRT assessment (DiCART™) in 20 healthy volunteers. Three different methods of CRT measurement were compared before and during dynamic circulatory changes induced by venous and arterial occlusion tests at both upper and lower limb levels: CRTCLIN corresponding to basic clinical assessment and considered as the reference method; CRTVIDEO corresponding to off-line videos reviewed by investigators recorded by DiCART™; and CRTDiCART corresponding to on-line videos analysed by a built-in proprietary mathematical algorithm included in DiCART™. Five subjects were excluded because of a DiCART™ dysfunction. ROCAUC to detect arterial occlusion test changes at the upper limb level were 1.00 (95%CI 1.00; 1.00), 0.96 (95%CI 0.88; 1.00), and 0.92 (95%CI 0.79; 1.00) for CRTCLIN, CRTVIDEO, and CRTDiCART, respectively. Precision of CRTCLIN and CRTVIDEO were significantly better than CRTDiCART (0.18 and 0.20 vs. 0.28; P < 0.05). Percentages of error were 76% and 87% for CRTVIDEO and CRTDiCART, respectively. DiCART™ had an excellent discrimination to detect major changes in CRT induced by arterial ischemia. However, the perfectible precision, the poor agreement with clinical assessment and numerous device dysfunctions give leads to the development of a further version of the prototype before promoting its use in clinical practice.Trial registration clinicaltrial.gov. Identifier: NCT04538612.

Effects of topical anaesthetics and fluorescein on the real-time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis.

BACKGROUND: The early microbiological diagnosis of corneal infections may prevent the condition from worsening. AIM: To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real-time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. METHODS: Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real-time PCR. RESULTS: The capacities of the real-time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). CONCLUSIONS: The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis.

New tool for the simultaneous detection of 10 different genotypes of Acanthamoeba available from the American Type Culture Collection.

BACKGROUND: Acanthamoeba keratitis (AK) is a sight-threatening infection, and none of the current diagnosis tests are able to detect in one reaction low levels of the vast majority of strains associated with pathology. The goal of this work was to validate a new tool for the detection of the American Type Cell Collection (ATCC) referenced Acanthamoeba monitoring simultaneously DNA extraction yields and PCR inhibitors. Performances were assessed on corneal scrapings. METHODS: Primers were selected in a region bracketing a 41 591 bp of the A castellanii mitochondrion gene. DNA extraction and PCR inhibitors were monitored by adding an internal control (virus). Acanthamoeba were detected and quantified by the real-time fast-duplex TaqMan PCR (f-d-real-t PCR) and negativity confirmed by SYBR Green real-time PCR. RESULTS: The f-d-real-t PCR detects 0.1 cyst/microl or less of the 10 referenced strains (sensitivity slightly lower for A astronyxis). Bacteria, fungi and herpesviruses do not cross-react. The specificity and sensitivity of the f-d-real-t PCR were higher than culture and other real-time PCR on 20 keratitis samples. CONCLUSION: The f-d-real t PCR detects in less than 2 h the Acanthamoeba strains available from the ATCC with a higher sensitivity and specificity than techniques previously reported. Larger trials are necessary to validate its usefulness for disease management and environmental studies.

Resistance of Acanthamoeba to classic DNA extraction methods used for the diagnosis of corneal infections.

AIMS: Sensitive diagnosis of Acanthamoeba infections may prevent the clinical condition from becoming worse. In order to improve the diagnosis tool performances, we studied the implication of the DNA extraction procedures on the detection of Acanthamoeba by real-time PCR. METHODS: Acanthamoeba cysts mixed with a tag virus were processed according to different DNA preparation procedures: heat, Proteinase K (ProtK), alkali lysis, QIAmp kit, MagNA Pure (DNA Mini kit, MagNA Pure Nucleic Acid isolation kit), ProtK+QIAmp and ProtK+MagNA Pure. Parasite-DNA loads were assessed by real-time PCR. RESULTS: The results show that the structures of Acanthamoeba cysts are resistant to reagents releasing the DNA from other cells and viruses. Heat, NaOH or ProtK did not allow the DNA extraction yields to be assessed or the inhibitors to be eliminated The QIAmp and the MagNA Pure partially improved the sensitivity of the PCR and eliminated the inhibitors. A significant increase in positive results was obtained with a ProtK treatment before commercial extraction kits. ProtK+MagNA Pure yielded the highest rates of positivity. CONCLUSION: To minimise false negative results, the nucleic-acid based Acanthamoeba diagnosis requires, first, the efficient lysis of cysts (without affecting the DNA) to make the DNA available for extraction and amplification, and, second, the elimination of PCR inhibitors. A significant increase in the detection rates is obtained by adding a ProtK treatment (10 min at 56 degrees C) before the commercial procedures. ProtK+MagNA Pure yielded the best results in 30 min, followed by ProtK+QIAmp (150 min).