Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and specific real-time PCR assays

Résumé : Un nombre croissant de cas de malaria chez les voyageurs et migrants a été rapporté. Bien que l'analyse microscopique des frottis sanguins reste traditionnellement l'outil diagnostic de référence, sa fiabilité dépend considérablement de l'expertise de l'examinateur, pouvant elle-même faire défaut sous nos latitudes. Une PCR multiplex en temps réel a donc été développée en vue d'une standardisation du diagnostic. Un ensemble d'amorces génériques ciblant une région hautement conservée du gène d'ARN ribosomial 18S du genre Plasmodium a tout d'abord été conçu, dont le polymorphisme du produit d'amplification semblait suffisant pour créer quatre sondes spécifiques à l'espèce P. falciparum, P. malariae, P. vivax et P. ovale. Ces sondes utilisées en PCR en temps réel se sont révélées capables de détecter une seule copie de plasmide de P. falciparum, P. malariae, P. vivax et P. ovale spécifiquement. La même sensibilité a été obtenue avec une sonde de screening pouvant détecter les quatre espèces. Quatre-vingt-dix-sept échantillons de sang ont ensuite été testés, dont on a comparé la microscopie et la PCR en temps réel pour 66 (60 patients) d'entre eux. Ces deux méthodes ont montré une concordance globale de 86% pour la détection de plasmodia. Les résultats discordants ont été réévalués grâce à des données cliniques, une deuxième expertise microscopique et moléculaire (laboratoire de Genève et de l'Institut Suisse Tropical de Bâle), ainsi qu'à l'aide du séquençage. Cette nouvelle analyse s'est prononcé en faveur de la méthode moléculaire pour tous les neuf résultats discordants. Sur les 31 résultats positifs par les deux méthodes, la même réévaluation a pu donner raison 8 fois sur 9 à la PCR en temps réel sur le plan de l'identification de l'espèce plasmodiale. Les 31 autres échantillons ont été analysés pour le suivi de sept patients sous traitement antimalarique. Il a été observé une baisse rapide du nombre de parasites mesurée par la PCR en temps réel chez six des sept patients, baisse correspondant à la parasitémie déterminée microscopiquement. Ceci suggère ainsi le rôle potentiel de la PCR en temps réel dans le suivi thérapeutique des patients traités par antipaludéens. Abstract : There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

Early detection of microcirculatory perfusion changes with a high resolution, real time laser Doppler imaging camera--frostbite case study.

A 41-year-old male presented with severe frostbite that was monitored clinically and with a new laser Doppler imaging (LDI) camera that records arbitrary microcirculatory perfusion units (1-256 arbitrary perfusion units (APU's)). LDI monitoring detected perfusion differences in hand and foot not seen visually. On day 4-5 after injury, LDI showed that while fingers did not experience any significant perfusion change (average of 31±25 APUs on day 5), the patient's left big toe did (from 17±29 APUs day 4 to 103±55 APUs day 5). These changes in regional perfusion were not detectable by visual examination. On day 53 postinjury, all fingers with reduced perfusion by LDI were amputated, while the toe could be salvaged. This case clearly demonstrates that insufficient microcirculatory perfusion can be identified using LDI in ways which visual examination alone does not permit, allowing prognosis of clinical outcomes. Such information may also be used to develop improved treatment approaches.

Breathhold three-dimensional coronary magnetic resonance angiography using real-time navigator technology.

The acquisition duration of most three-dimensional (3D) coronary magnetic resonance angiography (MRA) techniques is considerably prolonged, thereby precluding breathholding as a mechanism to suppress respiratory motion artifacts. Splitting the acquired 3D volume into multiple subvolumes or slabs serves to shorten individual breathhold duration. Still, problems associated with misregistration due to inconsistent depths of expiration and diaphragmatic drift during sustained respiration remain to be resolved. We propose the combination of an ultrafast 3D coronary MRA imaging sequence with prospective real-time navigator technology, which allows correction of the measured volume position. 3D volume splitting using prospective real-time navigator technology, was successfully applied for 3D coronary MRA in five healthy individuals. An ultrafast 3D interleaved hybrid gradient-echoplanar imaging sequence, including T2Prep for contrast enhancement, was used with the navigator localized at the basal anterior wall of the left ventricle. A 9-cm-thick volume, with in-plane spatial resolution of 1.1 x 2.2 mm, was acquired during five breathholds of 15-sec duration each. Consistently, no evidence of misregistration was observed in the images. Extensive contiguous segments of the left anterior descending coronary artery (48 +/- 18 mm) and the right coronary artery (75 +/- 5 mm) could be visualized. This technique has the potential for screening for anomalous coronary arteries, making it well suited as part of a larger clinical MR examination. In addition, this technique may also be applied as a scout scan, which allows an accurate definition of imaging planes for subsequent high-resolution coronary MRA.

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search.

BACKGROUND: The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated.We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system - specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. RESULTS: We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. CONCLUSIONS: In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization.

Multicenter, Prospective Clinical Evaluation of Respiratory Samples from Subjects at Risk for Pneumocystis jirovecii Infection by Use of a Commercial Real-Time PCR Assay.

Pneumocystis jirovecii pneumonia (PCP) is a common opportunistic infection. Microscopic diagnosis, including diagnosis using the Merifluor-Pneumocystis direct fluorescent antigen (MP-DFA) test, has limitations. Real-time PCR may assist in diagnosis, but no commercially validated real-time PCR assay has been available to date. MycAssay Pneumocystis is a commercial assay that targets the P. jirovecii mitochondrial large subunit (analytical detection limit, ≤3.5 copies/μl of sample). A multicenter trial recruited 110 subjects: 54 with transplants (40 with lung transplants), 32 with nonmalignant conditions, 13 with leukemia, and 11 with solid tumors; 9 were HIV positive. A total of 110 respiratory samples (92% of which were bronchoalveolar lavage [BAL] specimens) were analyzed by PCR. Performance was characterized relative to investigator-determined clinical diagnosis of PCP (including local diagnostic tests), and PCR results were compared with MP-DFA test results for 83 subjects. Thirteen of 14 subjects with PCP and 9/96 without PCP (including 5 undergoing BAL surveillance after lung transplantation) had positive PCR results; sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) were 93%, 91%, 59%, and 99%, respectively. Fourteen of 83 subjects for whom PCR and MP-DFA test results were available had PCP; PCR sensitivity, specificity, PPV, and NPV were 93%, 90%, 65%, and 98%, respectively, and MP-DFA test sensitivity, specificity, PPV, and NPV were 93%, 100%, 100%, and 98%. Of the 9 PCR-positive subjects without PCP, 1 later developed PCP. The PCR diagnostic assay compares well with clinical diagnosis using nonmolecular methods. Additional positive results compared with the MP-DFA test may reflect low-level infection or colonization.

Application of real-time PCR for total airborne bacterial assessment : comparison with epifluorescence microscopy and culture-dependent methods

Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count nonculturableor non-viable bacteria. Consequently, the quantitative assessment of bioaerosols is often underestimated. Use of the real-time quantitative polymerase chain reaction (Q-PCR) to quantify bacteria in environmental samples presents an alternative method, which should overcome this problem. The aim of this study was to evaluate the performance of a real-time Q-PCR assay as a simple and reliable way to quantify the airborne bacterial load within poultry houses and sewage treatment plants, in comparison with epifluorescencemicroscopy and culture-dependent methods. The estimates of bacterial load that we obtained from real-time PCR and epifluorescence methods, are comparable, however, our analysis of sewage treatment plants indicate these methods give values 270-290 fold greater than those obtained by the ''impaction on nutrient agar'' method. The culture-dependent method of air impaction on nutrient agar was also inadequate in poultry houses, as was the impinger-culture method, which gave a bacterial load estimate 32-fold lower than obtained by Q-PCR. Real-time quantitative PCR thus proves to be a reliable, discerning, and simple method that could be used to estimate airborne bacterial load in a broad variety of other environments expected to carry high numbers of airborne bacteria. [Authors]

Relationship between motion of coronary arteries and diaphragm during free breathing: lessons from real-time MR imaging.

OBJECTIVE: Diaphragmatic navigators are frequently used in free-breathing coronary MR angiography, either to gate or prospectively correct slice position or both. For such approaches, a constant relationship between coronary and diaphragmatic displacement throughout the respiratory cycle is assumed. The purpose of this study was to evaluate the relationship between diaphragmatic and coronary artery motion during free breathing. SUBJECTS AND METHODS: A real-time echoplanar MR imaging sequence was used in 12 healthy volunteers to obtain 30 successive images each (one per cardiac cycle) that included the left main coronary artery and the domes of both hemidiaphragms. The coronary artery and diaphragm positions (relative to isocenter) were determined and analyzed for effective diaphragmatic gating windows of 3, 5, and 7 mm (diaphragmatic excursions of 0-3, 0-5, and 0-7 mm from the end-expiratory position, respectively). RESULTS: Although the mean slope correlating the displacement of the right diaphragm and the left main coronary artery was approximately 0.6 for all diaphragmatic gating windows, we also found great variability among individual volunteers. Linear regression slopes varied from 0.17 to 0.93, and r2 values varied from .04 to .87. CONCLUSION: Wide individual variability exists in the relationship between coronary and diaphragmatic respiratory motion during free breathing. Accordingly, coronary MR angiographic approaches that use diaphragmatic navigator position for prospective slice correction may benefit from patient-specific correction factors. Alternatively, coronary MR angiography may benefit from a more direct assessment of the respiratory displacement of the heart and coronary arteries, using left ventricular navigators.

Submillimeter three-dimensional coronary MR angiography with real-time navigator correction: comparison of navigator locations.

Three-dimensional free-breathing coronary magnetic resonance angiography was performed in eight healthy volunteers with use of real-time navigator technology. Images acquired with the navigator localized at the right hemidiaphragm and at the left ventricle were objectively compared. The diaphragmatic navigator was found to be superior for vessel delineation of middle to distal portions of the coronary arteries.

Linking Real Time and Location in Scheduling Demand-Responsive Transit, September 1996

The role of rural demand-responsive transit is changing, and with that change is coming an increasing need for technology. As long as rural transit was limited to a type of social service transportation for a specific set of clients who primarily traveled in groups to common meal sites, work centers for the disabled, or clinics in larger communities, a preset calendar augmented by notes on a yellow legal pad was sufficient to develop schedules. Any individual trips were arranged at least 24 to 48 hours ahead of time and were carefully scheduled the night before in half-hour or twenty-minute windows by a dispatcher who knew every lane in the service area. Since it took hours to build the schedule, any last-minute changes could wreak havoc with the plans and raise the stress level in the dispatch office. Nevertheless, given these parameters, a manual scheduling system worked for a small demand-responsive operation.

Isothermal microcalorimetry: a novel method for real-time determination of antifungal susceptibility of Aspergillus species.

We evaluated microcalorimetry for real-time susceptibility testing of Aspergillus spp. based on growth-related heat production. The minimal heat inhibitory concentration (MHIC) for A. fumigatus ATCC 204305 was 1 mg/L for amphotericin B, 0.25 mg/L for voriconazole, 0.06 mg/L for posaconazole, 0.125 mg/L for caspofungin and 0.03 mg/L for anidulafungin. Agreement within two 2-fold dilutions between MHIC (determined by microcalorimetry) and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. This proof-of-concept study demonstrated the potential of isothermal microcalorimetry for growth evaluation of Aspergillus spp. and real-time antifungal susceptibility testing.

Real-time motion correction in navigator-gated free-breathing double-oblique submillimeter 3D right coronary artery magnetic resonance angiography.

RATIONALE AND OBJECTIVES: The purpose of this study was the investigation of the impact of real-time adaptive motion correction on image quality in navigator-gated, free-breathing, double-oblique three-dimensional (3D) submillimeter right coronary magnetic resonance angiography (MRA). MATERIALS AND METHODS: Free-breathing 3D right coronary MRA with real-time navigator technology was performed in 10 healthy adult subjects with an in-plane spatial resolution of 700 x 700 microm. Identical double-oblique coronary MR-angiograms were performed with navigator gating alone and combined navigator gating and real-time adaptive motion correction. Quantitative objective parameters of contrast-to-noise ratio (CNR) and vessel sharpness and subjective image quality scores were compared. RESULTS: Superior vessel sharpness, increased CNR, and superior image quality scores were found with combined navigator gating and real-time adaptive motion correction (vs. navigator gating alone; P < 0.01 for all comparisons). CONCLUSION: Real-time adaptive motion correction objectively and subjectively improves image quality in 3D navigator-gated free-breathing double-oblique submillimeter right coronary MRA.

Viscoelasticity in Achilles Tendonopathy: Quantitative Assessment by Using Real-time Shear-Wave Elastography.

Purpose To investigate the differences in viscoelastic properties between normal and pathologic Achilles tendons ( AT Achilles tendon s) by using real-time shear-wave elastography ( SWE shear-wave elastography ). Materials and Methods The institutional review board approved this study, and written informed consent was obtained from 25 symptomatic patients and 80 volunteers. One hundred eighty ultrasonographic (US) and SWE shear-wave elastography studies of AT Achilles tendon s without tendonopathy and 30 studies of the middle portion of the AT Achilles tendon in patients with tendonopathy were assessed prospectively. Each study included data sets acquired at B-mode US (tendon morphology and cross-sectional area) and SWE shear-wave elastography (axial and sagittal mean velocity and relative anisotropic coefficient) for two passively mobilized ankle positions. The presence of AT Achilles tendon tears at B-mode US and signal-void areas at SWE shear-wave elastography were noted. Results Significantly lower mean velocity was shown in tendons with tendonopathy than in normal tendons in the relaxed position at axial SWE shear-wave elastography (P < .001) and in the stretched position at sagittal (P < .001) and axial (P = .0026) SWE shear-wave elastography . Tendon softening was a sign of tendonopathy in relaxed AT Achilles tendon s when the mean velocity was less than or equal to 4.06 m · sec(-1) at axial SWE shear-wave elastography (sensitivity, 54.2%; 95% confidence interval [ CI confidence interval ]: 32.8, 74.4; specificity, 91.5%; 95% CI confidence interval : 86.3, 95.1) and less than or equal to 5.70 m · sec(-1) at sagittal SWE shear-wave elastography (sensitivity, 41.7%; 95% CI confidence interval : 22.1, 63.3; specificity, 81.8%; 95% CI confidence interval : 75.3, 87.2) and in stretched AT Achilles tendon s, when the mean velocity was less than or equal to 4.86 m · sec(-1) at axial SWE shear-wave elastography (sensitivity, 66.7%; 95% CI confidence interval : 44.7, 84.3; specificity, 75.6%; 95% CI confidence interval : 68.5, 81.7) and less than or equal to 14.58 m · sec(-1) at sagittal SWE shear-wave elastography (sensitivity, 58.3%; 95% CI confidence interval : 36.7, 77.9; specificity, 83.5%; 95% CI confidence interval : 77.2, 88.7). Anisotropic results were not significantly different between normal and pathologic AT Achilles tendon s. Six of six (100%) partial-thickness tears appeared as signal-void areas at SWE shear-wave elastography . Conclusion Whether the AT Achilles tendon was relaxed or stretched, SWE shear-wave elastography helped to confirm and quantify pathologic tendon softening in patients with tendonopathy in the midportion of the AT Achilles tendon and did not reveal modifications of viscoelastic anisotropy in the tendon. Tendon softening assessed by using SWE shear-wave elastography appeared to be highly specific, but sensitivity was relatively low. © RSNA, 2014.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.