Ultrasensitive Detection and Quantification of Toxins for Optimized Diagnosis of Clostridium difficile Infection

Recently developed ultrasensitive and quantitative methods for detection of Clostridium difficile toxins provide new tools for diagnosis and, potentially, for management of C. difficile infection (CDI). Compared to methods that detect toxigenic organism, ultrasensitive toxin detection may allow diagnosis of CDI with increased clinical specificity, without sacrificing clinical sensitivity; measurement of toxin levels may also provide information relevant to disease prognosis. This minireview provides an overview of these new toxin detection technologies and considers what these new tools might add to the field.     

Analysis of the population genetics of Opisthorchis viverrini sensu lato in the Nam Ngum River wetland,Lao PDR,by multilocus enzyme electrophoresis

A previous population genetics study of Opisthorchis viverrini from a locality in an endemic area in Thailand found little genetic variation over time and second intermediate fish host species. Since a similar comparative analysis is not available for Lao PDR, we conducted a study of O. viverrini from different endemic foci in Vientiane Province, Lao PDR, based on spatial, temporal and fish host species. A total of 620 adult O. viverrini originating from the Nam Ngum River wetland were analysed at five previously defined polymorphic enzyme loci. Of these worms, 252 were from six different localities (spatial samples), 162 worms from different years (temporal samples) and 206 worms from four different cyprinid fish species. Significant heterozygote deficiency was found in most O. viverrini populations with levels of genetic differentiation ranging between F _ST 0.0000 and 0.0197 suggesting that gene flow occurred at a variable rate. The role of temporal factors and fish host species had little influence on the level of genetic differentiation. As for O. viverrini from Thailand, these findings indicate that self-fertilization and/or a clonal distribution of O. viverrini occurs in Lao PDR. Unlike the results for O. viverrini from Thailand, spatial population substructuring may be the underlying population processes for O. viverrini in Lao PDR. These findings indicate that geographical variation may contribute to the transmission dynamics of the parasite with implications for parasite control. However, other host factors, such as snail intermediate hosts and mammal reservoir hosts, as well as human beings, may also play significant roles.     

Retinal Vessel Detection and Measurement for Computer-aided Medical Diagnosis

Since blood vessel detection and characteristic measurement for ocular retinal images is a fundamental problem in computer-aided medical diagnosis, automated algorithms/systems for vessel detection and measurement are always demanded. To support computer-aided diagnosis, an integrated approach/solution for vessel detection and diameter measurement is presented and validated. In the proposed approach, a Dempster–Shafer (D–S)-based edge detector is developed to obtain initial vessel edge information and an accurate vascular map for a retinal image. Then, the appropriate path and the centerline of a vessel of interest are identified automatically through graph search. Once the vessel path has been identified, the diameter of the vessel will be measured accordingly by the algorithm in real time. To achieve more accurate edge detection and diameter measurement, mixed Gaussian-matched filters are designed to refine the initial detection and measures. Other important medical indices of retinal vessels can also be calculated accordingly based on detection and measurement results. The efficiency of the proposed algorithm was validated by the retinal images obtained from different public databases. Experimental results show that the vessel detection rate of the algorithm is 100 % for large vessels and 89.9 % for small vessels, and the error rate on vessel diameter measurement is less than 5 %, which are all well within the acceptable range of deviation among the human graders.     

In vivo total retinal blood flow measurement by Fourier domain Doppler optical coherence tomography

There is considerable interest in new methods for the assessment of retinal blood flow for the diagnosis of eye diseases. We present in vivo normal human volumetric retinal flow measurement using Fourier domain Doppler optical coherence tomography. We used a dual-plane scanning pattern to determine the angle between the blood flow and the scanning beam in order to measure total flow velocity. Volumetric flow in each blood vessel around the optic nerve head was integrated in one cardiac cycle in each measurement. Measurements were performed in the right eye of one human subject. The measured venous flow velocity ranged from 16.26 mm/s to 29.7 mm/s. The arterial flow velocity ranged from 38.35 mm/s to 51.13 mm/s. The total retinal venous and arterial flow both added up to approximately 54 microl/min. We believe this is the first demonstration of total retinal blood flow measurement using the OCT technique.     

Glucose-6-Phosphate Dehydrogenase Deficiency and Diabetes Mellitus with Severe Retinal Complications in a Sardinian Population,Italy

Background: Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is one of the most common human genetic abnormalities, with a high prevalence in Sardinia, Italy. Evidence indicates that G6PD-deficient patients are protected against vascular disease. Little is known about the relationship between G6PD deficiency and diabetes mellitus. The purpose of this study was to compare G6PD deficiency prevalence in Sardinian diabetic men with severe retinal vascular complications and in age-matched non-diabetic controls and ascertain whether G6PD deficiency may offer protection against this vascular disorder.Methods: Erythrocyte G6PD activity was determined using a quantitative assay in 390 diabetic men with proliferative diabetic retinopathy (PDR) and 390 male non-diabetic controls, both aged ≥50 years. Conditional logistic regression models were used to investigate the association between G6PD deficiency and diabetes with severe retinal complications.Results: G6PD deficiency was found in 21 (5.4 %) diabetic patients and 33 (8.5 %) controls (P=0.09). In a univariate conditional logistic regression model, G6PD deficiency showed a trend for protection against diabetes with PDR, but the odds ratio (OR) fell short of statistical significance (OR=0.6, 95% confidence interval=0.35-1.08, P=0.09). In multivariate conditional logistic regression models, including as covariates G6PD deficiency, plasma glucose, and systemic hypertension or systolic or diastolic blood pressure, G6PD deficiency showed no statistically significant protection against diabetes with PDR.Conclusions: The prevalence of G6PD deficiency in diabetic men with PDR was lower than in age-matched non-diabetic controls. G6PD deficiency showed a trend for protection against diabetes with PDR, but results were not statistically significant.     

Detection and Grading of Hypertensive Retinopathy Using Vessels Tortuosity and Arteriovenous Ratio

Hypertensive retinopathy (HR) refers to changes in the morphological diameter of the retinal vessels due to persistent high blood pressure. Early detection of such changes helps in preventing blindness or even death due to stroke. These changes can be quantified by computing the arteriovenous ratio and the tortuosity severity in the retinal vasculature. This paper presents a decision support system for detecting and grading HR using morphometric analysis of retinal vasculature, particularly measuring the arteriovenous ratio (AVR) and retinal vessel tortuosity. In the first step, the retinal blood vessels are segmented and classified as arteries and veins. Then, the width of arteries and veins is measured within the region of interest around the optic disk. Next, a new iterative method is proposed to compute the AVR from the caliber measurements of arteries and veins using Parr–Hubbard and Knudtson methods. Moreover, the retinal vessel tortuosity severity index is computed for each image using 14 tortuosity severity metrics. In the end, a hybrid decision support system is proposed for the detection and grading of HR using AVR and tortuosity severity index. Furthermore, we present a new publicly available retinal vessel morphometry (RVM) dataset to evaluate the proposed methodology. The RVM dataset contains 504 retinal images with pixel-level annotations for vessel segmentation, artery/vein classification, and optic disk localization. The image-level labels for vessel tortuosity index and HR grade are also available. The proposed methods of iterative AVR measurement, tortuosity index, and HR grading are evaluated using the new RVM dataset. The results indicate that the proposed method gives superior performance than existing methods. The presented methodology is a novel advancement in automated detection and grading of HR, which can potentially be used as a clinical decision support system.

     

A comprehensive study of phenylalanine hydroxylase gene mutations in the Iranian phenylketonuria patients

Phenylketonuria (PKU) is a metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. After thalassemia, PKU is considered as the most common autosomal recessive diseases in the Iranian population. Therefore, an efficient diagnostic strategy is required to identify disease-causing mutations in this population. Following our first report in 2003, here we presented a comprehensive study on the mutation spectrum of the PAH gene in the Iranian population. This study was performed on 280 unrelated chromosomes from 140 Iranian patients with classic PKU. All 13 exons as well as exon-intron boundaries of the PAH gene were analyzed by direct DNA sequencing. Thirty four different mutations were identified by a mutation detection rate of 100%. IVS10-11G > A, p.P281L, R261Q, p.F39del and IVS11+1G > C were the most prevalent mutations with frequencies of 26.07%, 19.3%, 12.86%, 6.07 and 3.93%, respectively. All other mutations represented a relative frequency less than 3.5%. The data from this study provided a comprehensive spectrum of the PAH gene mutations which can facilitate carrier detection and prenatal diagnosis of PKU disease in the Iranian population.     

Enhanced Detection of Host Response Antibodies to Borrelia burgdorferi Using Immuno-PCR

Lyme disease is the fastest-growing zoonotic disease in North America. Current methods for detection of Borrelia burgdorferi infection are challenged by analysis subjectivity and standardization of antigen source. In the present study, we developed an immuno-PCR (iPCR)-based approach employing recombinant in vivo-expressed B. burgdorferi antigens for objective detection of a host immune response to B. burgdorferi infection. iPCR is a liquid-phase protein detection method that combines the sensitivity of PCR with the specificity and versatility of immunoassay-based protocols. Use of magnetic beads coated with intact spirochetes provided effective antigen presentation and allowed detection of host-generated antibodies in experimentally infected mice at day 11 postinoculation, whereas host-generated antibodies were detected at day 14 by enzyme-linked immunosorbent assay (ELISA) and day 21 by immunoblotting. Furthermore, magnetic beads coated with recombinant B. burgdorferi in vivo-expressed antigen OspC or BmpA demonstrated positive detection of host-generated antibodies in mice at day 7 postinoculation with markedly increased iPCR signals above the background, with the quantification cycle (C_q) value for each sample minus the mean background C_q plus 3 standard deviations (ΔC_q) being 4 to 10, whereas ΔC_q was 2.5 for intact spirochete-coated beads. iPCR demonstrated a strong correlation (Spearman rank correlation = 0.895, P < 0.0001) with a commercial ELISA for detection of host antibodies in human Lyme disease patient sera using the B. burgdorferi VlsE C6 peptide. In addition, iPCR showed potential applicability for direct detection of spirochetes in blood. The results presented here indicate that our iPCR assay has the potential to provide an objective format that can be used for sensitive detection of multiple host response antibodies and isotypes to B. burgdorferi infection.     

Klebsiella pneumoniae Induces an Inflammatory Response in an In Vitro Model of Blood-Retinal Barrier

Klebsiella pneumoniae has become an important pathogen in recent years. Although most cases of K. pneumoniae endogenous endophthalmitis occur via hematogenous spread, it is not yet clear which microbial and host factors are responsible for the ability of K. pneumoniae to cross the blood-retinal barrier (BRB). In the present study, we show that in an in vitro model of BRB based on coculturing primary bovine retinal endothelial cells (BREC) and primary bovine retinal pericytes (BRPC), K. pneumoniae infection determines changes of transendothelial electrical resistance (TEER) and permeability to sodium fluorescein. In the coculture model, bacteria are able to stimulate the enzyme activities of endothelial cytosolic and Ca²⁺-independent phospholipase A₂s (cPLA₂ and iPLA₂). These results were confirmed by the incremental expression of cPLA₂, iPLA₂, cyclo-oxygenase-1 (COX1), and COX2 in BREC, as well as by cPLA₂ phosphorylation. In supernatants of K. pneumoniae-stimulated cocultures, increases in prostaglandin E₂ (PGE₂), interleukin-6 (IL-6), IL-8, and vascular endothelial growth factor (VEGF) production were found. Incubation with K. pneumoniae in the presence of arachidonoyl trifluoromethyl ketone (AACOCF₃) or bromoenol lactone (BEL) caused decreased PGE₂ and VEGF release. Scanning electron microscopy and transmission electron microscopy images of BREC and BRPC showed adhesion of K. pneumoniae to the cells, but no invasion occurred. K. pneumoniae infection also produced reductions in pericyte numbers; transfection of BREC cocultured with BRPC and of human retinal endothelial cells (HREC) cocultured with human retinal pericytes (HRPC) with small interfering RNAs (siRNAs) targeted to cPLA₂ and iPLA₂ restored the pericyte numbers and the TEER and permeability values. Our results show the proinflammatory effect of K. pneumoniae on BREC, suggest a possible mechanism by which BREC and BRPC react to the K. pneumoniae infection, and may provide physicians and patients with new ways of fighting blinding diseases.     

Metastasis-associated protein 1 (MTA1) in gastric cancer tissues is positively associated with poorer prognosis

Background

The present study examined the clinical significance of metastasis-associated protein 1 (MTA1) in the progression and patient survival of gastric cancer.

New next-generation microwave thermosphere ablation for small hepatocellular carcinoma

Background/AimsIn July 2017, the Emprint™ next-generation microwave ablation system using thermosphere technology (Covidien, Boulder, CO, USA) was approved for use in Japan. This system can produce a predictable spherical ablation zone at higher temperatures than radiofrequency ablation (RFA). The aim of the present study was to elucidate whether this new microwave thermosphere ablation (MTA) could safely improve outcome compared to RFA, which is the standard of care for small hepatocellular carcinoma (HCC).MethodsThis retrospective study analyzed 513 patients with 630 HCCs (≤3 cm) who were performed by percutaneous RFA (174 patients, 214 HCCs) or MTA (339 patients, 416 HCCs) between January 2016 and March 2020.ResultsMedian ablation time was significantly shorter for MTA (240 seconds) than for RFA (721 seconds; P<0.001). A significant difference in 3-year local tumor progression rate was evident between the RFA group (22%) and MTA group (8%; P<0.001). Multivariable analysis revealed ablation procedure and tumor diameter as independent factors contributing to local tumor progression (MTA; P<0.001; hazard ratio, 0.565; 95% confidence interval, 0.437–0.731). In patients with primary HCC, a significant difference in overall survival was evident (RFA vs. MTA, 3-year, 77% vs. 95%, P=0.029). Ablation procedure and Child-Pugh score were independent factors contributing to survival. The total complication rate was significantly lower for MTA (8%) than for RFA (14%, P<0.05), particularly for bile duct injury (3% vs. 9%, respectively; P<0.05).ConclusionsNext-generation MTA for small HCC could provide safer, more curative treatment in a shorter ablation time than RFA.     

Recent Advances in Detection of Plasmodium ovale: Implications of Separation into the Two Species Plasmodium ovale wallikeri and Plasmodium ovale curtisi

Recent molecular studies indicate that Plasmodium ovale malaria is caused by two closely related species of protozoan parasites, thereby imposing new challenges for detection and species differentiation. This minireview explores the potential value of innovative methods for the molecular diagnosis of malaria with a strong emphasis on the discrimination and genotyping of P. ovale wallikeri and P. ovale curtisi as well as tools for the simultaneous detection of P. ovale sp. An update for the widely used NP-1993 to NP-2005 (SSU rRNA) protocols for all human malaria parasites is discussed.     

The fibular shaft axis and medial cortex of the proximal fibula are reliable landmarks for the mechanical axis of the tibia in patients with knee osteoarthritis

BackgroundThis study aimed to evaluate the validity of proximal fibular anatomic landmarks for measuring the coronal tibial mechanical axis in patients with knee osteoarthritis and to investigate individual factors associated with their reliability.MethodsA total of 106 knees in 96 patients were retrospectively reviewed. The angles between the tibial mechanical axis and fibular shaft axis (TFA), medial cortex of the proximal fibular shaft (MTA), and lateral cortex of the proximal fibular shaft (LTA) were measured from full-leg standing digital anteroposterior radiographs. An angle within three degrees was considered reliable. The association between the above three angles and individual factors, such as age, sex, body mass index (BMI), and varus–valgus knee malalignment, was determined to investigate individual factors associated with their reliability.ResultsThe median TFA, MTA, and LTA were 1.52°, 1.56°, and 2.62°, respectively. The reliability rates of TFA, MTA, and LTA were 73.6% (95% CI: 65.19–81.98%), 82.1% (74.77–89.38%), and 58.5% (49.11–67.87%), respectively. The reliability of TFA and MTA was not associated with individual variables. The reliability of LTA was associated with BMI. Among patients with BMI greater than 25.3 kg/m², LTA was considered reliable in 65.7%; this rate was significantly higher than that among patients with BMI less than 25.3 kg/m².ConclusionsThe fibular shaft axis and medial cortex of the proximal fibular shaft are reliable landmarks of the mechanical axis of the tibia. However, the reliability of the lateral cortex of the proximal fibular shaft is less satisfactory, especially in patients with BMI less than 25.3 kg/m².     

Continuous determination of cardiac output during exercise by the use of impedance plethysmography

A computer-based system has been developed that enables the continuous measurement of cardiac output at rest as well as during exercise. The respiratory and motion artefacts appearing in the first derivative of the transthoracic impedance change (dZ/dt) were eliminated by adopting an ensemble averaging technique. A sufficiently high correlation was observed between cardiac outputs determined by the impedance and CO₂ rebreathing methods. The system may facilitate the physiological investigation of cardiac function during exercise.     

Refining genome-wide linkage intervals using a meta-analysis of genome-wide association studies identifies loci influencing personality dimensions

Personality traits are complex phenotypes related to psychosomatic health. Individually, various gene finding methods have not achieved much success in finding genetic variants associated with personality traits. We performed a meta-analysis of four genome-wide linkage scans (N=6149 subjects) of five basic personality traits assessed with the NEO Five-Factor Inventory. We compared the significant regions from the meta-analysis of linkage scans with the results of a meta-analysis of genome-wide association studies (GWAS) (N∼17 000). We found significant evidence of linkage of neuroticism to chromosome 3p14 (rs1490265, LOD=4.67) and to chromosome 19q13 (rs628604, LOD=3.55); of extraversion to 14q32 (ATGG002, LOD=3.3); and of agreeableness to 3p25 (rs709160, LOD=3.67) and to two adjacent regions on chromosome 15, including 15q13 (rs970408, LOD=4.07) and 15q14 (rs1055356, LOD=3.52) in the individual scans. In the meta-analysis, we found strong evidence of linkage of extraversion to 4q34, 9q34, 10q24 and 11q22, openness to 2p25, 3q26, 9p21, 11q24, 15q26 and 19q13 and agreeableness to 4q34 and 19p13. Significant evidence of association in the GWAS was detected between openness and rs677035 at 11q24 (P-value=2.6 × 10⁻⁰⁶, KCNJ1). The findings of our linkage meta-analysis and those of the GWAS suggest that 11q24 is a susceptible locus for openness, with KCNJ1 as the possible candidate gene.     

Evaluation of the FilmArray Blood Culture Identification Panel: Results of a Multicenter Controlled Trial

Sepsis is a major cause of morbidity, mortality, and increased medical expense. Rapid diagnosis improves outcomes and reduces costs. The FilmArray blood culture identification panel (BioFire Diagnostics LLC, Salt Lake City, UT), a highly multiplexed PCR assay, can identify 24 etiologic agents of sepsis (8 Gram-positive, 11 Gram-negative, and 5 yeast species) and three antimicrobial resistance genes (mecA, vanA/B, and bla_KPC) from positive blood culture bottles. It provides results in about 1 h with 2 min for assay setup. We present the results of an eight-center trial comparing the sensitivity and specificity of the panel with those of the laboratories'' standard phenotypic identification techniques, as well as with molecular methods used to distinguish Acinetobacter baumannii from other members of the A. calcoaceticus-A. baumannii complex and to detect antimicrobial resistance genes. Testing included 2,207 positive aerobic blood culture samples, 1,568 clinical and 639 seeded. Samples were tested fresh or were frozen for later testing within 8 h after the bottles were flagged as positive by an automated blood culture system. At least one organism was detected by the panel in 1,382 (88.1%) of the positive clinical specimens. The others contained primarily off-panel organisms. The panel reported multiple organisms in 81 (5.86%) positive clinical specimens. The unresolved blood culture identification sensitivity for all target detections exceeded 96%, except for Klebsiella oxytoca (92.2%), which achieved 98.3% sensitivity after resolution of an unavoidable phenotypic error. The sensitivity and specificity for vanA/B and bla_KPC were 100%; those for mecA were 98.4 and 98.3%, respectively.     

Simultaneous Detection and Differentiation of Anti-Helicobacter pylori Antibodies by Flow Microparticle Immunofluorescence Assay

Helicobacter pylori is the key pathogen for gastroduodenal diseases. The clinical outcome of H. pylori infection is influenced by the presence of strain-specific virulence factors that are usually detected by the presence of specific anti-H. pylori antibodies in serum. Apart from the detection of these antibodies by enzyme-linked immunosorbent assay (ELISA), it is desirable to obtain additional information concerning the presence of certain virulence factors of H. pylori that are currently detected by immunoblot analysis. At present, the immunodiagnosis of an H. pylori infection includes two separate methods: ELISA and immunoblot analysis. Here, we report the development and evaluation of a new rapid flow microparticle immunofluorescence assay (FMIA) for detection of anti-H. pylori antibodies in human serum. The assay allows rapid qualitative and quantitative detection of anti-H. pylori antibodies by using crude antigen preparations as well as single recombinant antigens (urease A, urease B, CagA, and alkylhydroxy peroxide reductase) in the same sample with one measurement, and thus it combines the advantages of enzyme immunoassay and Western blot analysis. Seventy-five patient samples were analyzed by FMIA, ELISA, and Western blotting with respect to their immunoreactivity against crude H. pylori extracts and individual H. pylori antigens. Statistical analyses revealed an overall similarity of more than 90% among the results for FMIA, ELISA, and Western blot. Therefore, we conclude that FMIA is a powerful and time- and cost-saving assay system for the detection of antimicrobial antibodies, with higher sensitivity and a larger measurement range than ELISA.