Poor survival of dialysis patients with unhealed wounds because of critical limb ischemia: Limb salvage and survival requires wound healing.

Maintenance of blood flow in the wound area is required to heal wounds of critical limb ischemia (CLI) in dialysis patients. However, many dialysis patients have both a stenotic lesion in below-knee blood vessels and a cardiovascular event as complications, and thus, it may be difficult to ensure sufficient blood flow. Therefore, many deaths occur because of problems with wound healing. The aim of this study is to identify the optimal treatment, including revascularisation and amputation, from the perspective of wound healing by analysing the survival of hemodialysis patients with CLI who had healed or unhealed wounds in a lower extremity. The subjects were 52 patients who received maintenance dialysis at our clinic, including 27 with healed CLI wounds and 25 with unhealed CLI wounds. The Kaplan-Meier method was used to compare survival between the two groups. Multivariate analysis was conducted to examine the effect of an unhealed wound on mortality. The mean follow-up period was 1.7 ± 1.1 years. In the unhealed wound group, the 1-, 2-, and 3-year survival rates were 48%, 20%, and 12%, respectively. The overall survival rate was significantly lower in the unhealed wound group compared with the healed wound group (12% vs 63%, P = .0002 by log-rank test). In multivariate analysis, unhealed CLI wounds had a significant independent association with mortality (hazard ratio 3.32; 95% confidence interval [CI]: 1.41-8.77, P = .0054). In this study, the 3-year survival rate suggested a significantly poorer prognosis of hemodialysis patients with unhealed CLI wounds compared with those with healed wounds. An unhealed wound is an independent risk factor for mortality in hemodialysis patients with CLI.

Possible pathogenicity of commensal Entamoeba hartmanni revealed by molecular screening of healthy school children in Indonesia.

BACKGROUND: Although parasites are still endemic in developing areas, residents in those regions seem not to be affected by the presence of intestinal protozoans. This study aimed to investigate whether pathogenic and commensal protozoans are the causal agents of diarrhea via a school-based cross-sectional survey conducted in Indonesia, in September 2016. RESULTS: Molecular screening for intestinal protozoans in collected 144 stool samples from healthy students (age range 7-15 years) was carried out. The prevalence of protozoan parasites was as follows: Giardia intestinalis (56.3%), Entamoeba histolytica (0%), E. dispar (6.9%), E. moshkovskii (0%), E. hartmanni (31.3%), and E. coli (44.4%). Observational evaluation of stool conditions using the Bristol stool chart confirmed the loose stool rate (33.3-90.9%) in each age group. Logistic regression analysis of protozoan infection or colonization for loose stool (mild to severe diarrhea) as an outcome revealed no significant findings in examined protozoans including pathogenic G. intestinalis infection [adjusted odds ratio (AOR) 0.78, 95% confidence interval (CI) 0.36-1.67], except in E. hartmanni colonization (AOR 2.81, 95% CI 1.1-3.7, P = 0.026). CONCLUSIONS: The molecular survey of intestinal protozoans targeting healthy population with their stool form evaluation could address the pathogenicity of those parasites appropriately. In comparatively higher-age children at least 7 years of age or greater in the endemic area, G. intestinalis could regard commensal, while E. hartmanni seems to possess a certain pathogenicity as a causal agent of mild diarrhea.

Three monophyletic clusters in Retortamonas species isolated from vertebrates.

Retortamonas spp. has been reported as an intestinal parasite among various host organisms, including humans; however, its intra-genus molecular diversity has not yet been elucidated. Haplotypes of the 18S small subunit ribosomal RNA locus (1836-1899 bp) of Retortamonas spp. from humans (n = 8), pigs (n = 6), dogs (n = 1), goats (n = 16), water buffalos (n = 23), cattle (n = 7), rats (n = 3), and chickens (n = 5) were analyzed with references isolated from non-human mammals, amphibians, and insects. Phylogenetic and network analyses revealed a statistically supported three cluster formation among the vertebrate-isolated haplotypes, while insect-isolated haplotypes were independently clustered with Chilomastix. In the clade of vertebrate isolates, assemblage A (amphibian genotype), which included the amphibian references, was addressed as an out-group of the other clusters. Assemblage B (mammalian and chicken genotype) included most haplotypes from various mammals including humans with the haplotypes isolated from a chicken. Human isolates were all classified into this assemblage, thus assemblage B might correspond to R. intestinalis. Assemblage C (bovine genotype), which included specific haplotypes from water buffalos and cattle, was addressed as a sister lineage of assemblage B. Among the diversified haplotypes of assemblage B, a specific haplotype, which was identified from multiple host mammals (humans, dogs, pigs, cattle, water buffalos, elks, goats, and rats), indicates the potential zoonotic transmission of the Retortamonas among them. The genotyping classification of retortamonads could contribute to a better understanding of its molecular epidemiology, especially among humans and related host organisms.

Molecular phylogeny of Blastocystis isolates from wild rodents captured in Indonesia and Japan.

Blastocystis sp. is a common intestinal protist found worldwide in a variety of animals, including humans. Currently, 17 subtypes (STs) of Blastocystis isolates from mammalian and avian host species have been reported based on the small subunit ribosomal RNA gene (SSU rDNA). Among these, human Blastocystis were only identified among STs 1-9. Except ST9, all other STs comprised isolates from humans and other animal species. Entire sequence data of the SSU rDNA of nine Blastocystis isolates from laboratory rats or guinea pigs previously showed ST4, whereas Blastocystis isolates from wild rodents have not been addressed genetically. In this study, Blastocystis infection in wild rodents was surveyed in Indonesia and Japan, and 11 and 12 rodent Blastocystis parasites were obtained from Rattus exulans and R. novercious, respectively. All new Blastocystis isolates from wild rodents were identified as ST4 based on the SSU rDNA sequences. The best tree inferred with the entire sequences of the SSU rDNA of all ST4 isolates including 17 data registered in GenBank clearly showed monophyletic ST4A and ST4B clades. Although ST4 isolates from laboratory rats were separated into these two clades, all Blastocystis isolates from wild rodents in the present study were positioned into the clade ST4A and further separated into two sub-clusters within the clade ST4A according to the location of the host species. Considering the fact that laboratory rats were susceptible to both ST4A and ST4B, separation of the monophyletic sub-clusters of Blastocystis isolates from Indonesian Polynesian rats and Japanese brown rats may indicate the presence of geographical variations rather than a host-specific separation. In either way, the robust host preference to rodent species of ST4 Blastocystis was also confirmed.

Blastocystis phylogeny among various isolates from humans to insects.

Blastocystis is a common unicellular eukaryotic parasite found not only in humans, but also in various kinds of animal species worldwide. Since Blastocystis isolates are morphologically indistinguishable, many molecular biological approaches have been applied to classify these isolates. The complete or partial sequences of the small subunit rRNA gene (SSU rDNA) are mainly used for comparisons and phylogenetic analyses among Blastocystis isolates. However, various lengths of the partial SSU rDNA sequence have been used for phylogenetic inference among genetically different isolates. Based on the complete SSU rDNA sequences, consensus terminology of nine subtypes (STs) of Blastocystis sp. that were supported by phylogenetically monophyletic nine clades was proposed in 2007. Thereafter, eight additional kinds of STs comprising non-human mammalian Blastocystis isolates have been reported based on the phylogeny of SSU rDNA sequences, while STs 11 and 12 were only proposed on the base of partial sequences. Although many sequence data from mammalian and avian Blastocystis are registered in GenBank, only limited data on SSU rDNA are available for poikilotherm-derived Blastocystis isolates. Therefore, the phylogenetic positions of the reptilian/amphibian Blastocystis clades are unstable. The phylogenetic inference of various STs comprising mammalian and/or avian Blastocystis isolates was verified herein based on comparisons between partial and complete SSU rDNA sequences, and the phylogenetic positions of reptilian and amphibian Blastocystis isolates were also investigated using 14 new Blastocystis isolates from reptiles with all known isolates from other reptilians, amphibians, and insects registered in GenBank.

Molecular survey of Blastocystis sp. from humans and associated animals in an Indonesian community with poor hygiene.

Blastocystis sp. is a common parasite found in human and animal fecal samples. Currently, human Blastocystis isolates are classified into nine subtypes (STs) based on the phylogeny of their small subunit ribosomal RNA genes (SSU rDNAs). Since eight of the nine STs, except for ST9, have been reported in both humans and animals, these parasites are considered to be potentially zoonotic STs. To evaluate whether zoonotic transmissions play a main role in the lifecycle of Blastocystis, STs derived from humans, domestic pigs, domestic chickens, and wild rodents in a community with poor hygiene in Sumba Island, Indonesia were surveyed. Although fecal cross-contaminations between humans and animals were likely common at the investigation site, the confirmed major Blastocystis STs, which were detected as intense bands on gels following PCR targeting of the SSU rDNA, were different in each host species. STs 1-3 were found in resident children, while ST5, ST7, and ST4 were found in domestic pigs and chickens, and in wild rodents, respectively. Faint bands of STs 1, 2, and 7 were detected in samples from pigs, while no minor STs were observed in samples from the other host species. The distinct distributions of the major STs among the host animals examined, including humans, indicate host specificity in the lifecycle of Blastocystis. Considering the coprophagous nature of pigs, the presence of minor STs observed only in pigs could be explained by the mechanical passage of contaminated fecal materials.

Human Blastocystis subtyping with subtype-specific primers developed from unique sequences of the SSU rRNA gene.

The genus Blastocystis is one of the most genetically diverse parasites. Blastocystis isolates from humans and animals have been classified into subtypes (STs) based on the phylogeny of the small subunit rRNA gene (SSU rDNA). Although human Blastocystis isolates are limited to STs 1-9, the identification of all 9 STs remains challenging due to the lack of specific primers for several STs. The sequencing of partial SSU rDNA is therefore essential for the identification of several STs. In this study, we developed 9 sets of PCR primers to detect each of the 9 kinds of ST in humans. When these ST-specific primer pairs were examined reference Blastocystis for the 9 STs, all 9 amplified only the target ST even in a DNA mixture of all 9 STs. The specificities of the 9 primer sets were tested against several intestinal parasites and fungi found in human stool samples. No amplification with these common human intestinal eukaryotes was observed using the primer pairs for 8 STs, while the ST5 primer set gave only faint bands with some parasites. Since genomic DNA levels of these parasites extracted from Blastocystis-positive cultures are expected to be markedly lower than the pure or highly concentrated DNA samples tested, the cross-amplifications with these organisms are unlikely to be detected when DNA samples are extracted from Blastocystis-positive cultures. The PCR conditions for all 9 primer sets were the same, hence a one-step analysis by PCR amplification, followed by electrophoresis has potential as a simple tool for the subtyping of human Blastocystis isolates.

Prediction of Development of Critical Limb Ischemia in Hemodialysis Patients.

Hemodialysis (HD) patients with critical limb ischemia (CLI) suffer chronic inflammation and repeated infection, require intervention, and may have a protracted hospital stay. Therefore, early prediction is particularly important for management of CLI in patients with suspected peripheral artery disease. The purpose of this study is to develop a simple score for predicting the incidence of CLI in HD patients with suspected peripheral artery disease. The subjects were 139 asymptomatic patients receiving maintenance HD and with ABI <1.0. Multivariate logistic regression analysis was used to identify factors associated with development of CLI. These factors were subsequently weighted and integrated into a scoring system for the prediction of onset of CLI. Twenty-five patients had onset of CLI. Five factors selected from the multivariate model were weighted proportionally using their respective odds ratio (OR) for incidence of CLI (history of cerebral vascular disease, OR 6.42 [3 points]; diabetes, OR 3.92 [2 points]; hypoesthesia, OR 4.21 [2 points]; left ventricular ejection fraction <50%, OR 3.89 [2 points]; serum albumin <3.5 g/dL, OR 4.39 [2 points]). Three strata of risk were defined (low risk, 0 to 3 points; intermediate risk, 4 to 6 points; and high risk 7 to 11 points) with excellent prognostic accuracy for progression to CLI using the Kaplan-Meier method. Five factors were identified that increased the risk of progression to CLI in HD patients with suspected peripheral artery disease. A combination of those factors permitted establishment of three risk strata for accurate prediction of onset of CLI.

Molecular characterization of various trichomonad species isolated from humans and related mammals in Indonesia.

Trichomonad species inhabit a variety of vertebrate hosts; however, their potential zoonotic transmission has not been clearly addressed, especially with regard to human infection. Twenty-one strains of trichomonads isolated from humans (5 isolates), pigs (6 isolates), rodents (6 isolates), a water buffalo (1 isolate), a cow (1 isolate), a goat (1 isolate), and a dog (1 isolate) were collected in Indonesia and molecularly characterized. The DNA sequences of the partial 18S small subunit ribosomal RNA (rRNA) gene or 5.8S rRNA gene locus with its flanking regions (internal transcribed spacer region, ITS1 and ITS2) were identified in various trichomonads; Simplicimonas sp., Hexamastix mitis, and Hypotrichomonas sp. from rodents, and Tetratrichomonas sp. and Trichomonas sp. from pigs. All of these species were not detected in humans, whereas Pentatrichomonas hominis was identified in humans, pigs, the dog, the water buffalo, the cow, and the goat. Even when using the high-resolution gene locus of the ITS regions, all P. hominis strains were genetically identical; thus zoonotic transmission between humans and these closely related mammals may be occurring in the area investigated. The detection of Simplicimonas sp. in rodents (Rattus exulans) and P. hominis in water buffalo in this study revealed newly recognized host adaptations and suggested the existence of remaining unrevealed ranges of hosts in the trichomonad species.

Assessment of cyclic changes in the diameter of the aortic annulus using speckle-tracking trans-esophageal echocardiography.

It is uncertain whether dynamic variation in the diameter of the aortic annulus occurs during the cardiac cycle in humans. The purpose of this study was to analyze cyclic changes of the aortic annulus using speckle-tracking trans-esophageal echocardiography. The subjects were 40 patients with aortic stenosis and 40 controls. Absolute and relative changes in the diameter of the aortic annulus and the times at which the maximum and minimum diameters occurred during the cardiac cycle were determined using speckle-tracking trans-esophageal echocardiography. The maximum and minimum diameters were 22.9 ± 2.7 and 20.0 ± 2.9 mm, respectively, in controls. The change in diameter of the aortic annulus was 2.9 ± 0.7 mm, and the relative change was 12.9 ± 3.5%. The maximum aortic annulus diameter was reached at the onset of aortic valve opening, and the minimum diameter occurred in the rapid filling phase. The change in diameter of the aortic annulus was significantly smaller (2.2 ± 0.6 mm vs. 2.9 ± 0.7 mm, p < 0.0001), and the time to reach the maximum diameter was significantly longer (98.5 ± 17.5 ms vs. 83.4 ± 18.2 ms, p = 0.0004), in the aortic stenosis group than in the control group. The study found that dynamic changes of the aortic annulus occur in the cardiac cycle and can be measured using speckle-tracking trans-esophageal echocardiography. We also found that aortic stenosis has an effect on the extent and timing of these changes. This suggests that accurate assessment of aortic annulus diameter requires consideration of the timing of the cardiac cycle.

Genetic diversity of blastocystis in livestock and zoo animals.

Blastocystis is a common unicellular anaerobic eukaryote that inhabits the large intestine of many animals worldwide, including humans. The finding of Blastocystis in faeces in mammals and birds has led to proposals of zoonotic potential and that these hosts may be the source of many human infections. Blastocystis is, however, a genetically diverse complex of many distinct organisms (termed subtypes; STs), and sampling to date has been limited, both geographically and in the range of hosts studied. In order to expand our understanding of host specificity of Blastocystis STs, 557 samples were examined from various non-primate animal hosts and from a variety of different countries in Africa, Asia and Europe. STs were identified using 'barcoding' of the small subunit rRNA gene using DNA extracted either from culture or directly from faeces. The host and geographic range of several STs has thereby been greatly expanded and the evidence suggests that livestock is not a major contributor to human infection. Two new STs were detected among the barcode sequences obtained; for these, and for three others where the data were incomplete, the corresponding genes were fully sequenced and phylogenetic analysis was undertaken.

Subvalvular aortic stenosis diagnosed by 3D transesophageal echocardiography.

The patient was a 13-year-old male with chief complaints of exertional chest pain and dyspnea. Cardiac murmur was suspected in a medical checkup at 1 month old, at which time he was diagnosed with subvalvular aortic stenosis. He had subsequently been under follow-up observation at a nearby hospital for subvalvular aortic stenosis. He was admitted to our department for surgery due to aggravation of symptoms that had occurred over the previous year. Transthoracic echocardiography after admission showed an abnormal structure in the subvalvular aortic area, and the maximum pressure gradient between the left ventricle and aortic valve was 84 mmHg. The preoperative valve area was 0.71 cm(2), as measured by the Doppler method. Measurement of valve area by the trace method was difficult. Transesophageal echocardiography (TEE) showed a septum-like structure extending from the ventricular septum in the subvalvular area. On 3D TEE, the valve areas in the systolic and diastolic phases were 0.86 and 0.49 cm(2), respectively. Postoperative echocardiography showed resection of the structure in the subvalvular area, and the postoperative course was favorable.

Subtype identification of Blastocystis spp. isolated from patients in a major hospital in northeastern Thailand.

The present study is aimed to identify the prevalence of Blastocystis subtypes isolated from patients in a major hospital in northeastern Thailand. A total of 562 stool samples were examined by culture technique, and 56 Blastocystis-positive samples were analyzed further by the combination of restriction fragment length polymorphism (RFLP) followed by polymerase chain reaction with sequence-tagged site primers (PCR-STS). By RFLP profiles, Blastocystis genotypes were categorized into four groups: group A (12, 21.4%), group B (32, 57.1%), group C (10, 17.9%), and group D (2, 3.6%). By PCR-STS, only four subtypes were identified. All 12 (21.4%) isolates in group A were identified as subtype 1. Similarly, all 32 (57.1%) isolates in group B were subtype 3. In group C, 10 (17.9%) samples were all subtype 7, and two samples (3.6%) in group D were both subtype 6. Of 56 Blastocystis-positive patients, 31 (55.4%) were asymptomatic and 22 (39.4%) have gastrointestinal symptoms. No significant association was observed between the Blastocystis subtypes and the clinical features. Among the Blastocystis-positive patients, the most characteristic stool samples were loose (78.6%) and soft (17.9%). In conclusion, the most common Blastocystis spp. in northeastern Thailand was subtype 3 followed by subtype 1. Relatively minor subtypes, subtype 6 and subtype 7 which are considered as avian subtypes, were found for the first time in humans in Thailand.

Midwall ejection fraction for assessing systolic performance of the hypertrophic left ventricle.

BACKGROUND: In patients with left ventricular hypertrophy (LVH), LV midwall fractional shortening (FS) is used as a measure of LV systolic performance that is more physiologically appropriate than conventional FS. For evaluation of LV volume and ejection fraction (EF), 2-dimensional (2D) echocardiography is more accurate than M-mode echocardiography. The purpose of this study was to assess systolic performance by midwall EF using 2D speckle tracking echocardiography (STE). METHODS: Sixty patients were enrolled in the study. Patients were divided into two groups with LVH (n = 30) and without LVH (control group, n = 30). LV systolic function was compared between the two groups and the relationships of left ventricular mass index (LVMI) with LV systolic parameters, including midwall EF, were investigated. RESULTS: Midwall EF in the LVH group was significantly lower than that in the control group (42.8±4.4% vs. 48.1±4.1%, p <0.0001). Midwall FS was also significantly lower in the LVH group (13.4±2.8% vs. 16.1±1.5%, p <0.0001), but EF did not differ significantly between the two groups. There were significant correlations between midwall EF and LVMI (r=0.731, p <0.0001) and between midwall FS and LVMI (r=0.693, p <0.0001), with midwall EF having the higher correlation. CONCLUSIONS: These results show that midwall EF can be determined using 2D STE. Midwall EF can be used to monitor LV systolic dysfunction, which is not possible with conventional EF. Evaluation of midwall EF may allow assessment of new parameters of LV systolic function in patients with LV geometric variability.

Transcatheter atrial septal defect closure in a patient with paradoxical brain emboli: who should treat it and who should be treated?

This report presents the case of a 51-year-old female who was admitted to a local hospital because of a persistent headache. A diagnosis of multiple cerebral infarctions was thereafter made, but there was no evidence of either atherosclerosis or atrial fibrillation. The case was thought to be a cryptogenic stroke, however, Doppler ultrasonography of the lower extremities showed venous insufficiency. Transesophageal echocardiography revealed a secundum atrial septal defect (ASD) with a left to right shunt. Therefore, the final diagnosis was paradoxical brain emboli, and transcatheter ASD closure was successfully performed by cardiologists without any sequelae.

A case of paradoxical embolic ST-segment elevation myocardial infarction triggered by sleep apnea.

This report describes an obese 39-year-old man who experienced ST-segment elevation myocardial infarction with total thrombotic occlusion of the right coronary artery. Culprit vessel flow was improved by aspiration. Data suggested that myocardial infarction had resulted from paradoxical embolus via a patent foramen ovale triggered by the Mueller maneuver, which had induced negative intrathoracic pressure following an acute increase of right-heart volume in the context of obesity and sleep-disordered breathing (SDB). Obesity is increasing among younger populations and it represents a risk for SDB and thrombosis. Thus, this mechanism should be included within the differential diagnosis for myocardial infarction in young patients.

Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples.

Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial kits. In this study, we compared the detective sensitivity among five commercially available kits (MagNA Pure LC DNA Isolation Kit I, Roche; QuickGene SP Kit DNA, FujiFilm; NucleoSpin Plant II, Macherey-Nagel; QIAamp DNA Stool Mini Kit, Qiagen; ZR Fecal DNA Kit, Zymo Research) and fecal culture method. In a preliminary test, the DNA isolated with two kits (FujiFilm and Macherey-Nagel) showed negative PCR, while the other three kits showed positive PCR. Then, DNA from 50 clinical samples that was Blastocystis-positive in the examination of fecal culture method were isolated with the three kits and 1.1 kbp SSU rRNA gene was detected with PCR. The positive rates of the three kits (Roche, Qiagen, and Zymo Research) were 10, 48 and 94%, respectively. The present study indicated that there is different detective sensitivity among the commercial kits, and fecal culture method is superior in detection rate and cost performance than DNA-elution kits for diagnosis of Blastocystis sp. subtypes.