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.

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.

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.

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.

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.

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.

Impact of cilostazol on left ventricular geometry and function: assessment by tissue Doppler imaging and two-dimensional speckle-tracking echocardiography.

OBJECTIVES: Cilostazol, a type III phosphodiesterase inhibitor, is an antiplatelet agent with vasodilating properties. Positive inotropic and chronotropic effects are frequently observed with cilostazol, but there are few reports on the influence of cilostazol on left ventricular function. The aim of this study was to assess this effect using tissue Doppler imaging (TDI) and two-dimensional speckle-tracking echocardiography (2D-STE). METHODS: Thirty-five patients with normal left ventricular ejection fraction were enrolled in the study. Left ventricular cardiac function was assessed by TDI and 2D-STE before and after oral administration of cilostazol. Peak strain was defined using the peak radial strain (PRS), peak circumferential strain (PCS) and peak longitudinal strain (PLS). Time to peak strain was defined based on the times to PRS, PCS, and PLS, as T-PRS, T-PCS, and T-PLS, respectively. RESULTS: After cilostazol administration, there were significant decreases in the left ventricular end-diastolic and end-systolic diameters (47.3 ± 5.2 vs. 43.3 ± 4.9 mm, P < 0.0001; 29.3 ± 6.4 vs. 26.0 ± 5.5 mm, P < 0.0001, respectively), and significant increases in the left ventricular ejection fraction (70.6 ± 9.5 vs. 72.7 ± 7.8%, P = 0.0381) and peak systolic annular velocity (7.9 ± 1.7 vs. 9.5 ± 3.1 cm/sec, P < 0.0001). PRS, PCS, and PLS all increased significantly and T-PRS, T-PCS, and T-PLS all decreased significantly after cilostazol administration. CONCLUSION: Positive inotropic and chronotropic effects of cilostazol were found based on assessment by TDI and 2D-STE. We suggest that periodic echocardiographic assessment should be performed before and after oral administration of cilostazol.

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.

Molecular characterization of Blastocystis isolates from children and rhesus monkeys in Kathmandu, Nepal.

To investigate the possible transmission of Blastocystis organisms between local rhesus monkeys and children in Kathmandu, Nepal, we compared the subtype (ST) and sequence of Blastocystis isolates from children with gastrointestinal symptoms and local rhesus monkeys. Twenty and 10 Blastocystis isolates were established from 82 and 10 fecal samples obtained from children and monkeys, respectively. Subtype analysis with seven sequence-tagged site (STS) primers indicated that the prevalence of Blastocystis sp. ST1, ST2 and ST3 was 20%, 20% and 60% in the child isolates, respectively. In contrast to human isolates, ST3 was not found in monkey isolates and the prevalence of ST1 and ST2 was 50% and 70%, respectively, including three mixed STs1 and 2 and one isolate not amplified by any STS primers, respectively. Since Blastocystis sp. ST2 has been reported as the most dominant genotype in the survey of Blastocystis infection among the various monkey species, sequence comparison of the 150bp variable region of the small subunit rRNA (SSU rRNA) gene was conducted among ST2 isolates of humans and monkeys. Sequence alignment of 24 clones developed from ST2 isolates of 4 humans and 4 monkeys showed three distinct subgroups, defined as ST2A, ST2B and ST2C. These three subgroups were shared between the child and monkey isolates. These results suggest that the local rhesus monkeys are a possible source of Blastocystis sp. ST2 infection of humans in Kathmandu.

Blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in Ankara, Turkey.

Blastocystis infection has been reported to be associated with irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) and chronic diarrhoea. The availability of data on the subtypes of Blastocystis found in these patient groups would be of interest in understanding the significance of Blastocystis infection in chronic illness. In this study, we identify Blastocystis subtypes found in patients presenting with IBS, IBD, chronic diarrhoea and asymptomatic patients in Ankara, Turkey. Blastocystis was detected in 11 symptomatic patients by microscopy and 19 by stool culture. Stool culture was more sensitive than microscopy in identifying Blastocystis. Using standard nomenclature adopted in 2007, Blastocystis sp. subtype 3 was the most common in all groups, followed by Blastocystis sp. subtype 2. Identical subtypes of Blastocystis are found in patients with IBS, IBD and chronic diarrhoea. These particular subtypes show low host specificity and are carried by humans and some farm animals. The subtypes of Blastocystis that are commonly found in rodents and certain wild birds were not found in these patients. We suggest a model in which the severity of enteric protozoan infection may be mediated by host factors.

PCR-based subtyping of Blastocystis isolates from symptomatic and asymptomatic individuals in a major hospital in Ankara, Turkey.

The stool samples obtained from 94 patients with gastrointestinal symptoms and 109 asymptomatic individuals, who checked in due to other reasons, admitted at a major hospital in Ankara, Turkey were examined with native Lugol's iodine, trichrome, and Kinyoun's acid-fast stainings for parasitology examinations and with in vitro culture method for detection of Blastocystis. In a total of 203 stool samples tested, native Lugol's iodine and trichrome stainings could detect 12 (5.9%) and 20 (9.9%) positive samples for Blastocystis, respectively. Conversely, culture method could detect 66 (32.5%) positive samples, and this method was more sensitive compared to the both microscopic examinations (p < 0.001). Among 66 positive samples for Blastocystis, 27 were from symptomatic patients and 39 were from asymptomatic group. Subtypes (STs) were determined by PCR using seven different sequence-tagged site primers. ST3 was the most dominant in both symptomatic and asymptomatic groups and followed by ST1 or ST2. There were mixed infections with STs 1 and 2 or STs 1 and 3 in nine isolates. There was no statistical significance of the distribution of Blastocystis sp. subtypes between symptomatic and asymptomatic individuals (p > 0.05).

Expression of interferon gamma and proinflammatory cytokines in the cecal mucosa of rats experimentally infected with Blastocystis sp. strain RN94-9.

Blastocystis hominis is a zoonotic intestinal protozoan parasite whose pathogenic potential is still controversial. The aim of the present study was to clarify the pathogenicity of Blastocystis parasites in rats. Oral inoculation with 1 x 10(5) cysts of Blastocystis sp. strain RN94-9 in rats resulted in chronic infection in the cecum at least until 4 weeks after infection. Histological examination revealed neither mucosal sloughing nor inflammatory cell infiltration but showed a slight but significant increase in goblet cell numbers in the cecal mucosa 1-3 weeks post-infection. Differential staining of acidic and neutral mucins by the alcian blue-periodic acid-Schiff method showed that the predominantly increased cells were neutral mucin(+) but not acidic mucin(+) goblet cells. Reverse transcription real-time polymerase chain reaction studies demonstrated significant upregulation of the expression of interferon-gamma, interleukin (IL)-12, and tumor necrosis factor alpha, but not IL-6 or granulocyte-macrophage colony-stimulating factor, in the cecal mucosa at 2 and/or 3 weeks post-infection. The induction of local host responses, including mild goblet cell hyperplasia, and significant upregulation of type-1 and proinflammatory cytokines, suggest that Blastocystis sp. strain RN94-9 is a weakly pathogenic organism that could elicit proinflammatory as well as protective responses in local tissues.

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.

Terminology for Blastocystis subtypes--a consensus.

Blastocystis is a ubiquitous enteric protistan parasite that has extensive genetic diversity and infects humans and many other animals. Distinct molecular methodologies developed to detect variation and obtain information about transmission patterns and clinical importance have resulted in a confusing array of terminologies for the identification and designation of Blastocystis subtypes. In this article, we propose a standardization of Blastocystis terminology to improve communication and correlate research results. Based primarily on published small-subunit ribosomal RNA gene analyses, we propose that all mammalian and avian isolates should be designated Blastocystis sp. and assigned to one of nine subtypes.

Infectivity of different genotypes of human Blastocystis hominis isolates in chickens and rats.

Most Blastocystis hominis isolates from humans are believed to be potentially zoonotic. This is because B. hominis isolates found in a variety of other host species have been found to have identical or relatively similar genotypes to those found in human isolates. However, the transmission of human B. hominis isolates to other animals has not been confirmed experimentally. In this study, the infectivity associated with several unique human Blastocystis genotypes (subtypes 2, 3, 4 and 7) was therefore investigated by infecting chickens and rats with two isolates of each subtype experimentally. The results showed that one isolate of subtype 4 and one isolate of subtype 7 was capable of infecting both chickens and rats, while two isolates of subtype 2, another isolate of subtype 4, and another isolate of subtype 7 could only infect chickens. Conversely, two isolates of subtype 3 failed to infect either of the animals. These results confirmed that several genotypes from human isolates could infect chickens and/or rats, indicating that chickens and rats are suitable experimental animal models for studying the zoonotic potential of human Blastocystis isolates.

Molecular epidemiology of human Blastocystis in a village in Yunnan province, China.

The purpose of this study was to improve our understanding of the molecular epidemiology of human Blastocystis, focusing on 239 randomly selected individuals in a single village in Yunnan province, China. Emphasis was placed on the relative frequency of different Blastocystis subtypes and underlying risk factors. We used a cross-sectional study design, by employing a pre-tested questionnaire to obtain demographic data and behavioural risk factors, and collected faecal samples for culture and subsequent identification of Blastocystis. DNA was extracted from Blastocystis isolates and the subtypes were identified using 7 subtype-specific sequenced-tagged site (STS) primers. Overall, 78 faecal samples were Blastocystis culture-positive (32.6%, 95% confidence interval: 26.7-38.6%). The majority (n=73, 93.6%) were single infections with one of the known subtypes, whereas 2 isolates consisted of 2 concurrent subtypes. The remaining 3 isolates could not be identified with the currently known STS primers. Risk factors for a Blastocystis infection were drinking unboiled water, consumption of raw water plants and pig ownership. The consumption of raw water plants was positively associated with subtype 1 infections, and drinking unboiled water with subtype 3 infections. In conclusion, human Blastocystis was common in this village in southwest China, and different subtypes were associated with distinct transmission routes or sources of infection, and hence Blastocystis subtypes might be linked to specific environmental compartments.

Cross-sectional surveys and subtype classification of human Blastocystis isolates from four epidemiological settings in China.

The prevalence and geographical distribution of the intestinal protozoa Blastocystis in humans across China is unknown, and the relative importance of different subtypes has yet to be investigated. We assessed the community prevalence and relative frequencies of different Blastocystis subtypes in four epidemiological settings in China, i.e., Shanghai municipality, Yongjia county (Zhejiang province), Eryuan county, and Menghai county (both Yunnan province). Blastocystis infection was detected with the culture method, and the subtype was identified with polymerase chain reaction using a set of subtype-specific primers. The prevalence at the four study settings was 1.9, 5.9, 18.4, and 32.6%, respectively. People aged greater than or equal to 60 years had a higher prevalence in the former two settings, Shanghai and Yongjia, whereas the highest infection rate was found among individuals aged 10-17 years in the latter two settings, Eryuan and Menghai. A higher prevalence was found in men in the former two settings but in women in the latter two settings. Five different Blastocystis subtypes were identified from the 192 isolates. Subtype 3 was the predominant type, followed by subtype 1. In conclusion, the epidemiology of Blastocystis varies across China.

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.

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.