Genotypic Characterization of Enterocytozoon bieneusi in Specimens from Pigs and Humans in a Pig Farm Community in Central Thailand

We determined that 15.7% of pigs and 1.4% of humans in a pig farm community in central Thailand harbored Enterocytozoon bieneusi. Genotyping of E. bieneusi from pigs showed genotypes O, E, and H. However, only genotype A was found in human subjects. This indicates nonzoonotic transmission of E. bieneusi in this community.Enterocytozoon bieneusi is an opportunistic organism causing diarrhea in human immunodeficiency virus (HIV)-positive patients, in whom it has a prevalence of 2 to 50% (5). The infection not only has been reported to occur in immunocompromised hosts but also has been found in healthy individuals (14, 20). This organism can infect a broad range of animals (4, 12, 16, 18, 22, 23). Genotypes of E. bieneusi in humans and animals are differentiated using the polymorphisms of the internal transcribed spacer (ITS) sequence of the rRNA gene (4, 11). To date, at least 70 ITS genotypes have been reported to infect humans and animals (2, 6). The zoonotic nature of E. bieneusi was confirmed because ITS genotypes found in domestic and wild animals had been reported to occur in immunosuppressed hosts (22). In Thailand, we reported genotypes E, O, and PigEBITS 7, which have previously been identified in pigs (3, 4) and in Thai HIV-infected patients (9). This study aimed to identify the ITS genotypes of E. bieneusi in pigs and humans who worked in or lived near pig farms to investigate the transmission of E. bieneusi among these host species.A cross-sectional study of E. bieneusi infection was conducted in a community in Nakorn Pathom Province, Central Thailand, January 2005. This community is composed primarily of four pig farms, a residential area, and a school. The residential area, but not the school, was near the pig farms. Fecal specimens were collected from school children and those who were living in this community, including pig farmers. Fecal specimens were also collected from pigs of four farms and examined for E. bieneusi. The study was approved by the Ethics Committee of the Royal Thai Army, Medical Department. Informed consent was obtained from each adult individual and from parents of school children before enrollment in the study.Fecal specimens from pigs and humans were examined for microsporidian spores using gram-chromotrope staining under light microscopy (13). DNA was prepared from water-ethyl acetate-concentrated stool specimens using FTA filter paper (Whatman Bioscience, United Kingdom) as previously described (21). Amplification of the ITS region of the small-subunit rRNA gene was performed using primers under conditions described by Katzwinkel-Wladarsch et al. (8). For specimens with PCR-negative results, PCR amplification was repeated at least twice. DNA sequencing was conducted by Macrogen Inc., Seoul, Republic of Korea. Nucleotide sequences were determined using the Sequencher program (Gene Codes Corporation, Inc.), and multiple alignment was performed using Clustal X 1.83 for Windows (24). The genotype of each specimen was confirmed by determining the homology of the sequenced PCR product with the published sequence.A total of 268 pig fecal samples were collected. Pigs aged between 21 days and 22 months were examined for E. bieneusi infection. Microsporidial spores were found in 0.7% of pig fecal samples using gram-chromotrope staining, while a greater prevalence of E. bieneusi infection, 15.7%, was detected by PCR. The prevalences of E. bieneusi infection among the four farms and different age groups are presented in Table ​Table1.1. A significantly higher prevalence of E. bieneusi was found in pigs aged 2 to 3.9 months than in pigs of other age groups (chi-square test, P < 0.001). Multivariate analysis confirmed that pigs aged 2 to 3.9 months had a 5.3-times-greater risk of infection than pigs in other age groups (95% confidence interval, 2.6 to 10.6; P < 0.001). Of these 42 E. bieneusi-positive samples, 21 (50%) were successfully characterized by sequencing analysis, and the organism was identified as being of genotypes E (12 samples [57.1%]), O (8 samples [38.1%]), and H (1 sample [4.8%]).

TABLE 1.

Prevalence of E. bieneusi positivity in pig specimens as determined by PCR

Malignant craniopharyngioma; case report and review of the literature

A case of malignant craniopharyngioma in a 46‐year‐old woman presenting clinically with visual disturbance and bifrontal headache is reported. Histopathologic examination of the suprasellar mass showed a lesion characterized by nests of epithelial cells with a basaloid appearance, round‐to‐oval nuclei, moderate pleomorphism, hyperchromasia, increased nuclear cytoplastic ratio and high mitotic activity. Immunohistochemically, the tumor cells were positive for Ki‐67 (44.3%), p53 (98%), and p63 (100%), but negative for estrogen and progesterone receptors. Clinical and pathologic features with a brief review of the relevant literature for malignant craniopharyngioma as a novel member of tumors of the suprasellar region, is discussed.     

Microsatellite loci in the carcinogenic liver fluke,Opisthorchis viverrini and their application as population genetic markers

Opisthorchis viverrini is a carcinogenic foodborne trematode endemic in Southeast Asia especially in Thailand and the Lao People's Democratic Republic. Opisthorchiasis causes hepatobiliary diseases and cholangiocarcinoma (bile duct cancer). Currently there is substantial evidence on genetic variation of O. viverrini but the information on population genetic structure is lacking. Because microsatellite DNA of this parasite is not available, we for the first time isolated and utilized microsatellite DNA as genetic markers to examine genetic diversity and the population structure of O. viverrini. Partial genomic DNA libraries were constructed by conventional and enrichment methods which yielded microsatellite-containing clones of 0.18–0.25% and 16.84%, respectively. Within 41 microsatellite loci isolated 36.59% were perfect, 60.98% were interrupted and 2.44% were compound microsatellites. The CA repetitions were the most frequent, followed by GT and CAT. Primers specific to the flanking regions of 12 microsatellite loci were developed to genotype 150 O. viverrini individuals from geographical localities in Thailand and Lao PDR. Allele numbers per locus ranged from 2 to 15, with the mean expected heterozygosity of 0.03–0.66. Analyses of O. viverrini from 5 localities revealed a high level of genetic diversity and had significant deviation from Hardy–Weinberg equilibrium. Significant heterozygote deficiency as well as heterozygote excess was detected across all localities indicating the possibility of selfing (inbreeding) as a predominant reproductive mode. Significant genetic differentiation (F_ST) was also detected between worms from different localities with varying levels of genetic heterogeneity. We discuss our results in terms of what these novel microsatellite markers reveal about the epidemiology and transmission dynamics of this medically important parasite, both in terms of the current study and their potential for future comprehensive population genetic studies O. viverrini sensu lato in Southeast Asia.     

International reproducibility of single breathhold T2* MR for cardiac and liver iron assessment among five thalassemia centers

Purpose:

To examine the reproducibility of the single breathhold T2* technique from different scanners, after installation of standard methodology in five international centers.

Materials and Methods:

Up to 10 patients from each center were scanned twice locally for local interstudy reproducibility of heart and liver T2*, and then flown to a central MR facility to be rescanned on a reference scanner for intercenter reproducibility. Interobserver reproducibility for all scans was also assessed.

Results:

Of the 49 patients scanned, the intercenter reproducibility for T2* was 5.9% for the heart and 5.8% for the liver. Local interstudy reproducibility for T2* was 7.4% for the heart and 4.6% for the liver. Interobserver reproducibility for T2* was 5.4% for the heart and 4.4% for the liver.

Conclusion:

These data indicate that T2* MR may be developed into a widespread test for tissue siderosis providing that well‐defined and approved imaging and analysis techniques are used. J. Magn. Reson. Imaging 2010;32:315–319. © 2010 Wiley‐Liss, Inc.     

Visual function and inner retinal structure correlations in aquaporin-4 antibody-positive optic neuritis

Purpose

To investigate the correlation between visual function and thinning of the retinal nerve fiber layer (RNFL) and the macular ganglion cell-inner plexiform layer (GCIPL) as measured by optical coherence tomography (OCT) in eyes with aquaporin-4 IgG-positive optic neuritis (AQP4-IgG-positive ON).

Study design

Prospective study.

Methods

Patients with a history of ON were categorized into 2 groups: the AQP4-IgG-positive group and the AQP4-IgG-negative group. Patients with multiple sclerosis were excluded. All patients underwent ophthalmologic examination and OCT imaging at least 6 months after the last episode of acute ON. Visual function and inner retinal structure correlations were analyzed using Pearson correlation and regression analyses.

Results

Thirty-one previous ON eyes of 17 AQP4-IgG-positive patients and 21 previous ON eyes of 15 AQP4-IgG-negative patients were registered. Visual function, especially the visual field, was better correlated with RNFL than with macular GCIPL. The best correlation between visual function and RNFL was the linear model, whereas the best correlation between visual function and GCIPL was the nonlinear model (inverse regression). Regression models revealed worse visual function in AQP4-IgG-positive ON than in AQP4-IgG-negative ON, whereas no differences in RNFL and GCIPL were found between the 2 groups.

Conclusions

RNFL measured by OCT can be a useful retinal structure for estimating and monitoring visual field loss in AQP4-IgG-positive ON patients, particularly in patients whose visual field cannot be quantitated. The correlation between visual function and the inner retinal structure of eyes with AQP4-IgG is unique and differs from that of eyes without AQP4-IgG.

     

Missed appointments at a tuberculosis clinic increased the risk of clinical treatment failure

We investigated the charts of 381 new smear-positive tuberculosis patients at Khon Kaen Medical School during 1997-2001 using World Health Organization definitions to evaluate associations among treatment success or failure (defaulted, failed, died, or not evaluated) and tuberculosis clinic contact, demographics and clinical characteristics of the patients. Multinomial logistic regression was used for three-category outcome analysis: treatment success, transferred-out and clinical treatment failure. The treatment success and clinical treatment failure rates were 34.1% and 34.4%, respectively. About 46.5% and 85.8% of patients missed appointments at the tuberculosis clinic in the treatment success and treatment failure groups, respectively. The results show that patients who were absent from the tuberculosis clinic were 5.95 times more likely to have clinical treatment failure than treatment success, having adjusted for the effect of transfering-out and the effect of the treatment regimen and the sputum conversion status (adjusted odds ratio = 5.95; 95% CI: 2.99 to 11.84). The review showed that absence from the tuberculosis clinic was an independent risk factor for clinical treatment failure. We recommended that all new smear-positive tuberculosis patients should be followed closely at a tuberculosis clinic.     

3D-QSAR studies on chromone derivatives as HIV-1 protease inhibitors: application of molecular field analysis

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed for chromone derivatives against HIV-1 protease using molecular field analysis (MFA) with genetic partial least square algorithms (G/PLS). Three different alignment methods: field fit, pharmacophore-based, and receptor-based were used to derive three MFA models. All models produced good predictive ability with high cross-validated r(2) (r(2) (cv)), conventional r(2), and predictive r(2)(r(2)(pred)) values. The receptor-based MFA showed the best statistical results with r(2) (cv) = 0.789, r(2)= 0.886, and r(2)(pred) = 0.995. The result obtained from the receptor-based model was compared with the docking simulation of the most active compound 21 in this chromone series to the binding pocket of HIV-1 protease (PDB entry 1AJX). It was shown that the MFA model related well with the binding structure of the complex and can provide guidelines to design more potent HIV-1 protease inhibitors.     

A High‐yield Fall Risk and Adverse Events Screening Questions From the Stopping Elderly Accidents,Death, and Injuries (STEADI) Guideline for Older Emergency Department Fall Patients

Objectives

The objectives were to examine whether responses to the Stopping Elderly Accidents, Death, and Injuries (STEADI) questions responses predicted adverse events after an older adult emergency department (ED) fall visits and to identify factors associated with such recurrent fall.

Methods

We conducted a prospective study at two urban, teaching hospitals. We included patients aged ≥ 65 years who presented to the ED for an accidental fall. Data were gathered for fall‐relevant comorbidities, high‐risk medications for falls, and the responses to 12 questions from the STEADI guideline recommendation. Our outcomes were the number of 6‐month adverse events that were defined as mortality, ED revisit, subsequent hospitalization, recurrent falls, and a composite outcome.

Results

There were 548 (86.3%) patients who completed follow‐up and 243 (44.3%) patients experienced an adverse event after a fall within 6 months. In multivariate analysis, seven questions from the STEADI guideline predicted various outcomes. The question “Had previous fall” predicted recurrent falls (odds ratio [OR] = 2.45, 95% confidence interval [CI] = 1.52 to 3.97), the question “Feels unsteady when walking sometimes” (OR = 2.34, 95% CI = 1.44 to 3.81), and “Lost some feeling in their feet” predicted recurrent falls. In addition to recurrent falls risk, the supplemental questions “Use or have been advised to use a cane or walker,” “Take medication that sometimes makes them feel light‐headed or more tired than usual,” “Take medication to help sleep or improve mood,” and “Have to rush to a toilet” predicted other outcomes.

Conclusion

A STEADI score of ≥4 did not predict adverse outcomes although seven individual questions from the STEADI guidelines were associated with increased adverse outcomes within 6 months. These may be organized into three categories (previous falls, physical activity, and high‐risk medications) and may assist emergency physicians to evaluate and refer high‐risk fall patients for a comprehensive falls evaluation.

     

Comparison of white spot syndrome virus PCR-detection methods that use electrophoresis or antibody-mediated lateral flow chromatographic strips to visualize PCR amplicons

White spot syndrome virus (WSSV) PCR-detection methods that used electrophoresis or lateral flow chromatographic strips (LFCS) were to compare and visualize PCR amplicons. Real-time PCR was used to prepare a stock template solution containing 2.85 x 10 (6) copies WSSV/microl from WSSV-infected shrimp. Serial stock dilutions were used as templates for PCR amplification of a WSSV-specific DNA fragment that was detected either in ethidium bromide stained agarose electrophoresis gels or on a chromatographic strip where it interacted with antibody to markers labeled on hybridization complex. PCR amplification employed both 1-step PCR and semi-nested PCR methods. By using 1-step PCR, the LFCS method (100 copies) gave 10 times higher sensitivity than gel electrophoresis (10(3) copies). A combination of a semi-nested PCR with LFCS gave a comparable sensitivity to those with commercial kits for nested PCR (20 copies). In addition, LFCS confirmed amplicon identity, avoided handling of carcinogenic ethidium bromide and could be completed in approximately 20-30 min post-PCR compared with 1h for gel electrophoresis. The costs for the two methods were comparable. In conclusion, semi-nested PCR followed by LFCS is a safe and rapid alternative method for detection of WSSV that provides sensitivity similar to that obtained by standard nested PCR methods.