Evaluation of factors affecting real-time PCR performance for diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical stool samples.

Although PCR offers the potential for sensitive detection of parasites there are several pitfalls for optimal performance, especially when DNA is extracted from a complex sample material such as stool. With the aid of a sensitive inhibitor control in a duplex real-time PCR (qPCR) for identification of Entamoeba histolytica and Entamoeba dispar we have evaluated factors that influenced the performance of the qPCR and have suggested a rationale to be used in the analysis of clinical samples. Pre-PCR processing was found to be of outmost importance for an optimal amplification since inhibitors caused false-negative results when higher amounts of sample were used. Stool sampling with a flocked swab (ESwab, Copan), yielding on average 173 mg, gave positive qPCR results in samples with cysts of E. dispar that were negative in serially diluted stool samples. The degree of inhibition found varied between samples and was not an on-off phenomenon. Even low-grade inhibition, shown as an increase of two cycles in the qPCR for the inhibitor control, could lead to false negativity in samples with low amounts of parasites. Lack of amplification in the qPCR due to inhibition could be overcome by dilution of the extracted DNA by 1/10-1/20. We also describe the use of guanidinium thiocyanate buffer for transport and storage of samples as well as a time-saving semi-automated DNA extraction method in an Arrow instrument (Nordiag) preceded by bead beating.

Food and water security issues in Russia II: water security in general population of Russian Arctic, Siberia and Far East, 2000-2011.

BACKGROUND: Poor state of water supply systems, shortage of water purification facilities and disinfection systems, low quality of drinking water generally in Russia and particularly in the regions of the Russian Arctic, Siberia and Far East have been defined in the literature. However, no standard protocol of water security assessment has been used in the majority of studies. STUDY DESIGN AND METHODS: Uniform water security indicators collected from Russian official statistical sources for the period 2000-2011 were used for comparison for 18 selected regions in the Russian Arctic, Siberia and Far East. The following indicators of water security were analyzed: water consumption, chemical and biological contamination of water reservoirs of Categories I and II of water sources (centralized--underground and surface, and non-centralized) and of drinking water. RESULTS: Water consumption in selected regions fluctuated from 125 to 340 L/person/day. Centralized water sources (both underground and surface sources) are highly contaminated by chemicals (up to 40-80%) and biological agents (up to 55% in some regions), mainly due to surface water sources. Underground water sources show relatively low levels of biological contamination, while chemical contamination is high due to additional water contamination during water treatment and transportation in pipelines. Non-centralized water sources are highly contaminated (both chemically and biologically) in 32-90% of samples analyzed. Very high levels of chemical contamination of drinking water (up to 51%) were detected in many regions, mainly in the north-western part of the Russian Arctic. Biological contamination of drinking water was generally much lower (2.5-12%) everywhere except Evenki AO (27%), and general and thermotolerant coliform bacteria predominated in drinking water samples from all regions (up to 17.5 and 12.5%, correspondingly). The presence of other agents was much lower: Coliphages--0.2-2.7%, Clostridia spores, Giardia cysts, pathogenic bacteria, Rotavirus--up to 0.8%. Of a total of 56 chemical pollutants analyzed in water samples from centralized water supply systems, 32 pollutants were found to be in excess of hygienic limits, with the predominant pollutants being Fe (up to 55%), Cl (up to 57%), Al (up to 43%) and Mn (up to 45%). CONCLUSION: In 18 selected regions of the Russian Arctic, Siberia and Far East Category I and II water reservoirs, water sources (centralized--underground, surface; non-centralized) and drinking water are highly contaminated by chemical and biological agents. Full-scale reform of the Russian water industry and water security system is urgently needed, especially in selected regions.

Gene expression of estrogen receptors in PBMC from patients with Puumala virus infection.

The influence of estrogen signaling on infectious diseases is not fully known. Males seem to be more susceptible to infections than females. This has also been noted for the Scandinavian form of hemorrhagic fever with renal syndrome caused by Puumala hantavirus (PUUV). To investigate the differences in estrogen receptors in relation to sex and clinical severity, 20 patients (10 males, 10 females) with confirmed PUUV infection were studied. Real-time polymerase chain reaction was performed for analyzing mRNA expression of estrogen receptor-α (ERα), ERß, and ERß2 (ERß cx) in peripheral blood mononuclear cells from patients and healthy age- and sex-matched blood donors. Blood chemistry and peripheral blood mononuclear cells sampling were performed during the acute and convalescent phases. None or very small amounts of ERß were detected, and ERα and ERß2 mRNA were elevated in the patient group. The samples from the males were correlated with ERß2; the female samples, with ERα. Furthermore, the female and male samples are partly separated using multivariate statistic analysis (principal component analysis), supporting findings that clinical symptoms differ depending on sex.

An unbiased metagenomic search for infectious agents using monozygotic twins discordant for chronic fatigue.

BACKGROUND: Chronic fatigue syndrome is an idiopathic syndrome widely suspected of having an infectious or immune etiology. We applied an unbiased metagenomic approach to try to identify known or novel infectious agents in the serum of 45 cases with chronic fatigue syndrome or idiopathic chronic fatigue. Controls were the unaffected monozygotic co-twins of cases, and serum samples were obtained at the same place and time. RESULTS: No novel DNA or RNA viral signatures were confidently identified. Four affected twins and no unaffected twins evidenced viremia with GB virus C (8.9% vs. 0%, p = 0.019), and one affected twin had previously undetected hepatitis C viremia. An excess of GB virus C viremia in cases with chronic fatigue requires confirmation. CONCLUSIONS: Current, impairing chronic fatigue was not robustly associated with viremia detectable in serum.

Gene expression in peripheral blood leukocytes in monozygotic twins discordant for chronic fatigue: no evidence of a biomarker.

BACKGROUND: Chronic fatiguing illness remains a poorly understood syndrome of unknown pathogenesis. We attempted to identify biomarkers for chronic fatiguing illness using microarrays to query the transcriptome in peripheral blood leukocytes. METHODS: Cases were 44 individuals who were clinically evaluated and found to meet standard international criteria for chronic fatigue syndrome or idiopathic chronic fatigue, and controls were their monozygotic co-twins who were clinically evaluated and never had even one month of impairing fatigue. Biological sampling conditions were standardized and RNA stabilizing media were used. These methodological features provide rigorous control for bias resulting from case-control mismatched ancestry and experimental error. Individual gene expression profiles were assessed using Affymetrix Human Genome U133 Plus 2.0 arrays. FINDINGS: There were no significant differences in gene expression for any transcript. CONCLUSIONS: Contrary to our expectations, we were unable to identify a biomarker for chronic fatiguing illness in the transcriptome of peripheral blood leukocytes suggesting that positive findings in prior studies may have resulted from experimental bias.

Toxoplasmosis in immunocompromized patients.

Infection with the cosmopolitan parasite Toxoplasma gondii is often associated with severe consequences and a high mortality rate in immunocompromized patients. Non-specific symptoms make diagnosis challenging. Monitoring of patients at risk is of value. We here present 8 cases of toxoplasmosis in immunocompromized patients with suggestions for preventive monitoring.

Effect of supplement with lactic-acid producing bacteria on fatigue and physical activity in patients with chronic fatigue syndrome.

Disturbances in intestinal microbial ecology and in the immune system of the host have been implicated as a part of the pathogenesis in chronic fatigue syndrome. Probiotic lactic acid producing bacteria have been shown to prevent and alleviate gastrointestinal disturbances and to normalize the cytokine profile which might be of an advantage for patients suffering from chronic fatigue syndrome. The aim of the study was to evaluate the effect of Lactobacillus paracasei ssp. paracasei F19, Lactobacillus acidophilus NCFB 1748 and Bifidobacterium lactis Bb12 on fatigue and physical activity in CFS patients. Fifteen patients fulfilling the criteria set by international researchers in the field at the US Centre for Disease Control and Prevention in 1994 for chronic fatigue syndrome, were included in the study. The patients had high fatigue severity scores and high disability scores. During the first two weeks baseline observations without treatment were assessed, succeeded by four weeks of intake of a probiotic product and a four-week follow-up period. The fatigue, health and physical activity was assessed by the use of the Visual Analogue Scales and the SF-12 Health Survey. Faecal samples were collected and the normal microflora was analysed. Neurocognitive functions improved during the study period while there were no significant changes in fatigue and physical activity scores. No major changes occurred in the gastrointestinal microflora. At the end of the study 6 of 15 patients reported that they had improved according to the assessment described. The findings in this study that improvement of health is possible to achieve should encourage further studies with interventions with probiotics in patients with CFS.

A prospective study of diagnosis of Toxoplasma gondii infection after bone marrow transplantation.

Active infection with Toxoplasma gondii in immunocompromised transplant recipients can lead to toxoplasmosis, which may have a rapid disease course and in some cases be fatal. It is of paramount importance to diagnose toxoplasmosis at an early stage, and to initiate specific treatment to improve the outcome. Polymerase chain reaction (PCR) is today the primary diagnostic tool to diagnose toxoplasmosis in immunocompromised patients. Timely diagnosis may, however, be difficult if toxoplasmosis is at first asymptomatic. To investigate the magnitude of toxoplasmosis after bone marrow transplantation (BMT), we conducted a screening study by PCR where 21 autologous and 12 allogeneic BMT recipients were included. Peripheral blood samples were taken one week prior to BMT; thereafter, blood samples were drawn weekly for the first 6 months, and monthly up to one year after BMT. The samples were analyzed by conventional PCR and real-time PCR. T. gondii DNA was detected in peripheral blood from one patient 5 days post allogeneic BMT. There were no clinical signs of toxoplasmosis. Medical records were reviewed and showed a previously undiagnosed eye infection in another allogeneic BMT recipient. These two patients were seropositive for T. gondii. We concluded that monitoring for T. gondii DNA in peripheral blood samples using PCR might be a valuable method for identifying toxoplasma-seropositive stem cell transplant recipients.

"The Laundry Bag Project"--unequal distribution of dermatological healthcare resources for male and female psoriatic patients in Sweden.

BACKGROUND: Psoriasis and eczema are common dermatological diseases that occur with approximately equal frequency in men and in women. The aim of this study was to determine whether men and women with dermatological diseases in need of ultra-violet radiation (UV) treatment receive equal care. METHODS: We conducted a retrospective analysis of records of all patients referred to and seen at our clinic during 2003 with diagnoses of psoriasis or eczema. We performed a gender-based analysis of the number, type, and estimated cost of the treatments given to each patient. We evaluated similar data from a Swedish Psoriasis Association (SPA) treatment center and from the state pharmacy monopoly (Apoteksstatistiken). RESULTS: Men with eczema or psoriasis received more help with emollients than did women and were given a greater number of UV treatments. At our clinic and at the SPA center, women constituted 37 and 42%, respectively, of the individuals who received UV treatment; yet, they received only 34 and 36% of the treatments, respectively. Women were prescribed self-care more often than men, with 61% of prescriptions for emollients and 48% of specific topical treatments for psoriasis dispensed to women. CONCLUSIONS: We discovered previously unrecognized gender differences in standard dermatological treatment for common diagnoses at our hospital. To ensure optimal care for each patient, treatment disparity should be recognized and gender-based analyzes be carried out when planning dermatological health care.

beta-Alanine and gamma-aminobutyric acid in chronic fatigue syndrome.

BACKGROUND: Due to the occurrence of sleep disturbances and fatigue in chronic fatigue syndrome (CFS), an investigation was performed to examine if there is an abnormal excretion of gamma-aminobutyric acid (GABA) and/or its structural analogue beta-alanine in the urine from CFS patients. Both GABA and beta-alanine are inhibitory neurotransmitters in the mammalian central nervous system. METHODS: The 24 h urine excretion of GABA and beta-alanine was determined by isotope dilution gas chromatography mass spectrometry in 33 CFS patients and 43 healthy controls. The degree of symptoms in both patients and controls was measured by grading of three typical CFS symptoms using a Visual Analogue Scale. RESULTS: Men had a significantly higher excretion of both beta-alanine and GABA than women. Comparing CFS patients with healthy controls showed no significant difference in excretion of neither beta-alanine nor GABA. No correlation was found between the excretion of beta-alanine or GABA and any of the three characteristic CFS symptoms measured. However, two female and two male CFS patients excreted considerably higher amounts of beta-alanine in their 24 h urine samples than control subjects. CONCLUSIONS: Increased excretion of beta-alanine was found in a subgroup of CFS patients, indicating that there may be a link between CFS and beta-alanine in some CFS patients.

Reduced levels of oestrogen receptor beta mRNA in Swedish patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is an illness with unknown aetiology and pathophysiology. The difference in incidence by sex observed for CFS indicates a role for oestrogen and oestrogen receptors in disease development. Furthermore, an immunomediated pathogenesis has been suggested for CFS, providing an additional connection to oestrogen, which displays immunomodular functions. AIMS: To investigate a possible association of oestrogen receptor (ER) mRNAs and two ERbeta single-nucleotide polymorphisms (SNPs) with CFS. METHODS: Messenger RNA levels of ERalpha, ERbeta wt and ERbeta cx were investigated in peripheral blood mononuclear cells from 30 patients with CFS and 36 healthy controls by quantitative real-time polymerase chain reaction. Two ERbeta SNPs were scored in the same material. RESULTS: The CFS group showed significantly lower mRNA expression levels of ERbeta wt compared with the healthy control group. No differences were observed for ERalpha or ERbeta cx between patients and controls. There were no significant differences in frequency for the investigated ERbeta SNPs between cases and controls. CONCLUSIONS: The reduced ERbeta wt expression level observed in this study is consistent with an immune-mediated pathogenesis of CFS. Additionally, the observation that ERbeta wt expression is decreased in CFS could provide an entry point to identify interesting, potentially disease-causing, candidate molecules for further study. A possible connection between oestrogen, oestrogen receptors and CFS should be evaluated further.

Premorbid predictors of chronic fatigue.

CONTEXT: Chronic fatigue syndrome is a disabling problem characterized by persistent fatigue lasting at least 6 months with a number of ancillary symptoms. Although the etiology of chronic fatiguing illness is unknown, some evidence suggests that stress may confer increased risk for development of the disorder. Moreover, subjects with chronic fatiguing illness may have distinctive personality traits, although this finding could reflect confounding by other mechanisms. OBJECTIVE: To assess the prospective association of premorbid self-reported stress and personality with chronic fatigue-like illness. DESIGN: Prospective nested case-control study in a population-based sample. SETTING: General community. PARTICIPANTS: From the Swedish Twin Registry, 19,192 twins born between January 1, 1935, and December 31, 1958. MAIN OUTCOME MEASURES: Information about current chronic fatiguing illnesses was obtained from computer-assisted telephone interviews conducted between 1998 and 2002. Self-reported stress (based on a single question) and personality scales (emotional instability and extraversion in the Eysenck Personality Inventory) were measured from 1972 to 1973 by a mailed questionnaire. Relative risks were estimated with case-control analyses (matched for age and sex) and co-twin control analyses (comparing discordant pairs). RESULTS: Higher emotional instability and self-reported stress in the premorbid period were associated with higher risk for chronic fatigue-like illness in matched case-control analyses (odds ratios, 1.72 and 1.64, respectively). In co-twin control analyses, relative risk of emotional instability decreased to 1.02 whereas that of stress increased considerably to 5.81. There was no association between extraversion and fatigue. CONCLUSIONS: Elevated premorbid stress is a significant risk factor for chronic fatigue-like illness, the effect of which may be buffered by genetic influences. Emotional instability assessed 25 years earlier is associated with chronic fatigue through genetic mechanisms contributing to both personality style and expression of the disorder. These findings suggest plausible mechanisms for chronic fatiguing illness.

Chronic widespread pain and its comorbidities: a population-based study.

BACKGROUND: Chronic widespread pain (CWP), the cardinal symptom of fibromyalgia, is prevalent and co-occurs with numerous symptom-based conditions such as chronic fatigue syndrome, joint pain, headache, irritable bowel syndrome, and psychiatric disorders. Few studies have examined the comorbidities of CWP in the general population. Furthermore, little is known about the importance of familial (genetic and family environmental) factors in the etiology of co-occurrence. METHODS: Data were obtained from 44 897 individuals in the Swedish Twin Registry via computer-assisted telephone interview from 1998 through 2002 (age >/=42 years; 73.2% response rate). Screening for CWP was based on the American College of Rheumatology criteria without clinical evaluation. Measures for comorbidities were based on standard criteria when available. Odds ratios (ORs) were calculated in case-control and co-twin control designs to assess the effect of familial confounding in the associations. RESULTS: Considerable co-occurrences were found in CWP cases for chronic fatigue (OR, 23.53; 95% confidence interval [CI], 19.67-28.16), joint pain (OR, 7.41; 95% CI, 6.70-8.21), depressive symptoms (OR, 5.26; 95% CI, 4.75-5.82), and irritable bowel syndrome (OR, 5.17; 95% CI, 4.55-5.88). In co-twin control analyses, ORs were no longer significant for psychiatric disorders, whereas they decreased but remained significant for most other comorbidities. No changes in ORs were observed for headache. CONCLUSIONS: Associations between CWP and most comorbidities are mediated by unmeasured genetic and family environmental factors in the general population. The extent of mediation via familial factors is likely to be disorder specific.

Importance of genetic influences on chronic widespread pain.

OBJECTIVE: To estimate the relative importance of genetic and environmental factors in chronic widespread pain, and to assess whether there are sex differences in the type or magnitude of these influences. METHODS: Data were collected from a national sample of twins > or = 42 years of age, all of whom were participants in the Swedish Twin Registry. The presence of chronic widespread pain was assessed via computer-assisted telephone interviews, which were conducted between 1998 and 2002, using the American College of Rheumatology criteria for fibromyalgia. No clinical examinations were performed. In preliminary analyses, probandwise concordance rates and tetrachoric correlations were calculated. Structural equation modeling was then performed to estimate additive genetic, shared environmental, and nonshared environmental sources of variability in susceptibility for the development of chronic widespread pain. RESULTS: Of 61,355 eligible twins, 44,897 individuals (73.2%) responded to the interview. Both members of 15,950 pairs responded to the items regarding pain symptoms; of these pairs, 4,170 were monozygotic, 5,881 were same-sex dizygotic, and 5,755 were opposite-sex dizygotic. The prevalence of chronic widespread pain was 4.1%, and the ratio of women to men was 3.3 to 1. Probandwise concordance rates and tetrachoric correlations suggested modest genetic influences for both women and men. Genetic and shared environmental influences explained approximately half of the total variance, with no indication of sex differences in either the type or magnitude of these influences. CONCLUSION: Individual differences in the likelihood of developing chronic widespread pain reflect modest genetic influences. There are no significant sex differences in the type or expression of the genes responsible for chronic widespread pain or in the magnitude of the relative importance of these influences on chronic widespread pain.

Epidemiology of cancer-related fatigue in the Swedish twin registry.

BACKGROUND: Estimates of the prevalence of cancer-related fatigue (CRF) are wide, and data suggest that fatigue is more prevalent among cancer patients than among the general population. However, most studies examining the prevalence of CRF have been hospital-based or clinic-based studies, which often are subject to bias. METHODS: Point prevalence and prevalence odds ratios of fatigue were estimated using data from a large, population-based cohort that was screened for fatigue and linked with national registry-based data about cancer. Prevalence odds ratios and 95% confidence intervals were calculated using logistic regression with general estimating equations. RESULTS: Approximately 23% of cancer registrants reported abnormal fatigue in the previous 6 months, 19% reported abnormal fatigue that lasted for at least 1 month, 14% reported abnormal fatigue that lasted at least 6 months, and 11% reported abnormal fatigue that lasted at least 6 months and caused significant functional impairment. Individuals who were listed in the cancer registry within the last 5 years were more likely to report experiencing fatigue than individuals who were not listed. There was an elevated prevalence of fatigue among those who were registered with carcinomas of the lung, uterine cervix, colon-rectum, ovaries, and prostate. Both women and men who were listed recently in the cancer registry were more likely to experience any level of fatigue than the comparison group. However, a greater proportion of women experienced fatigue relative to men. CONCLUSIONS: A greater proportion of individuals who were listed in a national cancer registry reported experiencing fatigue compared with individuals in the general population.

The epidemiology of chronic fatigue in the Swedish Twin Registry.

BACKGROUND: Chronic fatigue syndrome (CFS) remains an idiopathic and controversial entity. METHOD: We screened 31405 individual members of the Swedish Twin Registry (aged 42-64 years) for the symptoms of fatiguing illness via a telephone questionnaire. We refined self-reported symptoms via data from several national registries and from physician review of all available medical records in order to approximate closely the dominant case definition of CFS. FINDINGS: The 6-month prevalence of CFS-like illness was 2.36% (95% CI 2.19-2.53) and was markedly higher in women than men, odds ratio 3.92 (95% CI 3.24-4.72) with no significant association with age or years of education. There was a highly significant association with occupation that disappeared after accounting for gender. INTERPRETATION: CFS-like illness may be more common that previously acknowledged. There is a marked increase in risk by gender. Previous reports that CFS is more prevalent in individuals in certain occupational categories were not confirmed and may have been due to confounding by gender.

Twin analyses of chronic fatigue in a Swedish national sample.

BACKGROUND: Chronic fatigue has infrequently been studied in twins. Data from twin studies can inform clinical and research approaches to the management and etiology of human complex traits. METHOD: The authors obtained telephone interview data on current chronic fatigue from 31406 individuals twins in the Swedish Twin Registry (aged 42-64 years, 75.68% response rate), from both members of 12407 pairs and from one member of 6592 pairs. Of the complete pairs, 3269 pairs were monozygotic, 9010 pairs dizygotic, and 128 pairs of unknown zygosity. Structural equation twin modeling was used to estimate the latent genetic architecture of varying definitions of fatiguing illness. RESULTS: Estimates of additive genetic effects, shared environmental effects, and individual-specific environmental effects were similar in males and females. No definition of current fatiguing illness (ranging from any fatigue to CFS-like illness) was strikingly distinctive. Individual-specific effects were the predominant source of variation, followed by modest genetic influences. We could not exclude a small but conceptually important contribution of shared environmental effects. CONCLUSIONS: Current fatiguing illness appears to be a complex trait resulting from both environmental and genetic sources of variation without pronounced differences by gender.

Chronic fatigue in a population sample: definitions and heterogeneity.

BACKGROUND: Numerous nosological decisions are made when moving from the common human symptom of unusual fatigue to the rare chronic fatigue syndrome (CFS). These decisions have infrequently been subjected to rigorous evaluation. METHOD: We obtained telephone interview data on fatiguing symptoms from 31406 individuals twins in the Swedish Twin Registry aged 42-64 years; 5330 subjects who endorsed fatigue and possessed no exclusionary condition formed the analytic group. We evaluated the definition and classification of CFS-like illness using graphical methods, regression models, and latent class analysis. RESULTS: Our results raise fundamental questions about the 1994 Centers for Disease Control criteria as (1) there was no empirical support for the requirement of four of eight cardinal CFS symptoms; (2) these eight symptoms were not equivalent in their capacity to predict fatigue; and (3) no combination of symptoms was markedly more heritable. Critically, latent class analysis identified a syndrome strongly resembling CFS-like illness. CONCLUSIONS: Our data are consistent with the 'existence' of CFS-like illness although the dominant nosological approach captures population-level variation poorly. We suggest that studying a more parsimonious case definition - impairing chronic fatigue not due to a known cause - would represent a way forward.

Physiological correlates of burnout among women.

OBJECTIVES: The purpose of this study was to investigate the immune, endocrine, and metabolic correlates of burnout among women. METHODS: Forty-three participants with high and 20 participants with low scores for the Shirom-Melamed Burnout Questionnaire were compared in terms of subjective symptoms, job strain, social support, plasma levels of prolactin, tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), C-reactive protein (CRP), neopterin, serum levels of dehydroepiandrosterone sulphate (DHEAs), progesterone, estradiol, cortisol, and glycated hemoglobin (HbA1C) in whole blood. RESULTS: Besides reporting more job strain, less social support at work, and higher levels of anxiety, depression, vital exhaustion (VE), and sleep impairments, participants with high burnout manifested higher levels of TNF-alpha and HbA1C, independent of confounders including depression. CONCLUSIONS: Among women, burnout seems to involve enhanced inflammatory responses and oxidative stress.