Sample preparation for arsenic speciation analysis in baby food by generation of substituted arsines with atomic absorption spectrometry detection.

A slurry sampling procedure for arsenic speciation analysis in baby food by arsane generation, cryogenic trapping and detection with atomic absorption spectrometry is presented. Several procedures were tested for slurry preparation, including different reagents (HNO3, HCl and tetramethylammonium hydroxide - TMAH) and their concentrations, water bath heating and ultrasound-assisted agitation. The best results for inorganic arsenic (iAs) and dimethylarsinate (DMA) were reached when using 3molL-1 HCl under heating and ultrasound-assisted agitation. The developed method was applied for the analysis of five porridge powder and six baby meal samples. The trueness of the method was checked with a certified reference material (CRM) of total arsenic (tAs), iAs and DMA in rice (ERM-BC211). Arsenic recoveries (mass balance) for all samples and CRM were performed by the determination of the tAs by inductively coupled plasma mass spectrometry (ICP-MS) after microwave-assisted digestion and its comparison against the sum of the results from the speciation analysis. The relative limits of detection were 0.44, 0.24 and 0.16µgkg-1 for iAs, methylarsonate and DMA, respectively. The concentrations of the most toxic arsenic species (iAs) in the analyzed baby food samples ranged between 4.2 and 99µgkg-1 which were below the limits of 300, 200 and 100µgkg-1 set by the Brazilian, Chinese and European legislation, respectively.

Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts.

Pneumocystis jirovecii is a major cause of life-threatening pneumonia in immunosuppressed patients including transplant recipients and those with HIV/AIDS, yet surprisingly little is known about the biology of this fungal pathogen. Here we report near complete genome assemblies for three Pneumocystis species that infect humans, rats and mice. Pneumocystis genomes are highly compact relative to other fungi, with substantial reductions of ribosomal RNA genes, transporters, transcription factors and many metabolic pathways, but contain expansions of surface proteins, especially a unique and complex surface glycoprotein superfamily, as well as proteases and RNA processing proteins. Unexpectedly, the key fungal cell wall components chitin and outer chain N-mannans are absent, based on genome content and experimental validation. Our findings suggest that Pneumocystis has developed unique mechanisms of adaptation to life exclusively in mammalian hosts, including dependence on the lungs for gas and nutrients and highly efficient strategies to escape both host innate and acquired immune defenses.

Outbreak of pneumocystis pneumonia in renal and liver transplant patients caused by genotypically distinct strains of Pneumocystis jirovecii.

BACKGROUND: An outbreak of 29 cases of Pneumocystis jirovecii pneumonia (PCP) occurred among renal and liver transplant recipients (RTR and LTR) in the largest Danish transplantation centre between 2007 and 2010, when routine PCP prophylaxis was not used. METHODS: P. jirovecii isolates from 22 transplant cases, 2 colonized RTRs, and 19 Pneumocystis control samples were genotyped by restriction fragment length polymorphism and multilocus sequence typing analysis. Contact tracing was used to investigate transmission. Potential risk factors were compared between PCP cases and matched non-PCP transplant patients. RESULTS: Three unique Pneumocystis genotypes were shared among 19 of the RTRs, LTRs, and a colonized RTR in three distinct clusters, two of which overlapped temporally. In contrast, Pneumocystis control samples harbored a wide range of genotypes. Evidence of possible nosocomial transmission was observed. Among several potential risk factors, only cytomegalovirus viremia was consistently associated with PCP (P=0.03; P=0.009). Mycophenolate mofetil was associated with PCP risk only in the RTR population (P=0.04). CONCLUSION: We identified three large groups infected with unique strains of Pneumocystis and provide evidence of an outbreak profile and nosocomial transmission. LTRs may be infected in PCP outbreaks simultaneously with RTRs and by the same strains, most likely by interhuman transmission. Patients are at risk several years after transplantation, but the risk is highest during the first 6 months after transplantation. Because patients at risk cannot be identified clinically and outbreaks cannot be predicted, 6 months of PCP chemoprophylaxis should be considered for all RTRs and LTRs.

Sequencing and characterization of the complete mitochondrial genomes of three Pneumocystis species provide new insights into divergence between human and rodent Pneumocystis.

Pneumocystis jirovecii is an important opportunistic pathogen associated with AIDS and other immunodeficient conditions. Currently, very little is known about its nuclear and mitochondrial genomes. In this study, we sequenced the complete mitochondrial genome (mtDNA) of this organism and its closely related species Pneumocystis carinii and Pneumocystis murina by a combination of sequencing technologies. Our study shows that P. carinii and P. murina mtDNA share a nearly identical number and order of genes in a linear configuration, whereas P. jirovecii has a circular mtDNA containing nearly the same set of genes but in a different order. Detailed studies of the mtDNA terminal structures of P. murina and P. carinii suggest a unique replication mechanism for linear mtDNA. Phylogenetic analysis supports a close association of Pneumocystis species with Taphrina, Saitoella, and Schizosaccharomyces, and divergence within Pneumocystis species, with P. murina and P. carinii being more closely related to each other than either is to P. jirovecii. Comparative analysis of four complete P. jirovecii mtDNA sequences in this study and previously reported mtDNA sequences for diagnosing and genotyping suggests that the current diagnostic and typing methods can be improved using the complete mtDNA data. The availability of the complete P. jirovecii mtDNA also opens the possibility of identifying new therapeutic targets.

HIV infection and antiretroviral therapy have divergent effects on mitochondria in adipose tissue.

BACKGROUND: Although human immunodeficiency virus (HIV) infection and antiretroviral therapy (ART) affect mitochondrial DNA (mtDNA) content and function, comprehensive evaluations of their effects on mitochondria in muscle, adipose tissue, and blood cells are limited. METHODS: Mitochondrial DNA quantification, mitochondrial genome sequencing, and gene expression analysis were performed on muscle, adipose tissue, and peripheral blood mononuclear cell (PBMC) samples from untreated HIV-positive patients, HIV-positive patients receiving nucleoside reverse transcriptase inhibitor (NRTI)-based ART, and HIV-negative controls. RESULTS: The adipose tissue mtDNA/nuclear DNA (nDNA) ratio was increased in untreated HIV-infected patients (ratio, 353) and decreased in those receiving ART (ratio, 162) compared with controls (ratio, 255; P < .05 for both comparisons); the difference between the 2 HIV-infected groups was also significant (P = .002). In HIV-infected participants, mtDNA/nDNA in adipose tissue correlated with the level of activation (CD38+ /HLA-DR+) for CD4+ and CD8+ lymphocytes. No significant differences in mtDNA content were noted in muscle or PMBCs among groups. Exploratory DNA microarray analysis identified differential gene expression between patient groups, including a subset of adipose tissue genes. CONCLUSIONS: HIV infection and ART have opposing effects on mtDNA content in adipose tissue; immune activation may mediate the effects of HIV, whereas NRTIs likely mediate the effects of ART.

Outbreaks of Pneumocystis pneumonia in 2 renal transplant centers linked to a single strain of Pneumocystis: implications for transmission and virulence.

BACKGROUND: There have been numerous reports of clustered outbreaks of Pneumocystis pneumonia (PCP) at renal transplant centers over the past 2 decades. It has been unclear whether these outbreaks were linked epidemiologically to 1 or several unique strains, which could have implications for transmission patterns or strain virulence. METHODS: Restriction fragment length polymorphism (RFLP) analysis was used to compare Pneumocystis isolates from 3 outbreaks of PCP in renal transplant patients in Germany, Switzerland, and Japan, as well as nontransplant isolates from both human immunodeficiency virus (HIV)-infected and uninfected patients. RESULTS: Based on RFLP analysis, a single Pneumocystis strain caused pneumonia in transplant patients in Switzerland (7 patients) and Germany (14 patients). This strain was different from the strain that caused an outbreak in transplant patients in Japan, as well as strains causing sporadic cases of PCP in nontransplant patients with or without HIV infection. CONCLUSIONS: Two geographically distinct clusters of PCP in Europe were due to a single strain of Pneumocystis. This suggests either enhanced virulence of this strain in transplant patients or a common, but unidentified, source of transmission. Outbreaks of PCP can be better understood by enhanced knowledge of transmission patterns and strain variation.

The longitudinal effects of behavioral, health, and socio-demographic factors on body mass index among older Chinese adults.

OBJECTIVES: To examine the effects of behavioral, health, and socio-demographic factors on being overweight or obese among older Chinese adults. METHODS: This research uses panel data from the China Health and Nutrition Survey, which was designed to examine how social and economic transformation affected the health and nutritional status of residents. For these analyses, we used all available information on adults aged 60 years or older surveyed in 1997, 2000, 2004, and 2006 (N = 3,591). Body mass index (BMI) was dichotomized as normal (18.5-24.9 kg/m(2)) and overweight (25.0-29.9 kg/m(2))/obese (≥30 kg/m(2)). Generalized estimating equations were used to estimate population-averaged (marginal) effects. RESULTS: The combined prevalence of overweight or obese was approximately 33%. Moderate or heavy non-leisure physical activities (OR = 0.39; 95% CI = 0.32-0.49) and smoking (OR = 0.69; 95% CI = 0.57-0.84) decreased the odds of being overweight or obese, while drinking alcohol (OR = 1.25; 95% CI = 1.05-1.50) increased the odds. For individuals in all income levels, the amount of non-leisure physical activity strongly affected the BMI among the older Chinese adults. CONCLUSIONS: Active lifestyle interventions may help counter what could otherwise be a growing obesity epidemic in China.

Predictors of body mass index among low-income community-dwelling older adults.

This study investigated demographic, behavioral, and functional predictors of overweight and obesity, using secondary data from 705 community-dwelling individuals aged 65 years and older receiving or seeking Medicaid personal care services. Half of the participants were obese, while an additional 28% were overweight. The relationships between body mass index (BMI) levels and selected independent variables were analyzed. Females were more likely to be obese, while those who were older (75 years or older), more cognitively impaired, and smoked were less likely to obese. Comparing obesity with being overweight, being female and reporting more pain symptoms increased the odds of being obese, whereas being older (75 years or older) and being more cognitively impaired decreased the odds. The especially high rates of obesity in Texas have a profound impact on personal health and may result in increased health care costs that threaten public programs as well.

Psychotropic medication discussions in older adults' primary care office visits: So much to do, so little time.

OBJECTIVES: To examine discussions of psychotropic medications during the older patient's visit to primary care physicians, identify how physician's competing demands influence these discussions, describe different scenarios physicians utilize to address mental health complaints of older adults, and recommend best practices for diagnosing and treating such patients. METHOD: Convenience sample of 59 videotapes of primary care office visits involving mental health discussions in the United States complemented by patient and physician surveys. Videotaped visits were examined using logistic regression for grouped-level data to explore contributions of physician's competing demands to the likelihood of having psychotropic medication discussions. Tape transcripts were selected to provide examples of prescribing and referral behaviors. RESULTS: One-third of these visits contained no psychotropic medication discussions despite its important role in treating mental illnesses. When prescribing psychotropic medicines, physicians presented information about the medication's purpose and brand name more often than adverse effects or usage. More competing demands (i.e., more topics discussed or more leading causes of disability addressed during the visit) were associated with less psychotropic medication discussions. Selected case scenarios illustrate the importance of acknowledging mental illness, prescribing psychotropic medications, explaining the medications, and/or referring patients to mental health providers to address their mental health complaints. CONCLUSION: Competing demands may constrain discussions of psychotropic medications. Given the seriousness of mental illness in late life, system-level changes may be needed to correctly diagnose mental illness, take more proactive actions to improve mental health, and enhance information exchange concerning psychotropic medication in a manner that meets patients' needs.

Nucleic acid amplification tests for diagnosis of smear-negative TB in a high HIV-prevalence setting: a prospective cohort study.

BACKGROUND: Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV. METHODS: We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD) test (Gen-Probe Inc, San Diego, CA). We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid), and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment. RESULTS: Of 211 patients enrolled, 170 (81%) were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203). Among HIV-seropositive patients, 94 (55%) reported taking co-trimoxazole prophylaxis and 29 (17%) reported taking antiretroviral therapy. Seventy-five patients (36%) had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28-51) and that of secA1 was 24% (95% CI 15-35). Both tests had specificities of 95% (95% CI 90-98). The MTD test correctly identified 18 (24%) TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate. CONCLUSIONS: The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of Mycobacterium tuberculosis in sputum, even currently available tests can provide substantial clinical impact in smear-negative populations.

Provision of counseling on diabetes self-management: are there any age disparities?

OBJECTIVE: To determine whether there are any age-related disparities in the frequency of provision of counseling and education for diabetes care in a large HMO in Central Texas. METHODS: EMR search from 13 primary care clinics on patients aged ≥18 years (n=1300) who had been diagnosed with type 2 diabetes. RESULTS: There were no significant age differences in the frequency of provision of counseling about HBGM, diet, smoking or diabetes education. However, there were significant age differences in the provision of exercise counseling. Patients aged ≥75 were significantly less likely to have been provided exercise counseling than those aged <65 (adjusted OR=0.60; 95% CI=0.37-0.98). The mean HbA1c for patients aged ≥75 and 65-74 were significantly lower than that of patients aged <65 (8.9 vs. 9.0 vs. 9.7; P<0.001). CONCLUSION: While age-related variations in self-management protocols were not found, the provision of formal diabetes education was low (29.4%). The persistence of key risk factors in later life (e.g., obesity) underscores the need for better self-management protocols for older adults. PRACTICE IMPLICATIONS: Additional efforts on strategies to increase counseling about lifestyle habits and diabetes self-management care by appropriate health care providers is needed. Diabetes counseling should be individually tailored in older population.

Disparities in diabetes management by race or ethnicity in a primary care clinic in central Texas.

We determined the nature and magnitude of extant health disparities in patients with type 2 diabetes (T2DM) by race and ethnicity. Data were abstracted from the electronic medical records and charts of all patients 18 years or older who had been diagnosed with T2DM and seen over a 1-year period in one primary care clinic. Data abstracted included patient demographics; provision of counseling on smoking cessation, diet, exercise, and home blood glucose monitoring (HBGM); health care utilization; laboratory measures; and clinical outcomes. No significant racial or ethnic differences were found in the rate of provision of counseling on smoking cessation, diet, exercise, and HBGM, which were all suboptimal according to American Diabetes Association recommendations. In addition, no significant differences were found in the mean number of hospital admissions, emergency room visits, and referrals for specialty care. However, the mean HbA1c levels for African Americans (9.9%) and Hispanics (9.0%) were significantly higher than that of whites (8.7%; P<.0001), even after controlling for body mass index and age. Explanation of the significant racial and ethnic differences found in HbA1c levels, despite similar diabetes self-management treatment protocols or health care utilization, calls for further research.

Restriction fragment length polymorphism typing demonstrates substantial diversity among Pneumocystis jirovecii isolates.

Better understanding of the epidemiology and transmission patterns of human Pneumocystis should lead to improved strategies for preventing Pneumocystis pneumonia (PCP). We have developed a typing method for Pneumocystis jirovecii that is based on restriction fragment length polymorphism (RFLP) analysis after polymerase chain reaction amplification of an approximately 1300 base-pair region of the msg gene family, which comprises an estimated 50-100 genes/genome. The RFLP pattern was reproducible in samples containing >1000 msg copies/reaction and was stable over time, based on analysis of serial samples from the same patient. In our initial analysis of 48 samples, we found that samples obtained from different individuals showed distinct banding patterns; only samples obtained from the same patient showed an identical RFLP pattern. Despite this substantial diversity, samples tended to cluster on the basis of country of origin. In an evaluation of samples obtained from an outbreak of PCP in kidney transplant recipients in Germany, RFLP analysis demonstrated identical patterns in samples that were from 12 patients previously linked to this outbreak, as well as from 2 additional patients. Our results highlight the presence of a remarkable diversity in human Pneumocystis strains. RFLP may be very useful for studying clusters of PCP in immunosuppressed patients, to determine whether there is a common source of infection.

Polymerase chain reaction of secA1 on sputum or oral wash samples for the diagnosis of pulmonary tuberculosis.

BACKGROUND: Nucleic acid amplification tests are sensitive and specific for identifying Mycobacterium tuberculosis in sputum smear-positive populations, but they are less sensitive in sputum smear-negative populations. Few studies have assessed their performance among patients infected with HIV, and no studies have assessed their performance with oral wash specimens, which may be easier to obtain than sputum samples. METHODS: We performed a prospective study involving 127 adults from 2 populations who were undergoing evaluation for respiratory complaints at Mulago Hospital in Kampala, Uganda. We obtained and tested sputum samples for Mycobacterium tuberculosis, and we simultaneously obtained oral wash specimens to test for M. tuberculosis DNA by polymerase chain reaction (PCR) amplification of a novel locus, the secA1 gene. A positive mycobacterial culture of sputum was used to define cases of tuberculosis; we calculated the sensitivity and specificity of the PCR assay with sputum or oral wash specimens in reference to the standard of sputum culture results. RESULTS: Tuberculosis (75 [59%] of 127 patients) and HIV infection (58 [46%] of 126 patients) were both common in the study population. PCR of sputum samples was highly sensitive (sensitivity, 99%; 95% confidence interval, 93%-100%) and specific (specificity, 88%; 95% confidence interval, 77%-96%) for detection of pulmonary tuberculosis and performed well among HIV-infected patients and among patients with negative sputum smear results. PCR of oral wash specimens was less sensitive (sensitivity, 73%; 95% confidence interval, 62%-83%) but also detected a substantial proportion of tuberculosis cases. CONCLUSIONS: PCR targeting the secA1 gene was highly sensitive and specific for identifying M. tuberculosis in sputum samples, independent of smear or HIV infection status. Oral washes showed promise as an easily obtained respiratory specimen for tuberculosis diagnosis. PCR of sputum for detection of the secA1 gene could be a rapid, effective diagnostic tool for tuberculosis referral centers.