Waterpipe tobacco smoking prevalence and illegal underage use in waterpipe-serving premises: a cross-sectional analysis among schoolchildren in Stoke-on-Trent.

OBJECTIVES: Waterpipe tobacco smoking has received little epidemiological and policy attention in the UK despite reports of increasing prevalence alongside an anecdotally non-compliant industry. This study aimed to determine how waterpipe tobacco smoking is changing among young people in the UK, both in terms of prevalence and sociodemographic correlates of use, and to quantify the extent of illegal underage use in waterpipe-serving premises in the UK. STUDY DESIGN: Repeat cross-sectional. METHODS: A secondary analysis of two cross-sectional surveys (total N = 3376), conducted in 2013 and 2015 among secondary school students aged 11-16 years in Stoke-on-Trent, measured lifetime (both surveys) and regular (at least monthly; 2015 survey only) waterpipe tobacco prevalence and location of usual use. Logistic regression models measured the association between independent variables (age, sex, ethnicity, presence of free school meals, cigarette smoking status) with lifetime and regular waterpipe tobacco use, and with illegal underage use; the latter defined as usually smoking waterpipe tobacco in a waterpipe-serving premise. RESULTS: Lifetime waterpipe tobacco prevalence remained similar in 2013 (13.7%, 95% confidence interval [CI] 12.0-15.4%) and 2015 (14.6%, 95% CI 12.8-16.4%), whereas regular use was measured at 2.9% (95% CI 2.1-3.8%) in 2015. Older, non-white, males who concurrently used cigarettes had higher odds of lifetime waterpipe tobacco use. Illegal underage use was reported among 27.1% of all regular users, correlates of which included increasing age and South Asian ethnicity. The presence of free school meals was not associated with lifetime or regular waterpipe tobacco prevalence, nor illegal underage use. CONCLUSIONS: Increased monitoring of waterpipe tobacco prevalence and patterns, including the underage policy compliance of waterpipe-serving premises, is needed to help inform policy decisions to control waterpipe tobacco use.

Can thyroid hormone mimics affect thyroid hormone measurement by immunoassay?

OBJECTIVES: This study aims to determine whether the environmental pollutant and thyroid mimic, tetrabromobisphenol A (TBBPA), interferes with thyroid hormone measurement by immunoassays. DESIGN & METHODS: Hormone-relevant concentrations of TBBPA were added to thyroid hormone-stripped human serum and subjected to 6 different thyroid hormone immunoassays. RESULTS: TBBPA was negative in all of the thyroid hormone immunoassays tested except at very high concentration (above that expected in serum of TBBPA-exposed workers) where it gave a marginally positive result in one immunoassay (in house T4 radioimmunoassay (RIA)). CONCLUSIONS: Serum TBBPA present as a result of workplace exposure or its use as a fabric flame retardant is very unlikely to give false positive results in thyroid hormone immunoassays.

Toll-like receptor expression in chronic hepatitis C: correlation with pro-inflammatory cytokine levels and liver injury.

BACKGROUND/AIMS: Toll-like receptors (TLR's) are critical receptors that promote innate immune responses to pathogen-associated molecular patterns. Activation of TLR's leads to production of pro-inflammatory cytokines such as tumour necrosis factor (TNF)-alpha. This study investigates whether peripheral blood monocyte expression of TLR's is disturbed in patients with chronic hepatitis C and whether levels of expression of these molecules are significantly correlated with hepatitis C virus (HCV) genotype, viral load, hepatic necroinflammatory activity, histological stage and circulating TNF-alpha concentrations. METHODS: In 18 non-cirrhotic patients with biopsy-proven, virologically-confirmed chronic hepatitis C and 32 controls, we measured expression of TLR2 and TLR4 on peripheral blood monocytes. HCV genotype, viral load, serum alanine aminotransferase (ALT) levels, histological stage of disease and circulating TNF-alpha and endotoxin levels were also determined. RESULTS: Peripheral blood monocyte expression of TLR2 and TLR4 were significantly increased in patients with chronic hepatitis C compared to controls, irrespective of HCV genotype or histological stage of disease. Circulating levels of TNF-alpha were also significantly increased in patients with chronic hepatitis C. In both the overall study cohort and patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, correlated significantly with serum TNF-alpha levels. In patients with chronic hepatitis C, monocyte expression of TLR2, but not of TLR4, also correlated significantly with serum ALT levels. Expression of TLR's was not significantly correlated with viral load. CONCLUSIONS: Up-regulation of peripheral blood monocyte expression of TLR2 and TLR4 occurs in patients with chronic hepatitis C. Increased monocyte expression of TLR2, but not of TLR4, correlates significantly with both increased circulating TNF-alpha levels and hepatic necroinflammatory activity in this disorder.

Correlates of placental infection with cytomegalovirus, parvovirus B19 or human herpes virus 7.

Vertical transmission of viruses is an important cause of morbidity in the fetus and neonate. Placental viral infection indicates risk of vertical transmission, but not always transmission to, or disease of the fetus. Specimens from mothers and babies from three groups-two prospective and one retrospective cohort-were tested for pathogens of teratogenic potential using multiplex PCR. Placental infection was present in 13% of the 105 samples collected. Assessment of the prospective cohorts showed cytomegalovirus (CMV) detected in 4% of placentae from unselected women, parvovirus B19 in 1% and Ureaplasma parvum in 1% of placentae. In a retrospective cohort of women at high risk of transmitting congenital infection due to seroconversion during pregnancy, miscarriage or stillbirth, CMV was detected in 64% and human herpes virus type 7 in 9% of placentae. Of 14 PCR-positive placentae, two were associated with the birth of a living symptomatic infant, two with stillbirth, one with miscarriage, and two with elective terminations of pregnancy. Directed laboratory assessment of women at high risk of transmitting congenital infection, on the basis of clinical or laboratory markers, is important for accurate diagnosis of adverse outcomes of pregnancy. However, routine screening for viruses in the placentae from women with a low-risk serological profile for transmitting congenital infection is unlikely to result in significant numbers of additional diagnoses and is confounded by inadequacy of current diagnostic methods. The major pathogen detected in all cases of placental infection associated with fetal death was human CMV.

Development of multiplex PCRs for detection of common viral pathogens and agents of congenital infections.

Potential causes of congenital infection include Toxoplasma gondii and viruses such as cytomegalovirus (CMV), enterovirus, hepatitis C virus, herpes simplex virus types 1 and 2 (HSV-1 and -2), human herpesvirus types 6, 7, and 8, lymphocytic choriomeningitis virus, parvovirus, rubella virus, and varicella-zoster virus. Testing for each of these agents using nucleic acid tests is time consuming and the availability of clinical samples such as amniotic fluid or neonatal blood is often limited. The aim of this study was to develop multiplex PCRs (mPCRs) for detection of DNA and RNA agents in the investigation of congenital infection and an mPCR for the viruses most commonly requested in a diagnostic virology laboratory (CMV, Epstein-Barr virus, enterovirus, HSV-1, HSV-2, and varicella-zoster virus). The assays were assessed using known pathogen-positive tissues (cultures, placentae, plasma, and amniotic fluid) and limits of detection were determined for all the agents studied using serial dilutions of plasmid targets. Nested PCR was performed as the most sensitive assay currently available, and detection of the amplicons using hybridization to labeled probes and enzyme-linked immunosorbent assay detection was incorporated into three of the four assays. This allowed detection of 10 to 10(2) copies of each agent in the samples processed. In several patients, an unexpected infection was diagnosed, including a case of encephalitis where HSV was the initial clinical suspicion but CMV was detected. In the majority of these cases the alternative agent could be confirmed using reference culture, serology, or fluorescence methods and was of relevance to clinical care of the patient. The methods described here provide useful techniques for diagnosing congenital infections and a paradigm for assessment of new multiplex PCRs for use in the diagnostic laboratory.

Dipeptidase 1: a candidate tumor-specific molecular marker in colorectal carcinoma.

The aim of this study was to identify tumor-specific markers for the detection of rare disseminated colorectal tumor cells in peripheral venous blood and in intra-peritoneal saline lavage samples collected before and after resection of colorectal tumors. Using cDNA micro-array screening, we found dipeptidase 1 (DPEP1) to be highly expressed in colon tumors compared to matched normal mucosa. Relative reverse transcriptase (RT)-PCR showed that DPEP1 was over-expressed by >/=2 fold in colon tumor compared to normal colonic mucosal tissue in 56/68 (82%) patients. Using immunobead RT-PCR, a technique that first enriches for epithelial cells, we found DPEP1 positive cells in intra-peritoneal lavage and venous blood samples from 15/38 (39%) colorectal cancer cases. This is the first report of DPEP1 as a marker for disseminated colon tumor cells.

Secondary organic aerosol formation from the irradiation of simulated automobile exhaust.

A laboratory study was conducted to evaluate the potential for secondary organic aerosol formation from emissions from automotive exhaust. The goal was to determine to what extent photochemical oxidation products of these hydrocarbons contribute to secondary organic aerosol (SOA) and how well their formation is described by recently developed models for SOA formation. The quality of a surrogate was tested by comparing its reactivity with that from irradiations of authentic automobile exhaust. Experiments for secondary particle formation using the surrogate were conducted in a fixed volume reactor operated in a dynamic mode. The mass concentration of the aerosol was determined from measurements of organic carbon collected on quartz filters and was corrected for the presence of hydrogen, nitrogen, and oxygen atoms in the organic species. A functional group analysis of the aerosol made by Fourier transform infrared (FTIR) spectroscopy indicated

Integrons and gene cassettes in the enterobacteriaceae.

Integrons were detected in 59 of 120 (49%) urinary isolates of Enterobacteriaceae by PCR using degenerate primers targeted to conserved regions of class 1, 2, and 3 integrase genes. PCR sequencing analysis of the cassette arrays revealed a predominance of cassettes that confer resistance to the aminoglycosides and trimethoprim.

Diagnosis of enteric pathogens in children with gastroenteritis.

The aim of this study was to determine the isolation trends of common and emerging pathogens in children over a 12-month period. The study group included 412 children under 6 years with diarrhoea who were either hospitalised, or seen in the outpatients department of The Sydney Children's Hospital. Pathogens were detected in 137 (33%) samples, with rotavirus most common (40%), followed by adenovirus (26%), astrovirus (12%), Campylobacter jejuni (12%), Salmonella spp. (10%) and Giardia lamblia (< 1 %). Giardia-specific antigen (GSA) was detected in 11 of 382 (3%) using an enzyme immunoassay (EIA), and this included four samples in which cysts of G. lamblia were detected by microscopy. Using electron microscopy (EM), viruses were detected in 29 of 120 (24%) samples from hospitalised children and 53 of 171 (31%) outpatients (P = 0.23). Amongst this subset, Norwalk-like viruses (NLVs) were detected by RT-PCR in 10 samples including six of 14 with small round viruses, one of seven with small viral-like particles (SVLPs), and three of 126 EM-negative samples. Lactoferrin, detected by EIA, was 59% more likely to be positive in samples infected with salmonella/campylobacter than in samples in which bacterial pathogens were not isolated. As an indicator for infection with these bacterial agents, the assay showed a sensitivity and specificity of 95 and 40.3%, respectively. A routine microbiological analysis of stools from children of this age group should include a screen for foodborne bacterial agents and rotavirus. Tests for adenovirus, astrovirus and NLVs should be secondary. The cost-effectiveness of including the EIAs for lactoferrin and G. lamblia in the routine testing protocol needs to be evaluated.

Small intestinal mucosal immunity and morphometry in luminal overgrowth of indigenous gut flora.

OBJECTIVE: The aim of this study was to investigate the separate effects of indigenous oropharyngeal- and colonic-type flora on small intestinal mucosal immunity and morphometry in small intestinal bacterial overgrowth (SIBO). METHODS: A duodenal aspirate and random biopsies of underlying mucosa were obtained from 52 adult subjects (age range, 18-90 yr; median, 60 yr) without disorders that may otherwise disturb small intestinal histology or mucosal immunity. Villus height, crypt depth, villus/crypt ratios, counts of intraepithelial lymphocytes (IELs) and lamina propria total mononuclear cells, IgA, IgM, and IgG plasma cells, mast cells, and B and T lymphocytes were determined in relation to the presence or absence of SIBO and the nature of the overgrowth flora in all subjects. CD4+ve and CD8+ve T-cell counts were determined in 24 subjects. RESULTS: SIBO was present in 26 of 52 (50%) subjects. Overgrowth flora included colonic-type bacteria in 20 subjects and oropharyngeal-type flora alone in 6 subjects. Lamina propria IgA plasma cell counts were significantly increased in subjects with SIBO, irrespective of whether the overgrowth flora comprised oropharyngeal-type flora alone or included colonic-type bacteria. Neither villus height, crypt depth, villus/crypt ratios, nor total or other mononuclear cell counts in lamina propria differed significantly between subjects with and without SIBO, irrespective of the nature of the overgrowth flora. IEL counts were significantly higher than in culture-negative subjects only when the overgrowth flora included colonic-type bacteria. Even then, IEL counts were within a range currently considered normal. A significant, inverse correlation between advancing age and IEL counts became apparent after adjusting for the effect of SIBO of colonic-type flora. CONCLUSIONS: SIBO of oropharyngeal- and colonic-type flora are associated with differing disturbances of local duodenal mucosa. Nonetheless, these would not be readily apparent during routine histological assessment. Old age independently influences duodenal IEL counts.

Evaluation of the rice breath hydrogen test for small intestinal bacterial overgrowth.

OBJECTIVES: The aims of this study were 1) to document the sensitivity, specificity, and predictive values of the rice breath hydrogen test for small intestinal bacterial overgrowth; 2) to determine the possible influence of concurrent gastric bacterial overgrowth and gastroduodenal pH on the efficacy of this test; and 3) to investigate whether reliability is limited by an inability of small intestinal luminal flora to ferment rice or its product of hydrolysis, maltose. METHODS: Twenty adult subjects were investigated with microbiological culture of proximal small intestinal aspirate and a 3-g/kg rice breath hydrogen test. Gastroduodenal pH, the presence or absence of gastric bacterial overgrowth, and the in vitro capability of small intestinal luminal flora to ferment rice and maltose, its product of hydrolysis, were determined. RESULTS: Sensitivity of the rice breath hydrogen test for small intestinal bacterial overgrowth was 33% and remained low even when subjects with small intestinal overgrowth with oropharyngeal-type (38%) and colonic-type flora (20%) and those with concurrent small intestinal and gastric bacterial overgrowth (40%) were considered separately. Sensitivity remained suboptimal despite favorable gastroduodenal luminal pH and documented ability of bacterial isolates to ferment rice and maltose in vitro. Specificity of the rice breath hydrogen test for small intestinal bacterial overgrowth was 91%. Positive predictive value, negative predictive value, and predictive accuracy were 75%, 63%, and 65%, respectively. CONCLUSIONS: Clinical value of the rice breath hydrogen test for detecting small intestinal bacterial overgrowth is limited. The rice breath hydrogen test is not a suitable alternative to small intestinal intubation and culture of secretions for the detection of small intestinal bacterial overgrowth.

Detection of astrovirus gastroenteritis in children.

A commercial enzyme immunoassay (EIA) for the detection of astrovirus antigen was used to detect the virus during a 12-month survey of enteric pathogens in children in outpatient (n = 238) and hospital (n = 176) settings. It was found to have a 100% sensitivity and 98.6% specificity. Nineteen astrovirus isolates were detected and confirmed by northern hybridization, cell culture, and RT-PCR. The virus was detected mainly amongst outpatients although a comparison of the detection rate with that in hospitalised children did not demonstrate a statistically significant difference (p = 0.1347). In contrast, there was a strong association between hospitalization and rotavirus infection (p = 0.0371), and a strong association between infection detected in outpatients and adenovirus infection (p = 0.0193). Strains of astrovirus were sequenced, genotyped and shown to be: type 1 (n = 11), type 3 (n = 1), and type 4 (n = 7). Maximum genetic variation in type 1 isolates was 8.6% and type 4 was 7.8%. Changes did not result in amino acid substitutions.

Characterisation of two new gene cassettes, aadA5 and dfrA17.

Escherichia coli INS33 was isolated from the urinary tract of an infected patient. It was resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfafurazole, tetracycline and trimethoprim. PCR screening revealed the presence of a class 1 integron that harboured two new gene cassettes, designated dfrA17 and aadA5. The new dfrA17 cassette was 91% identical to the known dfrA7 cassette. The aadA5 cassette was 95% identical over the first 830 bp to aadA4, but lacked the IS26 element found at the 3' end of this truncated cassette. Cloning and expression of the cassette region demonstrated that dfrA17 conferred high level resistance to trimethoprim but aadA5 conferred resistance to spectinomycin but not to streptomycin.

Serum immunoglobulin and soluble IL-2 receptor levels in small intestinal overgrowth with indigenous gut flora.

Murine studies have demonstrated that the presence of indigenous gut flora is crucial for the induction of systemic immune hyporesponsiveness to antigens initially encountered within the gastrointestinal lumen. This study investigated whether increased titers of such flora, as occur in human small intestinal bacterial overgrowth, may be associated with increased suppression of systemic immune responsiveness and the possible relation between systemic and mucosal immunity in this setting. Serum total immunoglobulin (Ig), immunoglobulin subclass, and soluble interleukin-2 receptor levels and lamina propria IgA plasma cell counts were determined in 50 consecutive subjects with (N = 30) and without (N = 20) small intestinal bacterial overgrowth. Luminal IgA levels were measured in 35 of these subjects. Serum concentrations of IgG3, but not of other immunoglobulin isotypes or soluble interleukin-2 receptors, were significantly reduced in subjects with bacterial overgrowth (P < 0.0005). Small intestinal lamina propria IgA plasma cell counts (P < 0.0005) and luminal IgA concentrations (P = 0.001) were significantly increased in this group. Serum IgG3 levels were significantly inversely correlated with luminal IgA levels (P < 0.01) and fell below the lower limit of normal (0.41 g/liter) in 17/30 (56.7%) subjects with bacterial overgrowth compared to 1/20 (5.0%) subjects without (P < 0.0005). These findings document an association between small intestinal bacterial overgrowth with indigenous gut flora and reduced serum IgG3 reactivity in humans, possibly via an interaction with mucosa-related immunoregulatory mechanisms. The possibility of underlying small intestinal bacterial overgrowth should be considered in patients with serum IgG3 deficiency, especially those with compatible symptoms and/or known predisposition.

Interleukin-6 and small intestinal luminal immunoglobulins.

Our aim was to determine the relationships between interleukin-6 and immunoglobulin levels within small intestinal luminal secretions. Twenty adult subjects with small intestinal bacterial overgrowth (N = 13), irritable bowel syndrome (N = 4), and nonulcer dyspepsia (N = 3) underwent endoscopic aspiration of secretions from the small intestinal mucosal surface for assessment of IL-6, IgA1, IgA2, IgM, IgG1, IgG2, IgG3, and IgG4 concentrations. Serum immunoglobulin concentrations and small intestinal histology were also determined. IgA2 and IgG3 were the predominant IgA and IgG subclasses in luminal secretions in 19/20 (95%) and 20/20 (100%) subjects, respectively. IgA1 and IgG1 predominated in serum in all subjects. No subject had villous atrophy. Luminal IL-6 concentrations correlated significantly with luminal IgA2, IgM, and IgG3 concentrations but not with IgA1 or any other IgG subclass levels. Conversely, luminal IL-6 or immunoglobulin concentrations did not correlate significantly with levels of any immunoglobulin isotype in serum. These observations suggest that important relationships exist between local IL-6 and IgA2, IgM, and IgG3 responses in human small intestinal luminal secretions. Local investigation is mandatory when assessing intestinal immune activity.

Liver damage in human small intestinal bacterial overgrowth.

OBJECTIVE: Some rodent strains with experimental small intestinal bacterial overgrowth (SIBO) unrelated to jejunoileal bypass are susceptible to hepatic damage, possibly because of increased small intestinal permeability to proinflammatory bacterial polymers. However, data on the prevalence of hepatic damage in human subjects with SIBO in this setting are lacking. This study addressed this issue. METHODS: Seventy adult subjects were investigated for possible SIBO and hepatic damage with bacteriological analysis of small intestinal aspirates and measurement of serum concentrations of alkaline phosphatase, gamma-glutamyl transpeptidase, aspartate aminotransferase, and alanine aminotransferase. Nutritional indices (serum albumin and anthropometry) and the urinary lactulose/mannitol ratio, an index of small intestinal permeability, were measured in all subjects with SIBO and liver damage. RESULTS: SIBO was present in 40 of 70 subjects (57.1%). Overgrowth flora included salivary-type bacteria alone in 11 subjects and colonic-type bacteria in 29 subjects (facultative anaerobes [Enterobacteriaceae] alone in 21 subjects and both facultative and obligate anaerobes [Enterobacteriaceae and Bacteroides spp] in eight subjects). Biochemical evidence of liver damage was found in zero of 30 subjects without SIBO, zero of 11 subjects with SIBO with salivary-type bacteria alone, zero of 21 subjects with SIBO with facultative but not obligate anaerobic colonic-type bacteria, and in one of eight subjects (12.5%) with SIBO with obligate anaerobic colonic-type bacteria, in whom serum alkaline phosphatase and gamma-glutamyl transpeptidase levels were elevated. Nutritional indices were normal in this patient. Small intestinal permeability was increased and, along with liver enzyme abnormalities, normalized after eradication of SIBO. Small intestinal permeability was also increased in three of six patients (50.0%) with SIBO with obligate anaerobic colonic-type bacteria who had no evidence of liver damage. CONCLUSIONS: SIBO per se is not a major risk factor for liver damage in humans, even when the overgrowth flora includes obligate anaerobes. Liver damage is not a necessary consequence of increased small intestinal permeability in this setting.