Influence of contaminant exposure on the development of aerobic ETBE biodegradation potential in microbial communities from a gasoline-impacted aquifer.

Aerobic biodegradation of ethyl tert butyl ether (ETBE) in a gasoline-impacted aquifer was investigated in laboratory microcosms containing groundwater and aquifer material from ETBE-impacted and non-impacted locations amended with either ETBE, or ETBE plus methyl tert butyl ether (MTBE). As sole substrate, ETBE was biodegraded (maximum rate of 0.54 day-1) without a lag in ETBE-impacted microcosms but with a lag of up to 66 days in non-impacted microcosms (maximum rate of 0.38 day-1). As co-substrate, ETBE was biodegraded preferentially (maximum rate of 0.25 and 0.99 day-1 in non-impacted and impacted microcosms, respectively) before MTBE (maximum rate of 0.24 and 0.36 day-1 in non-impacted and impacted microcosms, respectively). Further addition of ETBE and MTBE reduced lags and increased biodegradation rates. ethB gene copy numbers increased significantly (>100 fold) after exposure to ETBE, while overall cell numbers remained constant, suggesting that ethB-containing microorganisms come to dominate the microbial communities. Deep sequencing of 16S rRNA genes identified members of the Comamonadaceae family that increased in relative abundance upon exposure to ETBE. This study demonstrates the potential for ETBE biodegradation within the unsaturated and saturated zone, and that ETBE biodegrading capability is rapidly developed and maintained within the aquifer microbial community over extended timescales.

Biodegradation and fate of ethyl tert-butyl ether (ETBE) in soil and groundwater: A review.

This review summarises the current state of knowledge on the biodegradation and fate of the gasoline ether oxygenate ethyl tert-butyl ether (ETBE) in soil and groundwater. Microorganisms have been identified in soil and groundwater with the ability to degrade ETBE aerobically as a carbon and energy source, or via cometabolism using alkanes as growth substrates. Aerobic biodegradation of ETBE initially occurs via hydroxylation of the ethoxy carbon by a monooxygenase enzyme, with subsequent formation of intermediates which include acetaldehyde, tert-butyl acetate (TBAc), tert-butyl alcohol (TBA), 2-hydroxy-2-methyl-1-propanol (MHP) and 2-hydroxyisobutyric acid (2-HIBA). Slow cell growth and low biomass yields on ETBE are believed to result from the ether structure and slow degradation kinetics, with potential limitations on ETBE metabolism. Genes known to facilitate transformation of ETBE include ethB (within the ethRABCD cluster), encoding a cytochrome P450 monooxygenase, and alkB-encoding alkane hydroxylases. Other genes have been identified in microorganisms but their activity and specificity towards ETBE remains poorly characterised. Microorganisms and pathways supporting anaerobic biodegradation of ETBE have not been identified, although this potential has been demonstrated in limited field and laboratory studies. The presence of co-contaminants (other ether oxygenates, hydrocarbons and organic compounds) in soil and groundwater may limit aerobic biodegradation of ETBE by preferential metabolism and consumption of available dissolved oxygen or enhance ETBE biodegradation through cometabolism. Both ETBE-degrading microorganisms and alkane-oxidising bacteria have been characterised, with potential for use in bioaugmentation and biostimulation of ETBE degradation in groundwater.

Confirmation of BD ProbeTec Neisseria gonorrhoeae reactive samples by Gen-Probe APTIMA assays and culture.

BACKGROUND: Use of nucleic acid amplification tests (NAATs), such as strand displacement assay (SDA), for the detection of gonococcal infection in low prevalence populations is controversial because of the likelihood of false positive results. Use of supplementary NAATs with alternative target sites has been recommended for confirmation of primary NAAT results. AIM: To evaluate if SDA reactive specimens for Neisseria gonorrhoeae, which were either culture positive or negative, can be confirmed by alternative target NAATs such as transcription-mediated assays (TMA). METHODS: SDA reactive specimens were retested by TMA using APTIMA Combo 2 (AC2) and APTIMA GC (AGC) assays. Two different methods of specimen preparation were used to test the specimens. In method A, residual extract after SDA was retested and in method B, the original clinical specimen was re-extracted in TMA medium and then retested. Cervical or urethral swabs were requested to confirm the SDA results by culture. RESULTS: By method A, 26/49 (53.1%) of SDA positive specimens were positive by AC2 and/or AGC; 14/27 (51.8%) culture confirmed SDA positive tests were positive by AC2 and/or AGC. By method B, 38/39 (97.3%) SDA positive results were confirmed by both AC2 and AGC. All the 25 culture confirmed SDA positive tests were confirmed by both AC2 and AGC; 5/6 SDA positive tests that were culture negative were confirmed by both AC2 and/AGC. CONCLUSION: Alternative target site NAATs, such as AC2 and AGC, can be used to confirm SDA positive results using the same clinical specimen. There is high concordance between the three NAATs.

Shortening the voiding interval for men having chlamydia nucleic acid amplification tests.

Male patients are assessed for a sexually transmitted infection provided a considerable length of time has elapsed since last micturition. The current availability of highly sensitive nucleic acid amplification tests (NAATs) for Chlamydia trachomatis (CT) and Neisseria gonorrhoea such as APTIMA Combo2 (AC2) led us to investigate the impact of voiding interval on the positivity of urine tests for CT. Male patients attending a genitourinary medicine clinic at high clinical risk for CT infection and known CT positives returning for treatment were included. Two first-void urine (FVU) specimens were collected: the first sample in the standard manner and the second sample was collected 20 minutes later or as soon as possible thereafter. Fifty-two CT-positive males were included in the analysis. All of the second samples were also CT positive and none were in the equivocal range. Paired t-test analysis did not show a significant difference between relative light unit readings of the first and second urine samples (P = 0.127). Even in male patients who have recently passed urine, FVU tested by AC2 can still reliably detect CT. This provides us opportunity for more flexible and effective patient management.

Diagnosis, management and prevalence estimation of gonorrhoea: influences of Aptima Combo 2 assay with alternative target confirmation.

Case-notes and laboratory data were retrospectively reviewed for influences of dual testing by Aptima Combo 2 (AC2) for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) on the diagnosis, management and prevalence estimation of gonorrhoea in the genitourinary (GU) medicine clinic and community. NG positives by AC2 were confirmed by Aptima Gonococcus assay. Unconfirmed positives were rare. Our study showed that in the GU medicine clinic, AC2 detected about 20% extra cases of NG beyond culture. For best standard of care, NG culture and microscopy are still required in some patients to ensure that treatment is rapid and appropriate. Compared to self-referral at the GU medicine clinic, community tests made a substantial contribution to the overall number of NG cases found (40 community versus 35 Macclesfield GU medicine clinic). The ratio of female to male NG cases found was significantly higher (P = 0.002) in the community (13 M, 27 F) than at the GU medicine clinic (25 M, 10 F). In the community, over 60% of NG infections occurred in chlamydia-negative patients. The overall prevalence of NG in the GU medicine clinic was 1.3%, the true prevalence being much lower at 0.9% on primary test. Prevalence in the community was 0.4%. Data from dual testing in the community can clarify NG prevalence beyond the existing KC60 (sexually transmitted infections) reports.

The contribution of APTIMA Combo 2 assay to the diagnosis of gonorrhoea in genitourinary medicine setting.

For 929 female and 821 male patients attending a genitourinary clinic, samples intended for chlamydia diagnosis were dual tested by nucleic acid amplification for both chlamydia and Neisseria gonorrhoeae (NG). The assay used, Gen-probe APTIMA Combo 2 (AC2) detected all cases of NG found by conventional microscopy and culture. AC2 identified additional patients who had partners with NG, but were themselves negative by microscopy and culture. Few, if any, false-positive AC2 results were found. Use of AC2 increased the number of patients treated for NG. It can reduce the number of specimens required and may limit the need for multiple site testing.

Using chlamydia positivity to estimate prevalence: evidence from the Chlamydia Screening Pilot in England.

Studies have suggested that positivity can be used to estimate the prevalence of Chlamydia trachomatis in large-scale chlamydia screening programmes. A recent pilot of opportunistic screening in England estimated that the prevalence among 16-24-year-old women in Portsmouth and Wirral was 9.8% and 11.2%, respectively. This study assessed the continued validity of positivity as an approximate for prevalence. We re-analysed data from the Chlamydia Screening Pilot to estimate positivity,calculated as total positive tests divided by total tests, and compared these estimates with the previously reported prevalence, measured as the number of women testing positive divided by the total number of women screened. Overall positivity was 9.4% in Portsmouth and 11.0% in the Wirral; these estimates were not statistically different from prevalence, regardless of health-care setting, age group or symptoms. We conclude that positivity can be used as a proxy for prevalence.

Staphylococcal protein A; its preparation and an application to rubella serology.

Good yields of staphylococcal protein A are obtained by growing the staphylococcus Cowan type 1 on cellophane agar. The activity of these preparations in removing immunoglobulin G (IgG) from human serum can be readily measured by the Mancini radial-diffusion technique and the correct in-use dilution determined. Treatment with protein A of sera from women with a history of rubella may help in the identification of those having specific antibody in the IgM and IgA fractions. This relatively simple procedure may have worthwhile application in the diagnosis of rubella.

Cultural method for large-scale screening for Chlamydia trachomatis genital infection.

Idoxuridine-treated McCoy cells grown as monolayers in 96 well microplates provide a convenient method for the isolation of Chlamydia trachomatis. Staining of infected monolayers with periodic acid-Schiff reagent (PAS) allows easy recognition of C trachomatis inclusions without the need for dark-ground microscopy. By this method 384 clinical specimens can be examined concurrently. It is sufficiently sensitive to form the basis of a chlamydial culture service for patients attending Sexually Transmitted Diseases (STD) Clinics.

Associations between Mycoplasma genitalium, Chlamydia trachomatis and pelvic inflammatory disease.

OBJECTIVE: To evaluate the association between Mycoplasma genitalium, Chlamydia trachomatis, and pelvic inflammatory disease (PID) METHODS: A case-control methodology was used. Swab eluates were processed using the QIAamp DNA mini kit. Polymerase chain reaction (PCR) for M genitalium was carried out using a real time in-house 16S based assay. An endocervical swab was taken and tested for the presence of C trachomatis (ligase chain reaction, Abbott Laboratories), and a high vaginal swab was taken and tested for the presence of Neisseria gonorrhoeae and bacterial vaginosis. RESULTS: Of the PID cases 13% (6/45) had evidence of M genitalium infection compared to none of the controls (0/37); 27% (12/45) of the cases had C trachomatis infection compared to none of the controls; and 16% (7/45) of cases only had serological evidence of C trachomatis infection compared to 5% (2/37) of controls. Cases were more likely to present with M genitalium and/or C trachomatis than controls (p<0.001). CONCLUSIONS: This study indicates that there may be an association between M genitalium and PID, and that this relation is largely independent of C trachomatis. Future studies need to investigate the pathological basis of the relation between M genitalium and PID using samples from women with PID diagnosed using laparoscopy and endometrial biopsy. Little is known about the epidemiology of M genitalium: large scale epidemiological investigations are needed to determine the prevalence, incidence, and factors associated with this emerging infection.

Serological diagnosis with the Chlamydia Spot-IF test.

Using a set of sera for which full chlamydial micro-immunofluorescence results suggested a clear diagnosis, we have evaluated the Chlamydia Spot-IF test (bioMerieux), which allows a comparison of titres to Chlamydia trachomatis and C. psittaci antigens. A modification of the test in which the antigen slides were pre-treated with a monoclonal antibody to chlamydial lipopolysaccharide, improved its ability to differentiate infections with C. trachomatis from those with C. psittaci or C. pneumoniae.

Hepatitis B and hepatitis C virus infections: risk factors among drug users in Northwest England.

OBJECTIVES: to identify the risk factors for hepatitis B (HBV) and hepatitis C (HCV) virus infections in drug users attending two drug treatment centres in Northwest England, and to evaluate the effect of both needle exchange and hepatitis B vaccination on the prevalence of hepatitis B and hepatitis C infections. METHODS: a retrospective, cross-sectional study performed at the Regional Infectious Disease Unit and a Primary Care Centre for drug users in Liverpool. The study population included 773 drug users who had hepatitis serology performed between January 1992 and April 1996. Information on risk factors was obtained from clinical records; hepatitis serology data were obtained from the Liverpool Public Health Laboratory database. RESULTS: the overall seroprevalences of exposure markers for HBV (anti-HBc antibody) and HCV (anti-HCV antibody) were 48% and 67%, respectively. Duration of injecting drug use was the strongest predictor of HCV infection, with a crude odds ratio of 8.9 (95% confidence interval (CI): 4.5-17) for >10 compared to <3 years of injecting, and was also a strong predictor of HBV infection, with an adjusted odds ratio (controlled for the effects of HBV vaccination) of 5.7 (95% CI: 3.2-10) for >10 compared to <3 years' injecting. Vaccination against HBV was associated with greatly reduced HBV seroprevalence (crude odds ratio 0.11, 95% CI: 0.06-0.18). Overall, HCV was acquired earlier in the injecting career than HBV, but drug users who were not vaccinated against HBV acquired markers for HBV even more rapidly than for HCV. We found no independent protective effect for either anti-HBc or anti-HCV acquisition after the introduction of a needle-exchange scheme. CONCLUSIONS: hepatitis C is highly prevalent among Merseyside drug users and is likely to prove difficult to control because of rapid acquisition early in the injecting career. Vaccination against hepatitis B is the best means of protecting drug users from hepatitis B, and should be offered before injecting is commenced.

Evaluation of near patient testing for Chlamydia trachomatis in a pregnancy termination service.

AIM: To identify and evaluate advantages and disadvantages of a near patient test (NPT) for Chlamydia trachomatis, using Clearview Chlamydia MF (Unipath Ltd) in a British Pregnancy Advisory Service (BPAS) clinic. METHOD: The improved Clearview Chlamydia MF test was used to test endocervical swabs from 400 women attending BPAS clinic for termination of pregnancy. The results were compared with Ligase Chain Reaction (LCR), using Polymerase Chain Reaction (PCR) as the arbiter. RESULTS: Twenty-seven women tested positive by Clearview Chlamydia MF (24 confirmed by LCR) and 32 by LCR. COMMENT: NPT has potential advantages in specific situations where a quick result is required for optimal management of those testing positive. However, the current technology available for detection of Chlamydial infection results in time constraints, which limited its benefits in this study, where there was a high throughput of clients. A significant number of cases were missed by Clearview Chlamydia MF, though the sensitivity found is within the ranges reported for various enzyme immunoassays (EIA) - currently the most commonly used testing method. The study confirmed the high positivity in those attending for termination, especially in under 25-year-olds.

Management of Chlamydia trachomatis in a women's hospital: a review of current practice.

OBJECTIVE: To establish a measure of testing for Chlamydia trachomatis within the Liverpool Women's Hospital with a view to optimising both testing and management of infection. DESIGN: Prospective observational study to review the outcome of Chlamydia testing and subsequent management of patients between September 1997 - September 1998. RESULTS: It was observed that opportunities for detecting infection were missed and testing was undertaken predominantly for diagnostic purposes. RECOMMENDATION: Consideration be given to a centralised system for overview of positive results linking with audit/education to reduce sequelae of Chlamydia within gynaecology.

Biogeochemical characterisation of a coal tar distillate plume.

The distillation of acidified coal tars for up to 50 years has given rise to a phenol plume approximately 500 m long, 50 m deep and containing up to 15 g l(-1) dissolved organic carbon (DOC) in the Triassic Sandstones aquifer. A conceptual biogeochemical model based on chemical and microbiological analysis of groundwater samples has been developed as a preliminary to more detailed studies of the controls on natural attenuation. While the development of redox zones and the production of methane and carbon dioxide provide evidence of natural attenuation, it appears that degradation is slow. The existence of sulphate in the plume indicates that this electron acceptor has not been depleted and that consequently methanogenesis is probably limited. Based on a simple estimate of sulphate input concentration, a half-life of about 15 years has been estimated for sulphate reduction. Geochemical modelling predicts that increased alkalinity within the plume has not led to carbonate precipitation, and thus within the limits of accuracy of the measurement, alkalinity may reflect the degree of biodegradation. This implies a loss of around 18% of the DOC over a 30-year period. Despite limited degradation, microbial studies show that there are diverse microbial communities in the aquifer with the potential for both anaerobic and aerobic biodegradation. Microbial activity was found to be greatest at the leading edge of the plume where DOC concentrations are 60 mg l(-1) or less, but activity could still be observed in more contaminated samples even though cells could not be cultured. The study suggests that degradation may be limited by the high phenol concentrations within the core of the plume, but that once diluted by dispersion, natural attenuation may proceed. More detailed studies to confirm these initial findings are identified and form the basis of associated papers.

Microbiological analysis of multi-level borehole samples from a contaminated groundwater system.

A range of bacteriological, geochemical process-related and molecular techniques have been used to assess the microbial biodegradative potential in groundwater contaminated with phenol and other tar acids. The contaminant plume has travelled 500 m from the pollutant source over several decades. Samples were obtained from the plume using a multi-level sampler (MLS) positioned in two boreholes (boreholes 59 and 60) which vertically transected two areas of the plume. Activity of the microbial community, as represented by phenol degradation potential and ability to utilise a range of substrates, was found to be influenced by the plume. Phenol degradation potential appeared to be influenced more by the concentration of the contaminants than the total bacterial cell numbers. However, in the areas of highest phenol concentration, the depression of cell numbers clearly had an effect. The types of bacteria present were assessed by culture and DNA amplification by polymerase chain reaction (PCR). Bacterial groups or processes associated with major geochemical processes, such as methanogenesis, sulphate reduction and denitrification, that have the potential to drive contaminant degradation, were detected at various borehole levels. A comparative molecular analysis of the microbial community between samples obtained from the MLS revealed the microbial community was diverse. The examination of microbial activity complemented those results obtained through chemical analysis, and when combined with hydrological data, showed that MLS samples provided a realistic profile of plume effects and could be related to the potential for natural attenuation of the site.

Finding cases of Trichomonas vaginalis infection in England.

We describe the use of a new molecular assay for Trichomonas vaginalis (TV), the Gen-Probe Aptima TV (ATV) in female attendees at community clinics, a genitourinary (GU) medicine clinic and a prison GU medicine service. Positivity rates at community clinics and GU medicine were 0/382 (0%) and 3/358 (0.8%, 95% confidence interval [CI] 0-1.7%), respectively. Positivity was significantly higher, 29/269 (10.8%, 95% CI 7.1-14.5%), odds ratio (OR) 14.3 (4.11 < OR < 59.55), in those tested at the prison. A questionnaire survey of English GU medicine clinics and data from the UK Health Protection Agency (HPA) for England both demonstrated the large variation in case rates by region and testing methods employed. Higher rates were seen in women, in prison GU medicine services and in London GU medicine clinics. The ATV assay is now CE-marked (Conformité Européenne) and so a larger prospective study of its potential application is warranted.