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.

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.

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.

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.

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.

No evidence of the Chlamydia trachomatis variant in the UK.

OBJECTIVES: The discovery of a variant strain of Chlamydia trachomatis (Ct) in Sweden has raised awareness of its possible undetected spread in the UK. The assays that fail to detect this variant are widely used in this country. This study aimed to determine if this variant is circulating in the UK. METHOD: 1,680 genital specimens tested negative by the Roche assays were retested by Aptima Combo2. Discordant results were sequenced to check for the deletion variant. RESULTS: Of 1,680 specimens tested, 29 were candidates for sequencing: 16 were negative for the variant, 11 failed to amplify, and 2 were lost. DISCUSSION: No Ct deletion variants were found in the UK. If it is circulating, then the prevalence is low (0-0.77%), but even a low level cannot be ignored. The system we describe is simple and suitable for rapid response and phasing of surveillance to match an unknown level of threat if other variants emerge.

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.

Should we consider alternatives to combined cervical and urethral swabs for detection of Chlamydia trachomatis in females?

BACKGROUND: The optimum approach for detecting Chlamydia trachomatis (CT) is considered to be combined cervical and urethral testing. OBJECTIVE: To assess the contribution of female urethral swabs in CT diagnosis and to examine alternatives. METHOD: Urethral and endocervical samples for CT were performed on 757 sexually active female patients, >16 years, attending the genitourinary medicine clinic at Macclesfield District General Hospital from October 2005 to November 2006. Swabs were collected and transported to the laboratory in separate AC2 sample collection tubes and were tested by AC2 assay. RESULTS: Of the 757 patients tested simultaneously by both endocervical and urethral swab, a total of 90 had CT identified by either method giving a positivity rate of 11.9%. Results for urethral and endocervical swabs were concordant in 77 patients (85.6%). Eighty two infections (91.1%) would have been diagnosed by swabbing the cervix only but an additional 8 (8.9%) were picked up by urethral swab. Urethral symptoms had been mentioned by 1 of these 8 women. CONCLUSION: 8.9% infected women were positive only on urethral swab. One of these would have been picked up owing to presenting symptoms, hence reducing the extra yield to 7.8% and leaving only 7 positives on 757 urethral swabs with a detection rate of 1% of all urethral swabs. Considering the low yield and the discomfort of urethral swabbing, an additional urethral swab appears unwarranted on grounds of both cost and patient care. As a small number of cases were detected at the urethra but not the cervix, it may be worthwhile investigating the performance of AC2 when placing an endocervical swab in first catch urine. An effective and simpler approach may be a switch to testing vaginal swabs by AC2.

Testing specimens for Chlamydia trachomatis.

The introduction of NAATs has revolutionised chlamydial diagnostics and these tests are now the standard of care. However, as with all new technologies, they have also presented new challenges. This review attempts to answer some of the questions that have been raised, particularly by groups about to embark on implementing a screening programme. Laboratory tests are continually changing but it is hoped that the paper provides a useful update of the current situation.

Should Chlamydia trachomatis confirmation make you cross? Performance of collection kits tested across three nucleic acid amplification test platforms.

OBJECTIVE: To investigate the feasibility of confirming initially reactive nucleic acid amplification assays for Chlamydia trachomatis (CT) by cross testing on a second molecular platform. The three platforms investigated were Aptima Combo 2 assay (AC2), Cobas Amplicor CT test (PCR) and ProbeTec ET CT assay (SDA). METHODS: Serial dilutions of a CT culture were prepared in 0.9% saline; used to prepare simulated swab samples for all three platforms, and tested as in the manufacturer's instructions. For the cross testing investigation, 1 ml of the simulated swab samples prepared in each of the three collection kits was transferred into the appropriate collection kit for the second platform. RESULTS: AC2 demonstrated a higher analytical sensitivity than the SDA and PCR assays. Upon cross testing AC2 again demonstrated a superior performance to the SDA and PCR assays even when testing swab samples originally prepared in the SDA and PCR transport medium. The SDA assay was inhibited by the addition of transport medium from both the AC2 and PCR assays. CONCLUSION: The analytical sensitivity of the three assays is not identical, therefore confirming initially reactive samples on a second platform may prove to be difficult. However, the higher sensitivity of the AC2 assay could allow its use as a confirmatory assay for reactive swab samples collected in the SDA and PCR transport medium.

Risk factors associated with pelvic inflammatory disease.

OBJECTIVE: To investigate factors associated with pelvic inflammatory disease (PID). METHODS: A case-control study was used to investigate demographic and behavioural factors, and causative agents associated with PID. RESULTS: A total of 381 participants were recruited: 140 patients, and 105 and 136 controls in tubal ligation and general practice groups, respectively. When compared with a PID-free tubal ligation control group, increased risk of PID was associated with: age <25 years; age at first sexual intercourse <20 years; non-white ethnicity; not having had children; a self-reported history of a sexually transmitted disease; and exposure to Chlamydia trachomatis. When compared with a general practice control group, increased risk was associated with: age <25 years; age at first sexual intercourse <15 years; lower socioeconomic status; being single; adverse pregnancy outcome; a self-reported history of a sexually transmitted disease; and exposure to C trachomatis. Of the cases, 64% were not associated with any of the infectious agents measured in this study (idiopathic). CONCLUSIONS: A high proportion of cases were idiopathic. PID control strategies, which currently focus on chlamydial screening, have to be reviewed so that they can prevent all cases of PID. Behavioural change is a key factor in the primary prevention of PID, and potential modifiable risk factors were associated with PID.

Finding, confirming, and managing gonorrhoea in a population screened for chlamydia using the Gen-Probe Aptima Combo2 assay.

OBJECTIVES: To identify the prevalence of Neisseria gonorrhoeae (NG) within a population screened for Chlamydia trachomatis (CT). To monitor confirmatory microscopy, culture, and partner findings following reactive Aptima Combo2 assay (AC2) gonorrhoea screening tests. METHODS: Between June and December 2004, all gonorrhoea screening tests performed using AC2 for clients taking part in the Liverpool Chlamydia Screening Programme were monitored. Clients with AC2 NG reactive results were referred to a local genitourinary medicine (GUM) department for confirmatory microscopy, culture, treatment, and partner follow up. RESULTS: 47 (1%) of 4680 women and eight (1.7%) of 473 men had AC2 reactive gonorrhoea screening tests. Of those clients who agreed to follow up and were tested before any treatment, supportive evidence for a gonorrhoea diagnosis was found in 37 (97%) of 38 women and all five men. In the population opportunistically screened for chlamydia, CT prevalence rates were 12% for women and 15.7% for men. Although both women and men showed a higher relative risk for NG if chlamydia positive, of the 47 women who were reactive for NG by AC2, 55% (26) were negative for chlamydia. CONCLUSIONS: Sexually transmitted infections are rising in England and reduction of gonorrhoea rates is an objective of the Department of Health Sexual Health and HIV Strategy. AC2 tests provide an acceptable and accurate means of testing for gonorrhoea in an asymptomatic population in the community. AC2 had a higher positive predictive value than might be suggested by previous clinical trials in this low prevalence population. Although antibiotic sensitivity must be monitored, AC2 testing may offer a more acceptable alternative to microscopy and culture for NG in some populations.

Scrotal mass with fever and generalized lymphadenopathy in a young man secondary to Chlamydia trachomatis infection.

A young man presented with systemic upset and generalised lymphadenopathy. Later, it transpired that he was under investigation for a scrotal mass. Investigations were carried out to ascertain the cause of his symptoms including lymph node biopsy. Because of the presence of a scrotal mass in a sexually active male, a urinary Chlamydia ligase chain reaction (LCR) test was carried out. The result was positive and he was treated with doxycycline for 2 weeks. His symptoms settled and further, the urinary LCR was negative. We propose that Chlamydia trachomatis infection caused his illness and that urine PCR or LCR tests for Chlamydia is a convenient and useful investigation in sexually active males with generalised lymphadenopathy and fever of unknown origin.

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.

Liquid based cytology: examination of its potential in a chlamydia screening programme.

OBJECTIVE: To assess the feasibility of testing for chlamydia directly on a single liquid based specimen (ThinPrep test) collected for cervical screening. METHOD: Cervical smears were taken using a Cervex spatula and rinsed in the liquid based cytology collection vial. Following this, the conventional sample for chlamydia testing was taken from the endocervix using an Abbott Collection kit. Cytological specimens were prepared using an automated slide processor. Residual cellular material and the conventional samples were sent to the laboratory where both were tested for chlamydia by ligase chain reaction (LCR). The manufacturer's protocol for LCR urine testing was modified to substitute 1 ml of PreservCyt suspension. RESULTS: 581 women had both swab and cytology suspension tested for Chlamydia trachomatis with LCR. There were 19 concordant positive and 562 concordant negative reports. The stability of chlamydia in the cytology suspension was maintained for at least 5 months. CONCLUSION: The findings lead us to conclude that samples collected for liquid based cytology using the ThinPrep test collection vial provide a potential platform for chlamydia screening, though the study established several issues to be addressed to make this a practical proposition.

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.