bluuebunny/crossref_metadata_2025 huggingface dataset:
O3-S3.06 Rescreening for chlamydial infection using home-based, self-obtained vaginal swabs: a randomised controlled trial in family planning clinic clients
Authors: Xu F , Stoner B , Taylor S , Mena L , Tian L , Papp J , Hutchins K , Martin D , Markowitz L
Published: July 2011

Background

Family planning clinics provide contraceptive and preventive services for millions of low-income individuals. Screening and treatment for Chlamydia trachomatis infection in these clinics is a major part of the chlamydia control program in the USA. For women diagnosed with chlamydia, rescreening 3 months after treatment is recommended according to national guidelines. However, rescreening rates are low. The time and effort needed for patients to return to the clinic and the lack of access to follow-up care may contribute to the poor adherence to the rescreening recommendation.

Methods

We conducted a randomised controlled trial in family planning clinics in three cities. After informed consent, women/girls >16 years treated for laboratory-confirmed chlamydial infection were randomly assigned to the Home Group (mailed a vaginal swab kit for self collection at home) or the Clinic Group (made a clinic appointment) for rescreening at 3 months following treatment. Reminder calls were made about 2 weeks before scheduled rescreening. The endpoint was rescreening within a 7 week window, 1 week before to 6 weeks after, the scheduled rescreening date.

Results

404 women were enrolled and their group assignments were randomised by opening centrally stuffed envelops. Women assigned to the Home Group had higher rescreening rate than those in the Clinic Group: Overall, 40.8% of 196 in the Home Group and 20.7% of 208 in the Clinic Group were rescreened (p<0.001). The rescreening rates were 38.4% (Home) vs 19.8% (Clinic) among those living with parents, and 48.2% (Home) vs 21.2% (Clinic) among those with a history of chlamydia infection prior to the treated episode at enrolment (both p<0.001). Among women reached by a reminder call, rescreening rates were significantly higher in the Home Group (59.2% of 130) than in the Clinic Group (37.8% of 111) (p<0.001). Among 163 women not reached by the reminder call, the rescreening rate were low (<5%) in both groups. In the Home Group, 12 tested positive for chlamydia compared to 8 in the Clinic Group, and the rate of reinfection was 12.9% in the Home Group and 14.6% in the Clinic Group (p=0.8).

Conclusions

Use of home-based, self-obtained vaginal swabs resulted in a significant increase in rescreening rates compared to rescreening in the clinic. Our findings indicate a role for home-based specimen collection as an alternative to clinic-based rescreening for chlamydia in women.
O3-S4.02 Is single dose azithromycin adequate for asymptomatic rectal chlamydia?
Authors: Drummond F , Ryder N , Wand H , Guy R , Read P , McNulty A , Wray L , Donovan B
Published: July 2011

Background

Azithromycin is the recommended first-line therapy for asymptomatic rectal chlamydia. However a recent European study reported significant numbers of treatment failures, with higher failure rates in HIV positive men. In 2009, the Sydney Sexual Health Centre instituted a 6 week re-test policy for all cases of asymptomatic rectal chlamydia to assess the extent of azithromycin treatment failures.

Methods

We conducted a retrospective audit of all men who have sex with men (MSM) diagnosed with asymptomatic rectal chlamydia in 2009. MSM with anal symptoms were excluded from this analysis, due to the possibility of lymphogranuloma venereum. We then categorised the infections present at re-testing as probable re-infections (men reported ongoing sexual activity with an untreated partner) or probable treatment failures (men did not have any obvious ongoing exposure, either because they did not report any further anal sex with any existing partners or because condoms were used consistently with all partners).

Results

In the 12-month period there were 116 asymptomatic MSM treated for rectal chlamydia with 1 gram azithromycin as a single dose. Fourteen (12%) of the men were HIV positive. The median age was 33 years (range 20–64 years). Of the 116 men, 85 (73%) returned at varying times; median time of 10 weeks (78 days, range 21–372 days. Of the 85 men who returned, 11 (13%) were persistently positive and the median time to re-test was 11 weeks (78 days, range 47–209 days). Six of the 11 men were classified as probable re-infection and five as probable treatment failures, equating to an efficacy of 94%. None of the men classified as probable treatment failures were HIV positive.

Conclusions

Interpreted conservatively, the azithromycin treatment failure rate could have been as high as 13% in our study. However most of these cases could be explained by re-infection suggesting an actual treatment failure rate of 6%. There was no evidence azithromycin is an ineffective first-line therapy for asymptomatic rectal chlamydia in MSM, but prospective studies would be welcome.
O3-S5.01 Impact of AIC316, a novel antiviral helicase-primase inhibitor, on genital HSV shedding: randomised, double-blind, placebo-controlled trial
Authors: Wald A , Stoelben S , Tyring S , Warren T , Johnston C , Huang M L , Timmler B , Ruebsamen-Schaeff H , Corey L , Birkmann A
Published: July 2011

Background

Current treatments for HSV infection are imperfect, do not completely abrogate viral shedding or transmission, and do not interrupt HSV-2—HIV interactions. AIC316 is a helicase-primase inhibitor, a new class of anti-HSV compounds that has a distinct mode of action from currently available nucleoside analogues.

Methods

We investigated the safety and efficacy of AIC316 in patients with genital HSV-2 infection in a Phase 2, randomised, multicenter, parallel, double-blind, placebo-controlled trial of 4 different doses of AIC316 administered orally for 4 weeks. Participants were randomised 1:1:1:1:1 to one of the following dose groups: 5 mg given once daily (qd), 25 mg qd, 75 mg qd, 400 mg given once-a-week, or matching placebo. Participants in the once daily dose groups received a loading dose. During the trial period participants obtained a swab of genital secretions daily and maintained a diary of genital lesions. The primary endpoint was the rate of genital HSV shedding measured by HSV DNA PCR in the AIC316 groups vs placebo.

Results

156 (105 women and 51 men) healthy, immunocompetent, HSV-2 positive participants with a history of 1–9 recurrences per year prior to trial entry, or previous suppressive therapy, were randomised by seven US sites between May 2010 and October 2010. 147 completed the trial. Overall, about 9000 swabs for HSV PCR were collected and assayed for HSV DNA by a sensitive and accurate assay that can detect >150 copies/ml. The first results of these assessments will be presented.

Conclusion

The trial will provide insight into the antiviral activity of the novel agent AIC316 for genital HSV infections. This trial design presents a robust and efficient method for evaluating antiviral activity of candidate agents for mucocutaneous HSV infections. These initial efficacy and safety results will lead to selecting the dose for further trials with AIC316.
O3-S5.02 Frequent breakthrough genital HSV-2 shedding on standard and high dose valacyclovir
Authors: Johnston C , Saracino M , Kuntz S , Magaret A , Schiffer J T , Selke S , Huang Meei-li , Corey L , Wald A
Published: July 2011

Background

Short, rapidly cleared, subclinical shedding episodes are the predominant form of HSV-2 reactivation in the genital tract. Valacyclovir 500 mg once daily (SD-VAL) reduces the risk of sexual transmission of herpes simplex virus type 2 (HSV-2) by only 48%. We hypothesised that short HSV-2 shedding episodes occur frequently on SD-VAL and that high dose (HD)-VAL could suppress such episodes of genital HSV-2 shedding.

Methods

A randomised open-label crossover study using valacyclovir 500 mg daily (SD-VAL) vs valacyclovir 1 gm three times daily (HD-VAL) was conducted in HSV-2 seropositive, HIV seronegative persons with four or more genital herpes recurrences per year or laboratory confirmed primary genital HSV-2 infection in the previous 6 months. Each study arm lasted for 5 weeks, separated by 1 week wash out. Participants obtained genital swabs four times daily, which were assayed for HSV by quantitative PCR. The primary outcome was frequency of genital HSV shedding on each study arm; secondary outcomes included number and duration of HSV-2 shedding episodes and quantity of virus detected.

Results

Forty-three participants collected 9981 genital swabs during the study period. 292 (5.8%) of 5008 swabs had HSV detected during SD-VAL, compared to 164 (3.3%) of 4973 on HD-VAL (IRR=0.52, 95% CI=0.43% to 0.63%, p<0.001). Episodes were shorter on HD-VAL (median 7 h, compared to 10 h on SD-VAL, p=0.03) and the median maximum copy number was lower on HD-VAL (3.0 log10 copy/ml vs 2.5 log10 copies/ml, p=0.001). However, the annual episode rate was the same regardless of dose; there were 55 shedding episodes over 3.89 person-years of follow-up during SD-VAL (14.1 episodes/year) and 65 episodes over 3.93 person-years during HD-VAL (16.5 episodes/year, p=0.34).

Conclusion

Short bursts of genital HSV-2 reactivation persist during SD-VAL and HD-VAL. Compared to SD-VAL, HD-VAL decreased shedding frequency and episode duration but did not alter episode rate. These data may explain why risk of HSV transmission and HSV-specific genital inflammation persist even in the presence of antiviral therapy. More potent therapies are needed to completely suppress HSV-2 reactivation

ClinicalTrials.gov numberNCT00362297.
O1-S03.01 Antimicrobial resistance toNeisseria gonorrhoeain a cohort of young men in Kisumu, Kenya: 2002–2009
Authors: Mehta S , Maclean I , Ndinya-Achola J , Murugu R , Agunda L , Ronald A , Martin I , Bailey R , Moses S , Melendez J , Zenilman J
Published: July 2011

Background

We evaluated antimicrobial resistance inNeisseria gonorrhoeae(NG) isolated from men aged 18–24 enrolled in a randomised trial of male circumcision to prevent HIV.

Methods

Urethral specimens were obtained from men with discharge. These were inoculated in modified Thayer-Martin agar and incubated at 37°C in 5% CO2for 24–48 h, with confirmation of NG colonies by standard procedures. Minimum inhibitory concentrations (MICs) were determined by agar dilution. Clinical Laboratory Standards Institute criteria determined resistance: MIC≥2.0 μg/ml for penicillin, tetracycline, and azithromycin; ciprofloxacin MIC≥1.0 μg/ml; spectinomycin MIC≥128.0 μg/ml. Susceptibility to ceftriaxone and cefixime was MIC<0.25 μg/ml. We used PCR amplification to detect mutations in the parC and gyrA genes, associated with quinolone resistance.

Results

From 2002 to 2009, 168 NG isolates were obtained from 142 men. Plasmid mediated penicillin resistance (PPNG) was found in 65%, plasmid mediated tetracycline resistance (TRNG) in 97%, and 11% were ciprofloxacin resistant (QRNG). QRNG appeared November 2007, increasing from 9.5% in 2007 to 50% in 2009 see Abstract O1-S03.01 table 1. Resistance was not detected for spectinomycin, cefixime, ceftriaxone, and azithromycin, but MICs of cefixime (p=0.018), ceftriaxone (p<0.001), and azithromycin (p=0.097) increased over time. In a random sample of 51 men gentamicin MIC was assessed: 4 μg/ml (n=1), 8 μg/ml (n=49), 16 μg/ml (n=1). Increased MICs were associated with urban residence, multiple recent sex partners, not using condoms.

Abstract O1-S03.01 Table 1YearQuinolone resistance n/N (%)95% CI2002–20060/89 (0.0)–20072/21 (9.5)1.2 to 30.420086/22 (27.3)10.7 to 50.220097/14 (50.0)23.0 to 77.0p Value for trend<0.001

Conclusions

Quinolone resistance increased rapidly and alternative treatment, such as cefixime, is required for NG in this area. Systematic surveillance of antimicrobial resistance in NG is necessary for appropriate drug choice. Increases in MICs for oral cephalosporins add to growing concern for multi-drug resistant NG. The high prevalence of PPNG and TRNG suggest strong selective pressure from background antibiotic use.
O3-S5.04 The post-trial effect of periodic presumptive treatment for vaginal infections on the incidence of bacterial vaginosis and Lactobacillus colonisation
Authors: Balkus J , Jaoko W , Mandaliya K , Richardson B , Masese L , Gitau R , Kiarie J , Marrazzo J , Farquhar C , McClelland R S
Published: July 2011

Background

Bacterial vaginosis(BV) is a highly prevalent infection that frequently recurs following standard treatment. In a randomised controlled trial (RCT) of oral periodic presumptive treatment (PPT) to reduce vaginal infections among Kenyan women, we observed a decrease in BV and an increase in Lactobacillus colonisation among women randomised to receive 2 g metronidazole +150 mg fluconazole monthly for 12 months. After the trial, women were invited to continue follow-up in an open cohort study. These post-trial data were analysed to test the hypothesis that the treatment effect would persist in the absence of PPT.

Methods

Data were obtained from women who completed all 12 RCT visits and attended ≥1 cohort study visit within 120 days of their final RCT visit. We used Andersen-Gill proportional hazards models to estimate the post-trial effect of the intervention vs placebo on the incidence of BV by Gram stain (Nugent score ≥7) and Lactobacillus species by culture on Rogosa agar.

Results

The RCT enrolled 310 subjects (155 per arm), of whom 165 (83 active and 82 placebo) were included in this analysis. Included subjects were slightly older (median (IQR): 33 years (29–39) vs 30 years (26–35); p<0.001) and reported a longer duration of sex work (median (IQR): 6 years (2–11) vs 3 years (1–6); p<0.001) compared to those excluded. At the final RCT visit, which represented the baseline visit for this analysis, demographic and behavioural characteristics were similar by arm. The prevalence of BV at the final RCT visit was 16% in the active arm and 43% in the placebo arm (p<0.001). The post-trial incidence of BV was 260/100 person-years (p-yrs) in the active arm vs 358/100 p-yrs in the placebo arm (HR=0.76; 95% CI: 0.51% to 1.12%). The prevalence of Lactobacillus colonisation at the final RCT visit was 17% in the active arm and 18% in the placebo arm (p=0.81). The post-trial incidence of Lactobacillus colonisation was 180/100 p-yrs in the active arm vs 127/100 p-yrs in the placebo arm (HR=1.42; 95% CI: 0.85% to 2.71%).

Conclusions

Despite a decrease in BV and an increase in Lactobacillus colonisation during the RCT, the effect of PPT was not sustained during the 120 days following cessation of the intervention. New interventions that reduce BV recurrence and promote long-term Lactobacillus colonisation without the need for ongoing PPT or suppressive therapy are needed.
O3-S6.01 Improved diagnostics of bacterial vaginosis with molecular techniques
Authors: Speksnijder A , Gruteke P , Jonker D , de Vries H , van Dam A
Published: July 2011

Background

Bacterial vaginosis(BV) is a disturbance of the vaginal microflora. BV can cause discharge complaints and lead to pelvic inflammatory disease, ectopic pregnancy and premature birth. We evaluated a combination of PCR assay's and whole bacterial community analysis with the standard diagnostic algorithm to validate a molecular assay for the determination of BV.

Methods

160 women with vaginal discharge complaints were included. 80 women were classified as BV and 80 as non-BV according to Amsel criteria. Gram stains from vaginal smears were made for Nugent scoring. Vaginal swabs were tested with PCR assays forGardnerella vaginalis,Atopobium vaginae, BV associated bacterium type 2 (BVAB2) and Megaspheara type 1 (MS1). Whole bacterial community analysis was performed by fluorescent Terminal Restriction Fragment Length polymorphism (TRFLP) of 16S-rDNA. TRFLP patterns and predictive fragments of a number of BV associated bacteria were analysed with Bionumerics software (Applied Maths, Belgium).

Results

Compared to Amsel criteria, the highest sensitivity of 100% was achieved with a duplex PCR for G. vaginalis and/orA vaginaeand the highest specificity of 86% was found with a singleplex BVAB2 specific PCR. Best overall performance was shown using a duplex real time PCR for BVAB2 and/or MS1 with a sensitivity of 90% and a specificity of 78% with respect to Amsel criteria. Using Nugent criteria as a standard, this duplex PCR has a sensitivity of 84% and specificity of 86%. From TRFLP results, the presence of predictive fragments ofPrevotella, Aerococcus, Megaspheara, Mycoplasma, Peptostreptococcus, Leptotrichia, Eggerthella, Gardnerella, AtopobiumandDialisterwas most associated with BV positive samples. Cluster analysis of microbial profiles revealed clear differences between BV and non-BV and indicated possible intermediate or transition stages.

Conclusions

A combination of bacterial species are involved in BV. For molecular diagnostics a duplex PCR of Gardnerella en/of Atopobium can be used for initial screening confirmed by a BVAB2 specific PCR. A more effective alternative is a real time duplex PCR targeting BVAB2 and/or MS1. Microbial profiling supports most targets used in the PCR assays. Cluster analysis of microbial profiles can be used to interpret discordant validation results and possibly for diagnosis.
O3-S6.02 Screening forMycoplasma genitalium,Chlamydia trachomatisandBacterial vaginosisin a public hospital, pregnancy termination service
Authors: Garland S , Marceglia A , Tabrizi S , Costa A Maria
Published: July 2011

Garland SM1, Marceglia AH2, Tabrizi SN1Costa AM1 1 Microbiology Infectious Diseases, 2 Choices and Sexual Health Service, Royal Women's Hospital, Parkville, Victoria, Australia The Royal Women's Hospital is the largest public provider of therapeutic abortions in Victoria, Australia. Prior to their medical or surgical termination, all women presenting to the Pregnancy Advisory Service (PAS) have been screened forMycoplasma genitaliumutilising an in-house PCR assay 1 in addition toChlamydia trachomatisusing a commercial PCR and bacterial vaginosis (BV) by Gram stained smear of posterior fornix secretions. From August 2009 to December 2010, the prevalence forM genitaliumwas 4.6% (CI 3.5% to 5.6%),C trachomatis5.3% (CI 4.2% to 6.4%) and BV 16.2% (CI 14.4% to 18.0%). Most women had a normal genital tract on clinical examination. Of the women infected withC trachomatisandM genitalium, 42% and 34% respectively had abnormal genital tract signs. The average age of women attending the PAS clinic was 26.4 years, with 45.3% of the women being under 25. The average age for women withM genitaliumwas 24.6 years, whilst for those withC trachomatisit was 22.4 years. The 50 test of cures completed after treatment forM genitaliumto date have all been negative. This is in contrast to local treatment failure rates in similar aged males (symptomatic with nonspecific urethritis in a sexual health clinic) and females (screening within a general practitioner setting) of 28% and a population treatment failure rate of 12%. We are uncertain what role our direct observed patient treatment plays in this low failure rate. This presentation will report on the first 17 months of screening forM genitaliumin the PAS clinic and its implications for service provision within The Women's. Given the role ofM genitaliumin cervicitis, and the increasing evidence for its role in upper genital tract disease, screening for this bacterium should be considered, particularly with a surgical procedure such as termination of pregnancy, although the lack of a commercial test is problematic.
O3-S6.03 The diagnosis of lymphogranuloma venereum at one's fingertips
Authors: Crucitti T , Smet H
Published: July 2011

Background

Outbreaks of Lymphogranuloma venereum (LGV) in sexual networks of men who have sex with men (MSM) are reported in several countries in Europe. Although accurate laboratory diagnosis is required to provide adequate patient management, the laboratory identification of LGV can be problematic.

Objective

To establish a fast and reliable testing algorithm for the identification ofChlamydia trachomatisL serovars.

Methods

Previously, anal specimens from MSM suspected to be positive forC trachomatiswere tested with a testing algorithm using commercial molecular amplification assays. ConfirmedC trachomatissamples were then analysed in batches by RFLP to identify the L serovars. From September 2010 onwards, the Abbott CT/NG Real Time PCR has been used for the detection ofC trachomatisin biological specimens collected at or referred to the ITM for Chlamydial infection diagnosis. Furthermore, confirmation ofC trachomatisand identification of the L serovar types are performed with an in-house Real Time PCR assay. This assay uses DNA extract obtained with the Abbott assay. The selection of the primers and test procedure is based on the publication by Chen et al. and includes two specific probes for the detection of the L and the non L serovars.

Results

Out of a total of 940 samples tested with the new methods, we detected 58 (6.2%) positive samples forC trachomatisand of those 12 (20.7%) were L serovars. Eight were detected in specimens collected from the anus, two in urethral specimens, one in urine, and one in a vaginal specimen. All non vaginal specimens were collected from men. With the Abbott CT/NG Real Time PCR for screening and the in house RT PCR assay for confirmation, we were able to confirm positive results forC trachomatisand to distinguish the L serovar from the non L serovar types within 2 days after specimen reception. In addition the in house RT PCR assay was more sensitive, more discriminative and at least 4 times cheaper compared to the RFLP method.

Conclusion

The detection of L serovar ofC trachomatiscan be done on a routine basis at a very acceptable cost and test around time. The L serovar types may be more frequent in Belgium then initially thought, they are present in various biological specimens and possibly in women.

Abstract O3-S6.03 Table 1

LGVV Testing

Yearspecimens tested*L serovarnon L serovarnot typable2011 January19641102010 Sept-Dec74483502010 Jan-Aug231490200923175120081311†11200719135120061474320056213200411821*

Until September 2010 only male anal specimens or rectal biopsies were tested except for 2008 when 2 specimens from penile ulcers were tested.

†

Two penile ulcer specimens.
O3-S6.06 Self-collection vs physician-collection for the detection ofChlamydia trachomatis,Neisseria gonorrhoeae,Trichomonas vaginalisandMycoplasma genitalium
Authors: Kwatampora J , Senkomago V , Mugo N , Hill C , Ting J , Chitwa M , Patel S , Gukare H , Kimani J , Smith J
Published: July 2011

Background

To determine the efficacy of self-collection for primary screening of an array of sexually transmitted infections.

Methods

A cross-sectional baseline analysis was conducted on a cohort of 300 female sex workers (FSW) within an outpatient clinic from a suburban/slum area of Nairobi, from December 2009 to June 2010. APTIMA transcription-mediated amplification (TMA) assays (Gen-Probe Incorporated) were used for the detection ofChlamydia trachomatis(CT),Neisseria gonorrhoeae(GC),Trichomonas vaginalis(Trich) andMycoplasma genitalium(MGen) infections and, were analysed in San Diego, California. The APTIMA COMBO 2 assay (AC2) was used for CT/GC, the APTIMATrichomonas vaginalisassay for Trich, and an APTIMAMycoplasma genitaliumresearch assay for Mgen. FSW conducted a self-collected sample in privacy, based on standardised instructions, and then underwent a pelvic exam to obtain a physician-collected sample.

Results

A total of 299 FSW (mean age of 30) participated, of which 15% were HIV-seropositive. MGen was the most common infection (12.7% in physician; 20.7% in self-collection), followed by Trich (7.7%; 9.7%), CT (4.3%; 5.4%), and GC (2.7%; 3.3%). For all STIs examined, the self-collected samples detected more positive cases than the physician-collected samples. No self-collected sample missed an infection that was picked by the physician sample. κ for agreement between self-collected and physician-collected samples were high for CT: 0.89, GC: 0.89 and Trich: 0.87, and slightly lower for MGen: 0.73. Using physician sampling as the gold-standard, the sensitivity of self-collection was 100% for all STIs. The specificity for self-collection was high for CT, GC, and Trich (99.0%, 99.3% and 97.8% respectively) and slightly lower for MGen (91.6%).

Conclusion

A single self-collected sample appeared to perform with comparable sensitivity and specificity to that of physician-collected sampling for the detection ofChlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalisandMycoplasma genitalium.
O4-S1.02 Molecular cloning and expression of hydrogenosomal malate dehydrogenase ofTrichomonas vaginalis
Authors: Ardalan S , Lee C , Garber G
Published: July 2011

Background

Trichomoniasis, a sexually transmitted disease caused byTrichomonas vaginalis, is associated with adverse pregnancy outcomes, and increased risk of HIV acquisition. Malate dehydrogenase (MDH), which catalyses the interconversion of malate to oxaloacetate, has a pivotal role in the survival and pathogenicity of this amitochondrial protozoan. The objective of this study was to clone and express Malate dehydrogenase gene ofT vaginalis, and analyse the biological function of this hydrogenosomal enzyme.

Methods

The MDH gene from a clinical isolate ofT vaginaliswas amplified by PCR, and cloned into pET101/D-TOPO vector with a C-terminal 6XHis tag. Positive clones were screened and identified by restriction endonuclease digestion and sequence analysis. The plasmid pET101/D-MDH was then transformed into E.coli BL21(DE3) to express after IPTG induction. The expression product further analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The recombinant protein was purified with Ni-NTA agarose under native conditions. Western blot, using antibody raised against whole cellT vanginalis, was performed to determine the immunogenicity of purified recombinant protein.

Results

The recombinant plasmid pET101/D-MDH was constructed successfully. High homology (98%) of nucleotide sequence was revealed between the cloned MDH and the corresponding gene. The recombinant protein showed a high expression level when induced with 1 mM IPTG at 37° C for 4 h. SDS-PAGE analysis showed that the recombinant MDH protein with the correct molecular weight (about 60 kDa) was expressed in E.coli BL21 (DE3). Western blotting revealed that the purified recombinant protein was specifically recognised by sera from mice infected with whole cellT vaginalis.

Conclusions

A prokaryotic expression system ofT vaginalisMalate dehydrogenase gene has been established successfully. The immunogenicity of the recombinant protein has been tested. The present study shows that the recombinant MDH is specific and suitable for use as an antigen for detecting anti-Trichomonas vaginalisIgG antibodies. Our work has established a good foundation for future studies onT vaginalisvaccine construction.
O4-S1.03 Defining the in vitro functions of monoclonal antibodies developed to theHaemophilus ducreyitrimeric autotransporter DsrA
Authors: Leduc I
Published: July 2011

Background

The DsrA protein ofHaemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, is a multifunctional outer membrane trimeric autotransporter (TA) and a virulence factor. MostH ducreyistrains, which are grouped in two classes (I and II), express a DsrA protein. Although DsrA proteins from both classes ofH ducreyistrains share little identity in their N-terminal functional passenger domain, both are involved in serum resistance and function as adhesins to the extracellular matrix (ECM) proteins fibronectin (Fn) and vitronectin (Vn), as well as to fibrinogen (Fbg) and HaCat keratinocytes.

Methods

Monoclonal antibodies (mAbs) to DsrA were developed by immunising mice with full-length recombinant DsrA from class I strain 35 000HP (DsrAI). Western blots were used to determine the specificity of the mAbs and their ability to recognise multimers of DsrA. Whole-cell binding ELISAs were used to examine the capacity of the mAbs to bind the surface ofH ducreyi. The domains and the shortest nominal peptides of DsrA recognised by those mAbs were identified using a library of truncated DsrA proteins expressed in a dsrA mutant, a peptide library representing full-length DsrAI, as well as Surface Plasmon Resonance and mutagenesis studies. mAbs were tested for bactericidal activity and their ability to block binding of Fn, Vn and Fbg byH ducreyiusing bactericidal assays and whole cell blocking assays, respectively.

Results

Anti-DsrAI mAbs bound monomers and dimers of the DsrA protein from several class IH ducreyistrains but not the DsrAII protein. mAb 1.82 bound native DsrAI protein at the surface of a panel of class IH ducreyiisolates, but not the H .ducreyi strains that cause cutaneous chancroid. The DsrA protein from these strains was recognised by mAb 4.79, along with the DsrA protein from strain 35 000HP. Both 1.82 and 4.79 bound with high affinity to peptides in the N-terminal region of the DsrA translocator domain and the shortest nominal epitope for 1.82 is MEQNTHNINKLS. In whole cell blocking assays, mAb 1.82 partially blocked binding of Fn and Fbg by class IH ducreyistrain 35 000HP.

Conclusions

1.82 and 4.79 are DsrAI-specific mAbs that recognise multimers of DsrA and bind the surface of intactH ducreyi. Both mAbs bind an epitope in the N-terminal region of the translocator domain of DsrAI with high affinity. mAb 1.82 has modest Fn and Fbg blocking activity.
O4-S1.04 Rapid spread of herpes simplex virus-2 in the human genital tract
Authors: Schiffer J , Swan D , Magaret A , Johnston C , Selke S , Wald A , Corey L
Published: July 2011

Background

Genital herpes simplex virus-2 (HSV-2) infection consists of shedding episodes that correlate with transmission and genital lesion formation. Episodes are heterogeneous in terms of viral production and duration.

Methods

Using quantitative PCR data from 14 685 daily genital swabs performed daily in 531 persons, we summarised 1020 episodes according to their frequency, duration, peak HSV DNA copy number, first and last swab copy number, expansion and decay slopes, and morphology. We designed competing dynamical stochastic mathematical models and attempted to fully reproduce result ranges for each of these episode features. The models assumed the possibility of concurrent, spatially distinct plaques of viral infection and immunologic response.

Results

Our spatial model reproduced all kinetic features of the empirical shedding data. The model and parameter set that achieved best fit highlights three distinct mechanisms of viral spread. First, HSV-2 is constantly dispersed from neurons to epidermal cells throughout the genital tract at a rate of ∼100 HSV DNA copies/day. Approximately 75 times each year, a neuron-derived viral particle infects an epidermal cell, initiating an infectious plaque of epidermal cells and a detectable shedding episode. Second, within each plaque, cell-to-cell spread of HSV-2 particles occurs extremely rapidly between epidermal cells. One cell can produce 105 cell-associated HSV genomic copies/day, with 3% of viral particles infecting a neighbouring cell each day. In large plaques, >105 cells may be infected within ∼12 h producing up to 109 HSV DNA copies/ml. Third, cell-free viruses, despite being 3000-times less infectious than cell-associated viruses, decay more slowly and initiate spatially distinct secondary plaques. We use video simulations to display that episodes longer than 3 days, consist of dozens of concurrent viral plaques. Despite rapid cell-to-cell spread of HSV-2, infected cells are eliminated by localised CD8+ T-cells within 24 h of plaque initiation. Moreover, the extent of secondary plaque formation prior to episode termination is determined by spatial CD8+ T-cell density surrounding the site of infection.

Conclusions

Genital HSV-2 utilises three kinetically distinct methods of spread to initiate and sustain prolonged shedding episodes. The extent and severity of secondary plaque formation is determined by spatial immune cell density.
O4-S1.06 HPV 16 predicts clinical outcome in oral cancer patients treated by radiotherapy
Authors: Apolinario R M , Lara P C , Henriquez L A , Lloret M , Pinar B
Published: July 2011

Growing molecular and clinical evidence indicates that human papillomavirus (HPV) is involved in the aetiology of oral squamous cell carcinomas (SCCs). HPV(+) tumours appear to be clinically distinct from HPV(−) tumours, conferring improved survival outcomes for patients in oropharyngeal cancer but limited knowledge exist on oral cancer. Determination of the HPV status of tumours may assist in patient risk-stratification and ultimately guide optimum treatment. The primary aim of this study was to examine the distribution of HPV in oral SCCs as assessed in vitro amplification assays and correlated with clinical and demographic data. The secondary aim was to correlate the positivity of HPV tumours with clinical outcome in the largest series of oral cancer published with a long follow-up (up to 20 years).

Materials and Methods

One hundred thirty-one invasive oral SCCs were tested for HPV using laboratory-developed PCR assays for HPV16. P53 expression, tumour angiogenesis (CD-31 staining) and proliferation (MIB-1) were also assessed by immunohistochemistry in parafine embedded tissue. Patients mean age was 58.09±10.41, median 59 (116 men and 15 women). Clinical Stage distribution was: st I:17 cases; II: 56,III:32.IV:26. Most tumours were histological grade I (39) and II (74). Patients with pathological stage I-II were refereed to surgery (65 cases) and patients with Stage III-IVA were referred to surgery and postoperative radiation therapy (66 cases). Mean radiotherapy given doses were 62.13±7.74, median 65 Gy in 1.8-2 Gy fractions. No chemotherapy was used in any case.

Results

41 cases (31.3%) were HPV 16(+). No relation was found with age, gender, or tumour characteristics. In fact no relation was found to p53 expression, tumour proliferation or angiogenesis. 15-year DFS was 62.20% in HPV(+) patients was, compared to 37.3% in the HPV(-) group (p<0.076). In stage III-IV cases (treated by surgery and radiation therapy) this differences reached statistical signification (15 y DFS 72.4% vs 36.0% p<0.020). Similar results were found for Cause Especific Survival (15 y DFS 68.4% vs 26.2% p<0.054).

Conclusion

These data show that the HPV status is a good predictor of DFS and survival in patients treated with radical surgery and adjuvant radiotherapy in oral carcinomas. This prognostic advantage seem to be independent of tumour proliferation, p53 status or angiogenesis. Other molecular processes could be implicated in the different response to radiotherapy.
O4-S2.02 Seroprevalence of novel immunogens of chlamydia trachomatis and their cytokine response in PBMC Cells under in vitro conditions
Authors: Saluja D , Patel A , Mishra P , Sachdev D
Published: July 2011

Background

Chlamydia trachomatis (CT) is an obligate intracellular parasite which causes STD and trachoma. Despite major research into Chlamydial pathogenesis and host immune responses, immuno-protection has been hampered by the incomplete understanding of protective immunity in the genital tract. Characterised vaccine candidates in general have shown variable efficacy ranging from no protection to partial protection. It is therefore a research priority to identify novel Chlamydial antigens that may elicit protective immune responses.

Objectives

The goal of the present study was to assess the seroprevalence to pkn1and DNA j following natural CT infection in human. The prospects of pkn1 as a Type third secretion substrate and DNA j a non surface Chlamydial protein as potential antigen, prompted us to explore the immunogenic potential of both protein.

Methods

pkn, DNA j and ompA were cloned in bacterial expression vector pTrcHis. Ni+-NTA affinity chromatography was used to purify the recombinant proteins. Antigenic stretches of Pkn1, DNA j and OmpA were identified using Bcepred web server, designed for identification of subunit vaccine candidate by Bioinformatics Centre of IMTECH Chandigarh, India. To validate the bioinformatics based analysis, sera of human patient were used to determine seroreactivity of pkn1 and DNA j proteins. OmpA was used as a positive control during the study.

Results

Present study showed a high seroprevalence of antibodies against Pkn1 and OmpA (p<0.001) in sera of humans infected with CT. while, no antibodies were observed for DNA j. Our studies have shown an association between release of TNF-α and IFN-y levels upon stimulation of PBMC with Pkn1 and OmpA. Cytokine expression profiling (IL-1ß, TNF-α, IL-2 , IL10 and IFN-y) of Human PBMCs in response to Pkn1 stimulation demonstrate for the first time that Pkn1 is a novel immunodominant Chlamydial antigen that is capable of influencing both Th1 and Th2 immune responses by stimulating the release of both Humoural and Cell-mediated regulatory cytokines.

Conclusions

Our study demonstrated strong serological responses to Pkn1 and major outer membrane in natural human infection suggesting the role of pkn1 in immune response. Studies are in progress to check how the immunomodulation by Pkn1 alters host-pathogen interactions.
O4-S2.03 An anti-adhesive approach to prevention ofC trachomatisinfection
Authors: Campbell L A , Zidal S V , Patton D , Kuo Cho-chou
Published: July 2011

Background

We have reported that the major outer membrane ofC trachomatisis glycosylated and the glycan is a high mannose oligosaccharide. Cumulative studies have demonstrated that the glycan is important in attachment and infectivity through binding to the mannose receptor (MR). Glycan removal decreases infectivity in vitro and in vivo and simultaneous administration of mannan, a ligand of the MR, abrogatesC trachomatisinfection in a mouse model of pneumonitis. Thus, we are investigating the feasibility of an anti-adhesive therapy to preventC trachomatisinfection by identifying oligosaccharides that are effective in inhibiting infection in vitro and testing their efficacy in a mouse model of genital tract infection.

Methods

HeLa 229 cell monolayers were pretreated with serial concentrations of oligosaccharides prior to infection withC trachomatis. Neutralisation of infectivity was scored as >50% inhibition. For animal experiments, 8-week-old Swiss Webster female mice were primed with subcutaneous injections of Depo-Provera 1 week prior to challenge. Subsequently, mice were inoculated intravaginally with carbohydrates or PBS (n=5 mice per group) 30 min prior to infection withC trachomatis. Vaginal swab samples were obtained at 24, 48, and 72 h. post-infection, at peak times of shedding. Statistical significance was determined by the Student's t test.

Results

Carbohydrates have been tested in vitro in hapten inhibition experiments against three serovars (D, E, F) most frequently isolated from genital tract infection. At the highest concentration tested, 4-nitrophenyl-α-D-mannopyranoside inhibited infectivity by 91%–92%; α-D-mannose-PAA from 77 to 93%; hen ovalbumin by 85%–89%; mannan by 77%–83%; and the high mannose fraction prepared from ovalbumin by 59%–98%. To determine efficacy in vivo, Swiss Webster mice were inoculated with different concentrations of inhibitors. Of those tested thus far, shedding of organism was significantly decreased (p<0.05). The maximum inhibitions observed were: 4-nitrophenyl-α-D-mannopyranoside (86%), α-D-mannose-PAA (81%), mannan (93%), and the high mannose fraction from ovalbumin (94%).

Conclusions

These preliminary studies suggest the potential feasibility for developing an “anti-adhesive therapy” as an alternate topical microbicide approach to preventC trachomatisgenital tract infection.
O4-S2.04 Can a cervical barrier prevent chlamydia infection in the pigtailed macaque cervical challenge model?
Authors: Patton D , Sweeney Y Cosgrove , Moench T
Published: July 2011

Background

Numerous microbicides that inactivateChlamydia trachomatisin vitro have failed to prevent transmission of this pathogen in the pigtailed macaque cervical challenge model. SinceC trachomatisreplicates in endocervical columnar epithelium but not in the squamous vaginal epithelium, we tested whether a cervical barrier would improve protection when used in conjunction with an otherwise non-protective microbicide.

Methods

Miniature diaphragm-like cervical barriers were manufactured and provided by ReProtect, Inc. BufferGel, previously found ineffective in this model when used alone, was also provided by ReProtect, Inc. Twenty-four pigtailed macaques were randomly assigned to one of three study arms: cervical barrier device alone; cervical barrier with BufferGel; or no barrier device and no gel (control arm). Eight animals were enrolled in each arm. Each macaque underwent baseline exam, product administration if applicable, cervical challenge with C. trachomatis (E: 5×105 IFU), and weekly follow-up exams for 5 weeks. Chlamydia challenge occurred within 30 min of baseline exams. Cervical barrier devices were removed from all test macaques 18 h after insertion. Each exam included cervicovaginal colposcopy, vaginal pH and cervical swabs for chlamydia detection (culture and NAAT: GenProbe Aptima Combo2). Blood for serum antibody testing was collected at baseline and weeks 2 though five post-inoculation.

Results

Detection of chlamydial infection is detailed in the Abstract O4-S2.04 table 1 below. Colposcopic exams were comparable across each of the three test arms of the study, and thus did not detect toxic effects of the gel or the cervical barrier. Baseline vaginal pH was somewhat higher in the No Product arm than in either of the test arms. After chlamydial challenge and throughout the duration of the study, mean vaginal pH remained lowest in the barrier with BufferGel arm, but were similar (within 1 pH unit) in all three study arms.

Abstract O4-S2.04 Table 1

CxBar

Working definition of confirmed infection: Positive NAAT AND Positive culture and Positive IgG/IgM>1:16Working definition of probable infection: positive NAAT and positive culture & IgG/IgM<1:32 OR negative culture & negative culture IgG/IgM>1:32Working definition of no infection: negative or self-limited NAAT AND negative culture AND IgG/IgM<1:32Barrier with buffergel224Barrier only422No Product530

Conclusions

In this pilot study, a cervical barrier used alone provided little or no protection, but the barrier used with BufferGel reduced transmission by 50% (p=0.08). These results should encourage further study of the ability of a cervical barrier combined with a microbicide to provide greater protection against sexually transmitted infections than either used alone.
O5-S1.01 Employing school nurses as a healthcare point of contact for male high school students: a school-based intervention to prevent STD, HIV, and teen pregnancy
Authors: Dittus P , De Rosa C , Jeffries R , Afifi A , Cumberland W , Ethier K , Chung E , Martinez E , Loya R , Kerndt P
Published: July 2011

Background

Adolescent males tend to have lower levels of knowledge about sexual and reproductive health (SRH), and access health care less frequently than adolescent females. Innovative strategies are needed to reach males with accurate information and resources regarding their SRH needs. Such strategies may improve adolescent males' access to SRH services, including STD screening, treatment, and contraception.

Methods

A multi-level intervention was delivered and evaluated across 5 years in a large public school district in Los Angeles, California. One intervention component sought to improve students' awareness and utilisation of condom availability programs (CAPs) in schools by working with key school personnel, particularly nurses, to more effectively implement district CAP policies. Six intervention and six control high schools participated in the study . Analyses included survey data from 13 733 high school males across 5 years (T1–T5). A mixed model logistic regression analysis was used to test for an intervention effect on males' reports of services sought from the school nurse. Random effects on the student level were included to control for repeated measures on the same student.

Results

The sample was 80% Latino and 9% African American; the mean age was 16.3. In the intervention as compared to the control condition, statistically significant increases were observed across 5 years of intervention in respondents' reports of going to the school nurse for information about birth control, STDs, pregnancy, or sex (see Abstract O5-S1.01 figure 1), as well as reports of going to the school nurse for condoms. For example, 5.4% of intervention school males reported going to the nurse for condoms at T1; reports increased to 9.6% at T5 for this group, whereas an opposite trend was observed for control school males.

Abstract O5-S1.01 Figure 1

Adjusted ORs* for the change between time points in males going to the school nurse.

Conclusions

School district policy combined with promotion of a CAP increased the number of adolescent males who sought out SRH information and condoms from the school nurse. With few points of access to the healthcare system available to males, school nurses may be especially important in connecting males to the healthcare system for services such as STD screening and contraception. As budget shortfalls affect all areas of school administration, school nurses should be recognised and retained as key players in promoting access to SRH services.
O5-S1.03 Young adults' views on telemedicine consultations for sexual health in Australia
Authors: Garrett C C , Kirkman M , Hocking J , Chen M , Fairley C K
Published: July 2011

Background

Young adults in Australia face barriers to accessing sexual health services including concerns over confidentiality and privacy, cost, lack of transportation and limited options around medical providers. A possible solution to decreasing these barriers is the use of telemedicine.

Method

An online questionnaire examined young adults' (aged 16–24) views on using webcam and telephone consultations for sexual health in Australia. Descriptive statistics were used to describe the study sample and χ2was used to assess associations. Free text responses were analysed thematically.

Results

662 people completed the questionnaire. Overall, 23% (n=150) of participants were willing to have a sexual health consultation with a doctor using a webcam if no genital examination was necessary; this number decreased to 16% (n=105) if a genital examination over webcam was needed. Men were more willing than women to have a webcam consultation (28% vs 21%, p=<0.01). In addition, men with same sex partners were more willing to have webcam consultation, with an unknown doctor, than men without any same sex partners (48% vs 26%, p=0.04). Participants' top preference for consulting a doctor if asymptomatic and living 2 h from a doctor was telephone (51%, n=340, compared with 10% for webcam); if symptomatic, participants' top preference was in person (62%, n=412, compared with 16% for webcam) instead of having the consultation over a webcam. While it was hypothesised that webcam consultations would decrease privacy and confidentiality concerns by preventing people from having to present at a sexual health clinic, preliminary results suggest that webcam consultations may instead augment such concerns. Free text responses suggest that this may be due to the fact that online consultations can be recorded, stored and potentially, if security measures are breached, be retrievable and searchable online.

Conclusion

To our knowledge, no study has examined the use of webcam consultations between healthcare providers and clients for sexually transmitted disease care. Results suggest that webcam consultations are not yet an acceptable medium for sexual health consultations for youth in Australia. Concerns about trust, privacy and security around online medical consultations are likely to influence whether such technology is eventually adopted into routine medical care.