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Herein we show the development of a minimally instrumented paper-based molecular diagnostic for point of care detection of sexually transmitted infections caused by Chlamydia trachomatis. This new diagnostic platform incorporates cell lysis, isothermal nucleic acid amplification, and lateral flow visual detection using only pressure and heat sources, eliminating the need for expensive laboratory equipment. This paper-based test can be performed in less than one hour and has a clinically relevant limit of detection that is 100× more sensitive than current rapid immunoassays used for chlamydia diagnosis.

The Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) super-phylum contains, in addition to the three name-giving phyla, the Lentisphaerae and Kirimatiellaeota, as well as some uncultured candidate phyla, such as the Candidatus Omnitrophica. PVC bacteria have recently attracted attention from the scientific community for the peculiar characteristics that they display. For the third time, the PVC scientific community met recently at their bi-annual conference (http://pvcbacteria.org/pvcmeeting17/). In this special section, we would like to share some of the exciting results that were presented at this meeting.

The results of the Prevention Of Pelvic Infection (POPI) trial,1 published soon after criticism about the value for money of the chlamydia screening programme in England,2 should make us think even harder about how to investigate and control chlamydia and its associated morbidity. Should we be doing more of the same, but better? Should we be developing new approaches? Or do we need both? The innovative trial by Oakeshott et al in female college students in London provides new information about the effects on pelvic inflammatory disease (PID) of a single round of screening for Chlamydia trachomatis .1 By collecting specimens from all participants at baseline, Oakeshott and colleagues have provided the first data comparing the incidence of PID according to chlamydia infection status in women receiving immediate testing and treatment with those for whom testing and treatment were deferred by 12 months. The ethical decision to allow deferred testing is justified by the uncertainty about the incidence of PID in the community before the start of the trial, the low observed incidence in practice and the advice for all study participants about the availability of free chlamydia testing. The investigators randomised and followed 2529 women (mean age 20.9 years) and collected outcome data on 2377 (94%) from the women or their GPs. They obtained information from medical records of suspected PID cases and …

Chlamydia pecorum, an obligate intracellular bacterium, is associated with reproductive and systemic diseases in sheep, goats, pigs, cattle, and koalas. The main conditions include polyarthritis, conjunctivitis, enteritis, pneumonia, encephalomyelitis, orchitis, placentitis, and abortion. Even though there are several studies showing that C. pecorum infections are widely spread in the world, in Mexico there are no reports. During 2016, as part of a sheep restocking program in Mexico, sheep were imported from New Zealand. Briefly after their arrival in the herds in the State of Mexico, these sheep presented abortions during the last third of gestation. A total of 62 sheep vaginal swabs that had presented abortion from different municipalities of the State of Mexico were collected. Bacterial isolation was performed using L929 mouse fibroblasts, and molecular identification was achieved by 23S rRNA (Chlamydiaceae family) and ompA gene (species-specific) real-time polymerase chain reaction (PCR). In addition, the 16S rRNA subunit and ompA gene were amplified and sequenced. Seven of 62 samples were positive for C. pecorum by bacterial isolation, 23S rRNA, and ompA gene real-time PCR. The 16S rRNA subunit and ompA gene amplicons were purified and the nucleotide sequence was determined in both directions. The consensus sequences homology search was performed using BLASTn analysis and showed a 100% of homology with the C. pecorum 16S rRNA subunit and 99% with the C. pecorum ompA gene. The population structure analyses using ompA gene demonstrated 15 genetic populations or clusters of 198 sequences from GenBank and our sequences were in a particular genetic structure corresponding to genotype “O.” Herein, we describe the presence of C. pecorum in sheep imported from New Zealand into Mexico. Genetic analysis of the ompA gene showed that the isolates belong to genotype O and are related to strains isolated from sheep, cattle, and koalas. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-022-00682-9.

Context There are few UK data on the prevalence and clustering of risky behaviours in ethnically diverse adolescents. Objectives To investigate the prevalence of reported alcohol use, smoking and vaping, and explore whether these behaviours are associated with increased numbers of sexual partners. Design Questionnaire survey of ‘Test n Treat’ chlamydia screening trial participants. Setting and participants Sexually active students attending six London technical colleges completed confidential questionnaires and provided genitourinary samples. Results The median age of the 509 participants was 17 years (IQR: 16‐18), 47% were male, 50% were of black ethnicity, 55% reported ≥2 sexual partners in the past year (67% of males and 45% of females) and 6.2% had chlamydia infection and 0.6% gonorrhoea. Almost half (48%) reported getting drunk in the past month, 33% smoked cigarettes and 7% had ever vaped. A larger percentage of students with ≥2 sexual partners than 0‐1 partners reported getting drunk in the past month (53.7%, 144/268% versus 42.2% 94/223, adjusted prevalence ratio: 1.33, 95% confidence interval: 1.11‐1.61) and smoking cigarettes (36.6%, 100/273% versus 30.2%, 67/222, 1.34 (1.05‐1.70)). By contrast, multiple sexual partners were not associated with vaping or chlamydia infection, but numbers were small. Conclusions We found high prevalences of risky behaviour and an association between multiple sexual partners and smoking and/or getting drunk. Findings support the introduction of compulsory sex and relationship education in UK secondary schools, including information about the adverse effects of alcohol and smoking. Public contribution Participants helped with study design, conduct and interpretation.

The occurrence of keratoconjunctivitis infectiosa ovis (KIO) in the Netherlands was reported by Hofland et al. in 1969 for the first time. At present the disease is well known in the Netherlands. Nevertheless there are still questions about the causative agent and the most effective and easiest therapy. Most authors suppose that the disease is caused by Colesiota conjunctivae, although others mention infections by other chlamydia, Mycoplasma conjunctivae, Mycoplasma ovipneumoniae, Acholeplasma oculi and a wide variety of bacteria. The diagnosis can be made on the basis of the symptoms and the detection of the agent in conjunctival scrapings. The bacilliform bodies can be found in conjunctival smears in the cytoplasm (Giemsa, Stamp). Many therapies are used topically, parenterally or orally. Locally used eye-ointments must be effective against Colesiota; antimicrobial drugs administered by injection must be effective against the latter and also provide a sufficient cell tissue penetration with excretion into the lacrimal fluids. Injections have proved to be easier to administer, especially in serious outbreaks, but such outbreaks are exceptional (18). Some therapies have been evaluated in small scale experiments (28) as well as in field trials (17).

This study explored factors associated with interest in taking PrEP among men who have sex with men (MSM) attending HIV testing venues in Sweden. Data from 658 HIV-negative respondents, surveyed by a questionnaire at six sites, were analyzed descriptively and by univariable and multivariable logistic regression. A total of 453 (68.8%) of the respondents expressed interest in taking PrEP. Reporting self-perceived risk of HIV acquisition as moderate or high, reporting ≥ 5 partners for condomless anal intercourse during the past year, and reporting hard drug use during the past year were independently associated with interest in taking PrEP. However, an aggregated variable of self-reported rectal gonorrhea, rectal chlamydia, or syphilis infection during the past year was not associated with interest in taking PrEP. Overall, Swedish MSM were well-informed regarding PrEP, and interest in taking PrEP was positively associated with sexual risk indicators.

Masturbation is a common sexual practice in men, and saliva is often used as a lubricant during masturbation by men who have sex with men. However, the role of saliva use during masturbation in the transmission of chlamydia is still unclear. We developed population-level, susceptible-infected-susceptible compartmental models to explore the role of saliva use during masturbation on the transmission of chlamydia at multiple anatomical sites. In this study, we simulated both solo masturbation and mutual masturbation. Our baseline model did not include masturbation but included transmission routes (anal sex, oral-penile sex, rimming, kissing and sequential sexual practices) we have previously validated (model 1). We added masturbation to model 1 to develop the second model (model 2). We calibrated the model to five clinical datasets separately to assess the effects of masturbation on the prevalence of site-specific infection. The inclusion of masturbation (model 2) significantly worsened the ability of the models to replicate the prevalence of C. trachomatis. Using model 2 and the five data sets, we estimated that saliva use during masturbation was responsible for between 3.9% [95% confidence interval (CI) 2.0–6.8] and 6.2% (95% CI 3.8–10.5) of incident chlamydia cases at all sites. Our models suggest that saliva use during masturbation is unlikely to play a major role in chlamydia transmission between men, and even if it does have a role, about one in seven cases of urethral chlamydia might arise from masturbation.

Background The association between meeting partners on the web and sexual practices has been understudied in heterosexuals. Objective This study aims to examine the associations between the methods of meeting partners and sexual practices and HIV and sexually transmitted infections (STIs) in heterosexuals. Methods We conducted a survey among heterosexuals attending the Melbourne Sexual Health Centre in 2019. This survey asked about the methods through which the participants engaged in meeting their sexual partners, sexual practices, and intravenous drug use (IVDU) over the past 3 months. The participants’ HIV and STI (chlamydia, gonorrhea, and syphilis) status was obtained from clinical testing. Multivariable logistic regression was used to examine the association between each method of meeting and the participants’ sexual practices, IVDU, and STI status. Results A total of 698 participants (325 men and 373 women) were included in the study. Most of the participants reported using only one method to meet partners (222/325, 68.3% men; 245/373, 65.7% women; P=.05). The men met partners most commonly at social venues (eg, bar, pub, or party; 126/325, 38.8%), whereas the women met partners most commonly through friends or family (178/373, 47.7%). Paying for sex was associated with men meeting partners at sex venues (adjusted odds ratio [AOR] 145.34, 95% CI 26.13-808.51) and on the internet (AOR 10.00, 95% CI 3.61-27.55). There was no association between IVDU and methods of meeting. Social venues were associated with condomless vaginal sex among men (AOR 3.31, 95% CI 1.94-5.71) and women (AOR 2.58, 95% CI 1.61-4.13) and testing positive for STI among men (AOR 3.04, 95% CI 1.24-7.48) and women (AOR 3.75, 95% CI 1.58-8.89). Conclusions Heterosexuals who met partners at social venues had a more than threefold risk of testing positive for STIs, indicating that heterosexuals may benefit from health promotion campaigns that are delivered through a public setting.

Our research group has recently shown that Borrelia burgdorferi, the Lyme disease bacterium, is capable of forming biofilms in Borrelia-infected human skin lesions called Borrelia lymphocytoma (BL). Biofilm structures often contain multiple organisms in a symbiotic relationship, with the goal of providing shelter from environmental stressors such as antimicrobial agents. Because multiple co-infections are common in Lyme disease, the main questions of this study were whether BL tissues contained other pathogenic species and/or whether there is any co-existence with Borrelia biofilms. Recent reports suggested Chlamydia-like organisms in ticks and Borrelia-infected human skin tissues; therefore, Chlamydia-specific polymerase chain reaction (PCR) analyses were performed in Borrelia-positive BL tissues. Analyses of the sequence of the positive PCR bands revealed that Chlamydia spp. DNAs are indeed present in these tissues, and their sequences have the best identity match to Chlamydophila pneumoniae and Chlamydia trachomatis. Fluorescent immunohistochemical and in situ hybridization methods demonstrated the presence of Chlamydia antigen and DNA in 84% of Borrelia biofilms. Confocal microscopy revealed that Chlamydia locates in the center of Borrelia biofilms, and together, they form a well-organized mixed pathogenic structure. In summary, our study is the first to show Borrelia-Chlamydia mixed biofilms in infected human skin tissues, which raises the questions of whether these human pathogens have developed a symbiotic relationship for their mutual survival.

Background Sexual health (SH) services increasingly need to prioritise those at greatest risk of sexually transmitted infections (STIs). We used SH surveillance data to develop algorithms to triage individuals attending SH services within two high-risk populations: men who have sex with men (MSM) and young people (YP). Methods Separate multivariable logistic regression models for MSM and YP were developed using surveillance data on demographics, recent sexual history, prior STI diagnoses and drug/alcohol use from five clinics in 2015–2016 to identify factors associated with new STI diagnoses. The models were prospectively applied in one SH clinic in May 2017 as an external validation. Findings 9530 YP and 1448 MSM SH episodes informed model development. For YP, factors associated with new STI diagnosis (overall prevalence: 10.6%) were being of black or mixed white/black ethnicity; history of chlamydia diagnosis (previous year); and multiple partners/new partner (previous 3-months). The YPs model had reasonable performance (c-statistic: 0.703), but poor discrimination when externally validated (c-statistic: 0.539). For MSM, being of South Asian ethnicity; being born in Europe (excluding the UK); and condomless anal sex or drug use (both in previous 3-months) were associated with STI diagnosis (overall prevalence: 22.0%). The MSM model had a c-statistic of 0.676, reducing to 0.579 on validation. Interpretation SH surveillance data, including limited behavioural data, enabled triage algorithms to be developed, but its implementation may be problematic due to poor external performance. This approach may be more suitable to self-triage, including online, ensuring patients are directed towards appropriate services. Funding NIHR HTA programme (12/191/05)., Highlights • We report the development and validation of an algorithm predicting STI diagnosis among MSM and young people in England • Inclusion of sexual behaviour data, feasibly collected in clinical settings, was crucial to achieve reasonable model performance • External validation highlighted key limitations with automated triage based on predictive algorithms in sexual health settings

Prior studies have demonstrated that both bacterial vaginosis (BV) and sexually transmitted infections (STIs) are strong independent risk factors for subsequent STI. In observational studies of this biological enhancement (BE) hypothesis, it is important to adjust for the risk of STI exposure so that the independent effect of BE can be assessed. We sought to model if two markers of local sexual network (partner concurrency and cumulative number of STIs) represented residual confounding in the models of risk for subsequent infection in a study that screened 3620 women for STIs every 3 months for a year. Mixed-effects logistic regression was used to calculate the odds ratios for an incident diagnosis ofChlamydia trachomatis,Neisseria gonorrhoeae,Trichomonas vaginalisand BV following a diagnosis of any of these four at the prior visit, controlling for the cumulative number of STIs and partner concurrency variables. We found that partner concurrency and cumulative number of STIs were each associated with incident infection, and in general, controlling for these variables reduced the strength of the association between prior and incident infections. We conclude that the frequently found association between prior and incident STIs is associated with both BE and sexual network structure.

Background: Public health reporting is the cornerstone of public health practices that inform prevention and control strategies. There is a need to leverage advances made in the past to implement an architecture that facilitates the timely and complete public health reporting of relevant case-related information that has previously not easily been available to the public health community. Electronic laboratory reporting (ELR) is a reliable method for reporting cases to public health authorities but contains very limited data. In an earlier pilot study, we designed the Public Health Automated Case Event Reporting (PACER) platform, which leverages existing ELR infrastructure as the trigger for creating an electronic case report. PACER is a FHIR (Fast Health Interoperability Resources)-based system that queries the electronic health record from where the laboratory test was requested to extract expanded additional information about a case. Objective: This study aims to analyze the pilot implementation of a modified PACER system for electronic case reporting and describe how this FHIR-based, open-source, and interoperable system allows health systems to conduct public health reporting while maintaining the appropriate governance of the clinical data. Methods: ELR to a simulated public health department was used as the trigger for a FHIR-based query. Predetermined queries were translated into Clinical Quality Language logics. Within the PACER environment, these Clinical Quality Language logical statements were managed and evaluated against the providers’ FHIR servers. These predetermined logics were filtered, and only data relevant to that episode of the condition were extracted and sent to simulated public health agencies as an electronic case report. Design and testing were conducted at the Georgia Tech Research Institute, and the pilot was deployed at the Medical University of South Carolina. We evaluated this architecture by examining the completeness of additional information in the electronic case report, such as patient demographics, medications, symptoms, and diagnoses. This additional information is crucial for understanding disease epidemiology, but existing electronic case reporting and ELR architectures do not report them. Therefore, we used the completeness of these data fields as the metrics for enriching electronic case reports. Results: During the 8-week study period, we identified 117 positive test results for chlamydia. PACER successfully created an electronic case report for all 117 patients. PACER extracted demographics, medications, symptoms, and diagnoses from 99.1% (116/117), 72.6% (85/117), 70.9% (83/117), and 65% (76/117) of the cases, respectively. Conclusions: PACER deployed in conjunction with electronic laboratory reports can enhance public health case reporting with additional relevant data. The architecture is modular in design, thereby allowing it to be used for any reportable condition, including evolving outbreaks. PACER allows for the creation of an enhanced and more complete case report that contains relevant case information that helps us to better understand the epidemiology of a disease. Trial Registration

A number of different Chlamydia spp. have been detected in the class Amphibia with C. pneumoniae being the predominant species involved. Chlamydiae have been linked to mass mortality events, thereby representing significant pathogens that deserve attention with respect to worldwide amphibian decline. We here present six cases of chlamydiosis and asymptomatic chlamydial infections in different frog species from three ex situ amphibian conservation facilities. Clinical signs predominantly characterised by regurgitation, chronic wasting, lethargy and suspended breeding were associated with C. pneumoniae infection. Despite various treatment regimens, it was not possible to clear infections. However, intra vitam diagnostics succeeded from skin, faeces and urine for the first time.

Chlamydia spp. are a group of obligate intracellular pathogens causing a number of diseases in animals and humans. Avian chlamydiosis (AC), caused by Chlamydia psittaci (C. psittaci) as well as new emerging C. avium, C. gallinacea and C. ibidis, have been described in nearly 500 avian species worldwidely. The Crested Ibis (Nipponia nippon) is a world endangered avian species with limited population and vulnerable for various infections. To get a better understanding of the prevalence of Chlamydia spp. in the endangered Crested Ibis, faecal samples were collected and analysed. The results confirmed that 20.20% (20/99) of the faecal samples were positive for Chlamydiaceae and were identified as C. ibidis with co-existence of C. psittaci in one of the 20 positive samples. In addition, ompA sequence of C. psittaci obtained in this study was classified into the provisional genotype Matt116, while that of C. ibidis showed high genetic diversity, sharing only 77% identity with C. ibidis reference strain 10-1398/6. We report for the first time the presence of C. ibidis and C. psittaci in the Crested Ibis, which may indicate a potential threat to the endangered birds and should be aware of the future protection practice.

Introduction The aim of the study was to evaluate the frequency of occurrence of Chlamydia trachomatis (C.t.) DNA in the prostate material in the group of individuals with the chronic prostatitis. Material and methods The study included 65 males aged between 47 and 68 years of age, reporting for transrectal prostate biopsy because of the elevated serum prostate-specific antigen concentration and/or abnormalities detected in prostate palpation per rectum. The urethral smear collection was performed in all the patients in order to detect C.t. DNA. After that, the transrectal prostate biopsy was performed (histopathology tests, C.t. DNA). Additionally, the levels of anti-C.t. IgG antibodies and anti-C.t. IgA antibodies were checked in the serum. The DNA isolation from prostate specimens was conducted with the use of the Chelex method, while the C.t. DNA detection – with the ligase chain reaction. Specific antibodies were detected with the use of the ELISA method. Results C.t. DNA in the prostate gland was found in 7 out of 65 men (10.8%). In urethral smear, C.t. was found in none of the individuals. Anti-C.t. IgA antibodies were detected in the serum of 16/65 (24.6%), while anti-C.t. IgG antibodies in 6/65 (9.2%) of the examined males. IgA antibodies were found in two and IgG in one out of the 7 men who had C.t. infection in the prostate. Conclusions The presence of C.t. DNA in the prostate gland may be indicative of the role of chlamydia in the development of chronic prostatitis.

// Jiachao Cao 1 , Yumin Mao 1 , Bo Dong 1 , Wei Guan 1 , Jia Shi 1 and Suinuan Wang 1 1 Department of Neurosurgery, The Third Affiliated Hospital of Soochow University, Changzhou, China Correspondence to: Suinuan Wang, email: 292079658@qq.com Keywords: atherosclerosis, Chlamydia pneumonia, cytomegalovirus, specific antigen, immunohistochemistry detection Received: April 04, 2017 Accepted: June 04, 2017 Published: July 18, 2017 ABSTRACT To explore the relationship between certain pathogens, such as chlamydia pneumonia (Cpn) and cytomegalovirus (CMV), and carotid atherosclerosis (AS) in a Chinese population.Twenty-five carotid atherosclerotic stenosis patients from the Beijing Tiantan Hospital (affiliated with Capital Medical University) participated in the study. After undergoing digital subtraction angiography (DSA) and/or computed tomography angiography (CTA), the degree of carotid artery stenosis was over 70% in all cases, and the patients underwent carotid endarterectomy. Plaque specimens were obtained during surgery. The streptavidin-peroxidase (SP) method was used to test the Cpn and CMV antigens in the specimens, and the relationship between the Cpn and CMV pathogen infections and AS was analyzed based on the test results. In the group of 25 carotid atherosclerotic specimens, the detection rate of the Cpn-specific antigens was 84.0% (21/25). In the control group, the detection rate was 13.3% (2/15) in the ascending aortic intima. Thus, the between-group difference was significant (P

SUMMARYIdentification of priority populations such as men who have sex with men (MSM) is important in surveillance systems to monitor trends of sexually transmitted infections (STIs). We explored using routinely collected non-behavioural data as a means to establish MSM status in surveillance by assessing anorectal swab as a marker of male-to-male sexual exposure. We used chlamydia testing data from a sexual health clinic, 2007–2012. Men reporting any male sexual partner(s) in the previous 12 months were considered MSM. The dataset was split into development and validation samples to develop a univariate predictive model and assess the model fit. The dataset included 30 358 individual men and 48 554 episodes of STI testing; 45% were among reported MSM and an anorectal swab was performed in 40% of testing episodes. Anorectal swabbing had good diagnostic performance as a marker for MSM status (sensitivity = 87%, specificity = 99%, positive predictive value = 98·6%, negative predictive value = 90·3%). The model showed good fit against the internal validation sample (area under the curve = 0·93). Anorectal swabs are a valid marker of MSM behaviour in surveillance data from sexual health clinics, and they are likely to be particularly useful for monitoring STI trends among MSM with higher risk behaviour.

Il lavoro presentato in questa tesi di Dottorato è incentrato sullo sviluppo di strategie analitiche innovative basate sulla sensoristica e su tecniche di spettrometria di massa in ambito biologico e della sicurezza alimentare. Il primo capitolo tratta lo studio di aspetti metodologici ed applicativi di procedure sensoristiche per l’identificazione e la determinazione di biomarkers associati alla malattia celiaca. In tale ambito, sono stati sviluppati due immunosensori, uno a trasduzione piezoelettrica e uno a trasduzione amperometrica, per la rivelazione di anticorpi anti-transglutaminasi tissutale associati a questa malattia. L’innovazione di questi dispositivi riguarda l’immobilizzazione dell’enzima tTG nella conformazione aperta (Open-tTG), che è stato dimostrato essere quella principalmente coinvolta nella patogenesi. Sulla base dei risultati ottenuti, entrambi i sistemi sviluppati si sono dimostrati una valida alternativa ai test di screening attualmente in uso per la diagnosi della celiachia. Rimanendo sempre nel contesto della malattia celiaca, ulteriore ricerca oggetto di questa tesi di Dottorato, ha riguardato lo sviluppo di metodi affidabili per il controllo di prodotti “gluten-free”. Il secondo capitolo tratta lo sviluppo di un metodo di spettrometria di massa e di un immunosensore competitivo per la rivelazione di prolammine in alimenti “gluten-free”. E’ stato sviluppato un metodo LC-ESI-MS/MS basato su un’analisi target con modalità di acquisizione del segnale selected reaction monitoring per l’identificazione di glutine in diversi cereali potenzialmente tossici per i celiaci. Inoltre ci si è focalizzati su un immunosensore competitivo per la rivelazione di gliadina, come metodo di screening rapido di farine. Entrambi i sistemi sono stati ottimizzati impiegando miscele di farina di riso addizionata di gliadina, avenine, ordeine e secaline nel caso del sistema LC-MS/MS e con sola gliadina nel caso del sensore. Infine i sistemi analitici sono stati validati analizzando sia materie prime (farine) che alimenti (biscotti, pasta, pane, etc.). L’approccio sviluppato in spettrometria di massa apre la strada alla possibilità di sviluppare un test di screening multiplo per la valutazione della sicurezza di prodotti dichiarati “gluten-free”, mentre ulteriori studi dovranno essere svolti per ricercare condizioni di estrazione compatibili con l’immunosaggio competitivo, per ora applicabile solo all’analisi di farine estratte con etanolo. Terzo capitolo di questa tesi riguarda lo sviluppo di nuovi metodi per la rivelazione di HPV, Chlamydia e Gonorrhoeae in fluidi biologici. Si è scelto un substrato costituito da strips di carta in quanto possono costituire una valida piattaforma di rivelazione, offrendo vantaggi grazie al basso costo, alla possibilità di generare dispositivi portatili e di poter visualizzare il risultato visivamente senza la necessità di strumentazioni. La metodologia sviluppata è molto semplice, non prevede l’uso di strumentazione complessa e si basa sull’uso della isothermal rolling-circle amplification per l’amplificazione del target. Inoltre, di fondamentale importanza, è l’utilizzo di nanoparticelle colorate che, essendo state funzionalizzate con una sequenza di DNA complementare al target amplificato derivante dalla RCA, ne permettono la rivelazione a occhio nudo mediante l’uso di filtri di carta. Queste strips sono state testate su campioni reali permettendo una discriminazione tra campioni positivi e negativi in tempi rapidi (10-15 minuti), aprendo una nuova via verso nuovi test altamente competitivi con quelli attualmente sul mercato. The work presented in this PhD thesis is focused on the development of innovative analytical strategies based on sensor technology and mass spectrometry techniques in biological field and food safety. The first chapter deals on the study of methodological aspects and applications of sensing procedures for the identification and determination of biomarkers associated with celiac disease. In this context, a piezoelectric and amperometric immunosersor have been developed for the detection of anti-tissue transglutaminase antibodies associated with this disease. The innovation of these devices concerns the immobilization of the enzyme tTG in its open conformation (Open-tTG), which has been shown to be the one mainly involved in the pathogenesis. On the basis of the results, both systems are a good alternative to the screening tests currently in use for the diagnosis of celiac disease. In the context of the research theme aimed at developing new methods for the diagnosis of celiac disease, a further research objective of PhD thesis was the development of reliable methods for the control of gluten-free products. For this purpose, the second chapter deals on the development of mass spectrometry-based methods and of a competitive immunosensor for the detection of prolamines in gluten-free foods. A targeted proteomic LC-ESI-MS/MS-based method for simultaneous determination of gluten from different celiotoxic cereals on foods, was developed. In addition, the attention was focused on a competitive immunosensor as a screening rapid method for the detection of gliadin in flours. Both systems have been optimized using mixtures of rice flour fortified with gliadins, avenins, hordeins and secalins in case of LC-MS/MS analysis and just with gliadins in case of the sensor. Finally, these systems have been validated by analyzing both raw materials (flours) and food samples (biscuits, pasta, bread, etc.). The mass spectrometry approach opens the way to the possibility to perform a MS-based multiplex screening for the assessment of gluten-free products safety, but further studies should be conducted to investigate extraction conditions compatible with the competitive immunoassay that so far can just be used for the analysis of flours extracted with ethanol. The third chapter of this thesis concerns the development of new methods for the detection of HPV, Chlamydia and Gonorrhoeae in biological fluids. It has been chosen a strip of paper as a substrate which can constitute a valid detection platform, offering advantages due to the low cost, the ability to generate mobile devices and allows the visualization of results without instrumentations. The developed methodology is very simple, does not require the use of complex instrumentation and is based on the use of isothermal rolling circle amplification to amplify the target. Furthermore, of fundamental importance, is the use of nanoparticles with intrinsic coloration functionalized with a DNA sequence complementary to the amplified target deriving from RCA, because they allow the visualization by naked eye through the use of paper filters. These strips were tested on real samples allowing discrimination between positive and negative samples in a short time (10-15 minutes), opening the way to new and highly competitive assays with those currently available.

Background Sexually transmitted infections (STIs) pose a significant public health challenge in the United States. Traditional surveillance systems are adversely affected by data quality issues, underreporting of cases, and reporting delays, resulting in missed prevention opportunities to respond to trends in disease prevalence. Search engine data can potentially facilitate an efficient and economical enhancement to surveillance reporting systems established for STIs. Objective We aimed to develop and train a predictive model using reported STI case data from Chicago, Illinois, and to investigate the model’s predictive capacity, timeliness, and ability to target interventions to subpopulations using Google Trends data. Methods Deidentified STI case data for chlamydia, gonorrhea, and primary and secondary syphilis from 2011-2017 were obtained from the Chicago Department of Public Health. The data set included race/ethnicity, age, and birth sex. Google Correlate was used to identify the top 100 correlated search terms with “STD symptoms,” and an autocrawler was established using Google Health Application Programming Interface to collect the search volume for each term. Elastic net regression was used to evaluate prediction accuracy, and cross-correlation analysis was used to identify timeliness of prediction. Subgroup elastic net regression analysis was performed for race, sex, and age. Results For gonorrhea and chlamydia, actual and predicted STI values correlated moderately in 2011 (chlamydia: r=0.65; gonorrhea: r=0.72) but correlated highly (chlamydia: r=0.90; gonorrhea: r=0.94) from 2012 to 2017. However, for primary and secondary syphilis, the high correlation was observed only for 2012 (r=0.79), 2013 (r=0.77), 2016 (0.80), and 2017 (r=0.84), with 2011, 2014, and 2015 showing moderate correlations (r=0.55-0.70). Model performance was the most accurate (highest correlation and lowest mean absolute error) for gonorrhea. Subgroup analyses improved model fit across disease and year. Regression models using search terms selected from the cross-correlation analysis improved the prediction accuracy and timeliness across diseases and years. Conclusions Integrating nowcasting with Google Trends in surveillance activities can potentially enhance the prediction and timeliness of outbreak detection and response as well as target interventions to subpopulations. Future studies should prospectively examine the utility of Google Trends applied to STI surveillance and response.

SUMMARYRepeat rectal chlamydia infection is common in men who have sex with men (MSM) following treatment with 1 g azithromycin. This study describes the association between organism load and repeat rectal chlamydia infection, genovar distribution, and efficacy of azithromycin in asymptomatic MSM. Stored rectal chlamydia-positive samples from MSM were analysed for organism load and genotyped to assist differentiation between reinfection and treatment failure. Included men had follow-up tests within 100 days of index infection. Lymphogranuloma venereum and proctitis diagnosed symptomatically were excluded. Factors associated with repeat infection, treatment failure and reinfection were investigated. In total, 227 MSM were included – 64 with repeat infections [28·2%, 95% confidence interval (CI) 22·4–34·5]. Repeat positivity was associated with increased pre-treatment organism load [odds ratio (OR) 1·7, 95% CI 1·4–2·2]. Of 64 repeat infections, 29 (12·8%, 95% CI 8·7–17·8) were treatment failures and 35 (15·4%, 95% CI 11·0–20·8) were reinfections, 11 (17·2%, 95% CI 8·9–28·7) of which were definite reinfections. Treatment failure and reinfection were both associated with increased load (OR 2·0, 95% CI 1·4–2·7 and 1·6, 95% CI 1·2–2·2, respectively). The most prevalent genovars were G, D and J. Treatment efficacy for 1 g azithromycin was 83·6% (95% CI 77·2–88·8). Repeat positivity was associated with high pre-treatment organism load. Randomized controlled trials are urgently needed to evaluate azithromycin's efficacy and whether extended doses can overcome rectal infections with high organism load.

SUMMARYChlamydiae are frequently encountered intracellular Gram-negative bacteria. In pigs, these bacteria in combination with other pathogens contribute to the induction of a multi-aetiological syndrome. One of the major characteristics of Chlamydia spp. is their ability to cause prolonged, often subclinical infections. While the economic consequences of Chlamydia spp. infections in pig farms are not fully established, we know that reproductive disorders and other syndromes correlated with Chlamydia infection can lead to financial loss as a result of a reduction in pork production. Additionally, Chlamydia spp. presents a potential zoonotic hazard, therefore determining the prevalence of Chlamydia in pig populations is critical. In the present study 97 pig herds from Poland were involved. To determine the prevalence of Chlamydia PCR and CFT tests were used. In total 797 vaginal samples, 797 conjunctival samples, and 235 serum samples were collected and tested. The study took place from 2011 to 2014. We found Chlamydia spp. present in 71·2% of all tested farms. The percentage of animals testing positive on any given farm varied from 20% to 100%.

There has been some concern among parents and in the media that vaccinating children against human papillomavirus could be seen as giving children permission to engage in risky sexual behaviors (also known as sexual disinhibition). Several studies have found this concern to be unfounded but there have been no attempts to synthesize the relevant studies in order to assess if there is evidence of sexual disinhibition. The aim of this study was to synthesize recent literature examining sexual behaviors and biological outcomes (e.g., sexually transmitted infections) post-HPV vaccination. We reviewed literature from January 1, 2008-June 30, 2015 using PubMed, CINAHL, and Embase with the following search terms: [(sex behavior OR sex behavior OR sexual) AND (human papillomavirus OR HPV) AND (vaccines OR vaccine OR vaccination)] followed by a cited reference search. We included studies that examined biological outcomes and reported behaviors post-vaccination in both males and females. Studies were reviewed by title and abstract and relevant studies were examined as full-text articles. We identified 2,503 articles and 20 were eventually included in the review. None of the studies of sexual behaviors and/or biological outcomes found evidence of riskier behaviors or higher rates of STIs after HPV vaccination. Instead, the studies found that vaccinated compared to unvaccinated individuals were less likely to report vaginal intercourse without a condom (OR = 0.5; 95%CI = 0.4–0.6) and non-use of contraception (OR = 0.27; 95%CI = 0.15–0.48) and unvaccinated participants had higher rates of Chlamydia (OR = 2.3; 95%CI = 1.06–5.00). These results should be reassuring to parents and health care providers.

INTRODUCTION Immune response to genital Chlamydia trachomatis infection is involved in both immunity and pathology. The cytokine profile during infection has been implicated in the disease outcome, either resolution or severe sequelae. Serum cytokines of Chlamydia positive Nigerian women with tubal infertility were assessed to determine their possible relationship with tubal occlusion. MATERIAL AND METHODS One hundred and fifty age-matched consenting women (100 fertile and 50 with tubal infertility) were recruited based on C. trachomatis antibody positivity and grouped into infertile Chlamydia positive (CTpos) women (n = 50), fertile Chlamydia positive women (n = 50) and fertile Chlamydia negative (CTneg) women as controls (n = 50). High vaginal swabs and endo-cervical swabs were collected for microscopy, culture and gram staining. Cytokines [transforming growth factor β1 (TGF-β1), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), interleukin (IL)-4, IL-10 and IL-17A] were estimated by ELISA in sera. Data were analyzed using ANOVA, χ (2) and Spearman's correlation at p = 0.05. RESULTS Lower IFN-γ levels were observed in infertile women compared to fertile women. Fertile CTneg women had significantly higher TNF-α, and TGF-β1 compared to fertile and infertile CTpos women, respectively. Lower IL-10 levels were seen in fertile CTpos women compared to the infertile CTpos group. Vaginal discharge was negatively correlated with TNF-α and IFN-γ and positively with IL-4 in Chlamydia positive women. CONCLUSIONS Chlamydia positive women with tubal infertility have higher IL-10 and lower IFN-γ levels than controls, which may contribute to their development of tubal pathology.

Chlamydia trachomatis (Ct) is the leading cause of sexually transmitted diseases worldwide. There is no safe and effective vaccine to control the spread of Ct. In development of Ct vaccine, selection of appropriate candidate antigens and an effective delivery system may be the main challenges. Multi-epitope of major outer membrane protein (MOMPm) is the most suitable candidate for a Ct vaccine, while hepatitis B virus core antigen (HBcAg) has unique advantages as vaccine delivery system. Therefore, in this study, we evaluated the immunogenicity and protective immune response of a novel candidate vaccine in a murine model of chlamydial genital infection. This candidate vaccine comprises MOMPm peptide delivered with HBcAg. Our results of Ct-specific serum IgG and secretory IgA assay, cytokine assay, and cytotoxic T-lymphocyte assay revealed that immunogenicity of the candidate vaccine was much better than that of the corresponding synthetic MOMPm peptide. Furthermore, the protective effect of the candidate vaccine was also shown much better than that of the synthetic peptide by calculating the isolation of Chlamydia from vaginal swabs and histopathological analysis. Taken together, our results indicate that HBcAg carrying Ct MOMPm could be an effective immune prophylactic for chlamydial infection.

SUMMARYSeveral infections have been linked to telomere shortening and in some cases these associations have varied by sex. We assessed the association between seropositivity to four persistent pathogens (cytomegalovirus (CMV), herpes simplex virus-1,Helicobacter pylori,Chlamydia pneumoniae), and total pathogen burden on leukocyte telomere length in a diverse US sample. Data came from the Multi-Ethnic Study of Atherosclerosis, a population-based cohort study. We utilized cross-sectional survey data, and biological samples from participants tested for pathogens and telomere length (N= 163). Linear regression was used to examine the association between seropositivity for individual pathogens as well as total pathogen burden and telomere length, adjusting for various confounders. CMV seropositivity and increased total pathogen burden level were significantly associated with shorter telomere length among females (β= −0·1204 (standard error (s.e.) 0·06),P= 0·044) and (β= −0·1057 (s.e.= 0·05),P= 0·033), respectively. There was no statistically significant association among males. Our findings suggest that prevention or treatment of persistent pathogens, in particular CMV, may play an important role in reducing telomere shortening over the life course among women. Future research is needed to confirm these novel findings in larger longitudinal samples.

The intracellular human bacterial pathogen Chlamydia trachomatis pursues effective strategies to protect infected cells against death-inducing stimuli. Here, we show that Chlamydia trachomatis infection evokes 3-phosphoinositide-dependent protein kinase-1 (PDPK1) signaling to ensure the completion of its developmental cycle, further leading to the phosphorylation and stabilization of MYC. Using biochemical approaches and imaging we demonstrate that Chlamydia-induced PDPK1-MYC signaling induces host hexokinase II (HKII), which becomes enriched and translocated to the mitochondria. Strikingly, preventing the HKII interaction with mitochondria using exogenous peptides triggers apoptosis of infected cells as does inhibiting either PDPK1 or MYC, which also disrupts intracellular development of Chlamydia trachomatis. These findings identify a previously unknown pathway activated by Chlamydia infection, which exhibits pro-carcinogenic features. Targeting the PDPK1-MYC-HKII-axis may provide a strategy to overcome therapeutic resistance of infection., Highlights • MYC oncogene expression is induced and stabilized in Chlamydia-infected cells via activation of PDPK1. • Activation of the PDPK1-MYC axis enhances hexokinase II expression. • HKII-mitochondria association prevents apoptosis to support chlamydial growth. Activation of oncogenes promotes cell survival and metabolic reprogramming. Intracellular pathogens target oncogenes to exploit cellular machineries and inhibit apoptosis. Al-Zeer et al. now highlight the role of the oncogene MYC for inducing hexokinase II activation during Chlamydia infection, linking cell survival to energy metabolism.

Chlamydia is an obligate intracellular bacterial pathogen that replicates solely within a membrane-bound vacuole termed an inclusion. Chlamydia seems to perturb multiple cellular processes of the host, such as, rearrangement of the membrane trafficking system for its intracellular multiplication, and inhibition of host cell apoptosis for persistent infection. In an attempt to clarify host factor involvement in apoptosis regulation, we found that inhibition of Caspase-9 restricted, while Apaf-1 promoted, Chlamydia pneumoniae infection in HEp-2, HeLa, and mouse epithelial fibroblast (MEF) cells. These opposition contributions to the chlamydial infection were confirmed using caspase-9 (-/-) and apaf-1 (-/-) MEFs. Similar phenomena also appeared in the case of infection with Chlamydia trachomatis. Interestingly, caspase-9 in apaf-1 (-/-) MEFs was activated by chlamydial infection but during the infection caspase-3 was not activated. That is, caspase-9 was activated without support for multiplication and activation by Apaf-1, and the activated caspase-9 may be physically disconnected from the caspase cascade. This may be partially explained by the observation of caspase-9 accumulation within chlamydial inclusions. The sequestration of caspase-9 by chlamydia seems to result in apoptosis repression, which is crucial for the chlamydial development cycle. Because Apaf-1 shares domains with intracellular innate immune receptor NOD1, it may play a key role in the strategy to regulate chlamydial infection.