Orogenital and anal infection by Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, and other sexually transmitted infections in men who have sex with men in Lisbon.

BACKGROUND: Men who have sex with men (MSM) are at risk for sexually transmitted infections (STIs), but more data on extragenital carriage are needed. AIM: We assessed the genital and extragenital prevalence of bacterial and other STIs in MSM in a Lisbon sexual health clinic. METHODS: We screened oral, anal, and urine samples of MSM visiting the GAT-CheckpointLX clinic June 2017-December 2021 for Chlamydia trachomatis (including lymphogranuloma venereum, LGV), Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Mycoplasma hominis, Ureaplasma urealyticum, and U. parvum. Ano-oro-genital lesions were tested for LGV, Treponema pallidum, and Herpes Simplex Virus. Blood was tested for HIV and T. pallidum antibodies. RESULTS: N. gonorrhoeae was found in 16.6% of the MSM followed by C. trachomatis (13.2%), M. genitalium (10.3%) and T. vaginalis (0.2%). The most frequent occurrence was anorectal (C. trachomatis, M. genitalium) and oral (N. gonorrhoeae). We found high carriage of U. urealyticum (36.1%) and M. hominis (22.1%). LGV was detected in 21.8% of chlamydia-positive anorectal swabs. Syphilis was detected in 22.6% of tested MSM, while 13.8% had HIV. Gonorrhoea and chlamydia were significantly more prevalent in MSM with concomitant HIV or syphilis. CONCLUSION: The substantial extragenital prevalence of bacterial STIs in MSM, and HIV and syphilis coinfections, suggest screening has value in identifying hidden carriage and in contributing for providing better care.

Demographic and Clinical Characteristics of Mpox Patients Attending an STD Clinic in Lisbon.

Mpox is a viral disease caused by the monkeypox virus, which marked the year of 2022 with a global outbreak. While previously considered to be a zoonosis of almost exclusive animal-to-human transmission, the current outbreak has been attributed to human-to-human transmission, particularly sexual transmission. As a new sexually transmissible disease, we studied the epidemiological and clinical features, as well as the concomitant occurrence of other sexually transmissible diseases, treatment approach, and outcome of our 291 patients, in the current outbreak. We found a total of 169 concomitant sexually transmissible infections of bacterial and viral origins, corresponding to 107 patients. Neisseria gonorrhoeae was the most common agent, particularly in the anal location. With this work, we emphasize the need for a thorough epidemiological and medical history, as well as a concomitant complete laboratorial screening for other STIs in patients with confirmed or suspected mpox.

Viral genetic clustering and transmission dynamics of the 2022 mpox outbreak in Portugal.

Pathogen genome sequencing during epidemics enhances our ability to identify and understand suspected clusters and investigate their relationships. Here, we combine genomic and epidemiological data of the 2022 mpox outbreak to better understand early viral spread, diversification and transmission dynamics. By sequencing 52% of the confirmed cases in Portugal, we identified the mpox virus sublineages with the highest impact on case numbers and fitted them into a global context, finding evidence that several international sublineages probably emerged or spread early in Portugal. We estimated a 62% infection reporting rate and that 1.3% of the population of men who have sex with men in Portugal were infected. We infer the critical role played by sexual networks and superspreader gatherings, such as sauna attendance, in the dissemination of mpox virus. Overall, our findings highlight genomic epidemiology as a tool for the real-time monitoring and control of mpox epidemics, and can guide future vaccine policy in a highly susceptible population.

Chlamydial and Gonococcal Genital Infections: A Narrative Review.

Sexually transmitted infections (STIs) constitute one of the leading causes of disease burden worldwide, leading to considerable morbidity, mortality, health expenditures, and stigma. Of note are the most common bacterial STIs, chlamydial and gonococcal infections, whose etiological agents are Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), respectively. Despite being usually asymptomatic, in some cases these infections can be associated with long-term severe complications, such as pelvic inflammatory disease, chronic pelvic pain, infertility, ectopic pregnancy, and increased risk of other STIs acquisition. As the symptoms, when present, are usually similar in both infections, and in most of the cases these infections co-occur, the dual-test strategy, searching for both pathogens, should be preferred. In line with this, herein we focus on the main aspects of CT and NG infections, the clinical symptoms as well as the appropriate state-of-the-art diagnostic tests and treatment. Cost-effective strategies for controlling CT and NG infections worldwide are addressed. The treatment for both infections is based on antibiotics. However, the continuing global rise in the incidence of these infections, concomitantly with the increased risk of antibiotics resistance, leads to difficulties in their control, particularly in the case of NG infections. We also discuss the potential mechanism of tumorigenesis related to CT infections. The molecular bases of CT and NG infections are addressed, as they should provide clues for control or eradication, through the development of new drugs and/or effective vaccines against these pathogens.

Learning and assessment strategies to develop specific and transversal competencies for a humanized medical education.

Introduction: Medical education should promote the development of skills and abilities that can be applied to real-world work performance. The aim of this study is to evaluate technical and methodological knowledge, as well as physician-patient communication skills, as one of the most important transversal competencies that a good physician should acquire; all this in a reliable, accurate and objective way. Methods: We present a rubric specifically designed and implemented for the evaluation of specific and transversal competencies in the physiology practical sessions, during the second year of the medical degree. The assessment consists in two evaluation tests: 1) a theoretical test that consists of multiple-choice questions. Students must demonstrate that they have acquired adequate theoretical knowledge (specific competency "to know"); 2) a practical test, in which students are evaluated by the rubric through the simulation of a medical consultation. Thus, demonstrating their ability to execute/apply what they have learned in class (specific competency "to know how to do"). They are also evaluated on the transversal competencies that we call "communication with the patient" (transversal competency "to know how to be there") and "dealing with the patient" (transversal competency "to know how to be"). Results: We evaluated whether there were differences in the grades obtained by students when the transversal competencies were not assessed (academic years 2017-2018 and 2018-2019; n = 289), and when the transversal competencies were assessed by applying the rubric in the academic years 2019-2020, 2021-2022, and 2022-2023 (n = 526). Furthermore, we present a student perception that supports the use of clinical simulation and our rubric as a good method within the competency learning process. Discussion: The acquisition of these competencies, starting from the first courses of undergraduate education, helps to raise the students' awareness in the development of a more humanized medicine, allowing a better response to the patients' needs. Our rubric, which clearly indicate the performance criteria, have become an excellent method to carry out the assessment of competencies, both for students and teachers, since they allow to obtain clear evidence of the level of acquisition and application of knowledge.

Global gene expression analysis of Streptococcus agalactiae at exponential growth phase.

A comparative overview of the global gene expression levels of S. agalactiae reference strain NEM316 at the exponential growth phase was done through RNA-sequencing. The expression levels of 47 genes potentially linked to virulence evidenced that: i) the major nuclease, GBS_RS03720/gbs0661, presented higher mean expression values than the remainder of DNase genes; ii) the genetic pilus island PI-2a genes presented higher mean expression values than PI-1 coding genes; and, iii) three virulence-associated genes ranked among the top-100 most expressed genes (GBS_RS07760, GBS_RS09445 and GBS_RS03485). Among this top-100, genes encoding proteins involved in "Translation, ribosomal structure and biogenesis" represented 46%. Curiously, genes with no assigned function were grouped in the category of highly expressed genes. As very little is known about the molecular mechanisms behind the release of DNases, preliminary assays were developed to understand whether direct DNA exposure would affect gene expression at the exponential growth phase. No differentially expressed genes were detected, indicating that follow-up studies are needed to disclose the complex molecular pathways (and stimuli) triggering the release of DNases. In general, our insights on the global expression levels of NEM316 at exponential growth phase with and without DNA exposure should open novel research lines to decipher S. agalactiae puzzling adaptation and virulence mechanisms, such as DNase production.

The Type III Secretion Effector CteG Mediates Host Cell Lytic Exit of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular bacterium causing ocular and urogenital infections in humans that are a significant burden worldwide. The completion of its characteristic infectious cycle relies on the manipulation of several host cell processes by numerous chlamydial type III secretion effector proteins. We previously identified the C. trachomatis CteG effector and showed it localizes at the host cell plasma membrane at late stages of infection. Here, we showed that, from 48 h post-infection, mammalian cells infected by wild-type C. trachomatis contained more infectious chlamydiae in the culture supernatant than cells infected by a CteG-deficient strain. This phenotype was CteG-dependent as it could be complemented in cells infected by the CteG-deficient strain carrying a plasmid encoding CteG. Furthermore, we detected a CteG-dependent defect on host cell cytotoxicity, indicating that CteG mediates chlamydial lytic exit. Previous studies showed that Pgp4, a global regulator of transcription encoded in the C. trachomatis virulence plasmid, also mediates chlamydial lytic exit. However, by using C. trachomatis strains encoding or lacking Pgp4, we showed that production and localization of CteG are not regulated by Pgp4. A C. trachomatis strain lacking both CteG and Pgp4 was as defective in promoting host cell cytotoxicity as mutant strains lacking only CteG or Pgp4. Furthermore, CteG overproduction in a plasmid suppressed the host cell cytotoxic defect of CteG- and Pgp4-deficient chlamydiae. Overall, we revealed the first chlamydial type III secretion effector involved in host cell lytic exit. Our data indicates that CteG and Pgp4 participate in a single cascade of events, but involving multiple layers of regulation, leading to lysis of host cells and release of the infectious chlamydiae.

Ongoing monkeypox virus outbreak, Portugal, 29 April to 23 May 2022.

Up to 27 May 2022, Portugal has detected 96 confirmed cases of monkeypox. We describe 27 confirmed cases (median age: 33 years (range: 22-51); all males), with an earliest symptom onset date of 29 April. Almost all cases (n = 25) live in the Lisbon and Tagus Valley health region. Most cases were neither part of identified transmission chains, nor linked to travel or had contact with symptomatic persons or with animals, suggesting the possible previously undetected spread of monkeypox.

Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus.

The largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1-6). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018-2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus.

Clinical and Epidemiological Characterization of Lymphogranuloma Venereum in a Sexually Transmitted Diseases Clinic in Lisbon, 2001 to 2020.

ABSTRACT: Between 2001 and 2020, 54 LGV cases were diagnosed in a sexually transmitted disease clinic in Lisbon, most in men who have sex with men (87%), HIV negative (63%), from the anorectal mucosa (72.2%). Cases among heterosexuals were also identified (13%). Surveillance programs irrespective of sexual orientation and HIV status are needed to avoid the morbidity associated with LGV.

Genomic insights on DNase production in Streptococcus agalactiae ST17 and ST19 strains.

Streptococcus agalactiae evasion from the human defense mechanisms has been linked to the production of DNases. These were proposed to contribute to the hypervirulence of S. agalactiae ST17/capsular-type III strains, mostly associated with neonatal meningitis. We performed a comparative genomic analysis between ST17 and ST19 human strains with different cell tropism and distinct DNase production phenotypes. All S. agalactiae ST17 strains, with the exception of 2211-04, were found to display DNase activity, while the opposite scenario was observed for ST19, where 1203-05 was the only DNase(+) strain. The analysis of the genetic variability of the seven genes putatively encoding secreted DNases in S. agalactiae revealed an exclusive amino acid change in the predicted signal peptide of GBS0661 (NucA) of the ST17 DNase(-), and an exclusive amino acid change alteration in GBS0609 of the ST19 DNase(+) strain. Further core-genome analysis identified some specificities (SNVs or indels) differentiating the DNase(-) ST17 2211-04 and the DNase(+) ST19 1203-05 from the remaining strains of each ST. The pan-genomic analysis evidenced an intact phage without homology in S. agalactiae and a transposon homologous to TnGBS2.3 in ST17 DNase(-) 2211-04; the transposon was also found in one ST17 DNase(+) strain, yet with a different site of insertion. A group of nine accessory genes were identified among all ST17 DNase(+) strains, including the Eco47II family restriction endonuclease and the C-5 cytosine-specific DNA methylase. None of these loci was found in any DNase(-) strain, which may suggest that these proteins might contribute to the lack of DNase activity. In summary, we provide novel insights on the genetic diversity between DNase(+) and DNase(-) strains, and identified genetic traits, namely specific mutations affecting predicted DNases (NucA and GBS0609) and differences in the accessory genome, that need further investigation as they may justify distinct DNase-related virulence phenotypes in S. agalactiae.

Transcontinental Dissemination of the L2b/D-Da Recombinant Chlamydia trachomatis Lymphogranuloma venereum (LGV) Strain: Need of Broad Multi-Country Molecular Surveillance.

Previously, we identified a Chlamydia trachomatis lymphogranuloma venereum (LGV) recombinant strain possessing a non-LGV ompA genotype. Here, culture-independent genome sequencing confirms its circulation in Europe, Middle East, and North America, and unveils emergence of antibiotic resistance. Broad surveillance is needed.

Neisseria gonorrhoeae clustering to reveal major European whole-genome-sequencing-based genogroups in association with antimicrobial resistance.

Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted disease gonorrhoea, has shown an extraordinary ability to develop antimicrobial resistance (AMR) to multiple classes of antimicrobials. With no available vaccine, managing N. gonorrhoeae infections demands effective preventive measures, antibiotic treatment and epidemiological surveillance. The latter two are progressively being supported by the generation of whole-genome sequencing (WGS) data on behalf of national and international surveillance programmes. In this context, this study aims to perform N. gonorrhoeae clustering into genogroups based on WGS data, for enhanced prospective laboratory surveillance. Particularly, it aims to identify the major circulating WGS-genogroups in Europe and to establish a relationship between these and AMR. Ultimately, it enriches public databases by contributing with WGS data from Portuguese isolates spanning 15 years of surveillance. A total of 3791 carefully inspected N. gonorrhoeae genomes from isolates collected across Europe were analysed using a gene-by-gene approach (i.e. using cgMLST). Analysis of cluster composition and stability allowed the classification of isolates into a two-step hierarchical genogroup level determined by two allelic distance thresholds revealing cluster stability. Genogroup clustering in general agreed with available N. gonorrhoeae typing methods [i.e. MLST (multilocus sequence typing), NG-MAST (N. gonorrhoeae multi-antigen sequence typing) and PubMLST core-genome groups], highlighting the predominant genogroups circulating in Europe, and revealed that the vast majority of the genogroups present a dominant AMR profile. Additionally, a non-static gene-by-gene approach combined with a more discriminatory threshold for potential epidemiological linkage enabled us to match data with previous reports on outbreaks or transmission chains. In conclusion, this genogroup assignment allows a comprehensive analysis of N. gonorrhoeae genetic diversity and the identification of the WGS-based genogroups circulating in Europe, while facilitating the assessment (and continuous monitoring) of their frequency, geographical dispersion and potential association with specific AMR signatures. This strategy may benefit public-health actions through the prioritization of genogroups to be controlled, the identification of emerging resistance carriage, and the potential facilitation of data sharing and communication.

Biofilm formation by ST17 and ST19 strains of Streptococcus agalactiae.

Bacterial biofilms are an important virulence factor with a vital role in evasion from the host immune system, colonization and infection. The aim of the present study was to evaluate in vitro the effects of three environmental factors (H+, glucose and human plasma) in biofilm formation, by carrier and invasive Streptococcus agalactiae strains of ST17 and ST19 sequence types, including DNase producers and non-producers. Bacteria ability to assemble biofilms was classified based on crystal violet assay. Biofilm formation was also monitored by scanning electron microscopy. Depending on the growth medium used, each bacterial isolate could fit in different biofilm production categories. Our data showed that optimal conditions for S. agalactiae biofilm assembly were reached after 48 h incubation at pH 7.6 in the presence of glucose and inactivated human plasma. In the presence of inactivated human plasma, the biofilm biomass of ST19 strains experienced a higher increase than ST17 strains. The composition of the extracellular polymeric matrix of the three strongest biofilm producers (all from ST17) was accessed by enzymatic digestion of mature biofilms and proteins were shown to be the predominant component. The detailed identification of the extracellular protein components should contribute to the development of new therapeutic strategies to fight S. agalactiae infections.

Cephalosporin-Resistant Neisseria gonorrhoeae Isolated in Portugal, 2019.

We report a multidrug-resistant Neisseria gonorrhoeae exhibiting resistance to ceftriaxone and cefixime, isolated in Portugal in 2019. Whole-genome sequencing was performed for typing and identification of genetic determinants of antimicrobial resistance. Because of its antimicrobial susceptibility profile, awareness should be raised for the circulation of this strain.

Fifteen years of a nationwide culture collection of Neisseria gonorrhoeae antimicrobial resistance in Portugal.

Neisseria gonorrhoeae antimicrobial resistance (AMR) and gonorrhea disease burden remain major public health concerns worldwide. To contribute to the supranational demands to monitor and manage the spread of antimicrobial-resistant N. gonorrhoeae, the Portuguese NIH promoted the creation of the National Laboratory Network for Neisseria gonorrhoeae Collection (PTGonoNet). The present study reports the N. gonorrhoeae major AMR trends observed from 2003 up to 2018. All isolates described in the present study constitute the opportunistic ongoing N. gonorrhoeae isolate collection supported by the National Reference Laboratory for Sexually Transmitted Infections of the Portuguese NIH, enrolling strains isolated in 35 different public and private laboratories. Minimum inhibitory concentrations were determined using E-tests for azithromycin, benzylpenicillin, cefixime, ceftriaxone, ciprofloxacin, gentamicin, spectinomycin and tetracycline. Molecular typing was determined using NG-MAST. AMR data of 2596 country-spread isolates show that 87.67% of all N. gonorrhoeae isolates presented decreased susceptibility to at least one antimicrobial. A continuous decreased susceptibility and resistance to penicillin, tetracycline and ciprofloxacin can be observed along the years. However, no decreased susceptibility to cephalosporins was observed until 2018, while for azithromycin, this was always low. The most common observed NG-MAST genogroups were G1407, G7445, G225, G2, and G1034. This study evidences the advantages of a nationwide collection of isolates and of centralized AMR testing to respond to supranational (EURO-GASP) requirements while providing unprecedented data on AMR in the context of 15 years of surveillance.