Epidemiological profile and genetic resistance of Neisseria gonorrhoeae infection in women in a poor region of São Paulo, Brazil.

BACKGROUND AND AIM: Gonorrhea is a bacterial infection in the urogenital tract, transmitted by sexual or perinatal contact, caused by Neisseria gonorrhoeae, a gram-negative diplococcus. The present study evaluates the frequency of N. gonorrhoeae in women treated at Hospital Wladimir Arruda in poor area of São Paulo and also verifies the presence of genetic resistance against three antimicrobials of different classes: Tetracycline, Azithromycin and Ciprofloxacin. METHODS: This is an observational and descriptive study with a quantitative approach. Samples were collected at Hospital Escola Wladimir Arruda. The volunteers are women from 16 to 65 years of age. Sociodemographic, gynecological, sexual and health data are collected through a questionnaire, their symptoms/clinical manifestation were requested by the medical records, and then the participant is referred for collection of samples of cervical vaginal smear. The samples were screened for N. gonorrhoeae (dcmH gene) and tested for resistance genes to Tetracycline, Azithromycin and Ciprofloxacin through PCR. RESULTS: In the total of 127 samples analyzed by Real-Time PCR, 23 were positive and correspond to a general prevalence of a gonococcal infection in the studied population of 17% (CI:95%), and the participants were married (43.4%), had active sexual life (56.5%) and did not use any type of condom during sexual intercourse (52.1%). The resistance to the tetM ribosomal gene was found in 14 samples, prevalence of 60% (CI= 95%). CONCLUSIONS: We have described a concerning frequency of N. gonorrhoeae infection in females attended in an outcare patient. Also, most of the strains detected presented resistance to one or more antimicrobials.

Functional characterization of the gonococcal polyphosphate pseudo-capsule.

Neisseria gonorrhoeae is an exclusively human pathogen able to evade the host immune system through multiple mechanisms. Gonococci accumulate a large portion of phosphate moieties as polyphosphate (polyP) on the exterior of the cell. Although its polyanionic nature has suggested that it may form a protective shield on the cell surface, its role remains controversial. Taking advantage of a recombinant His-tagged polyP-binding protein, the presence of a polyP pseudo-capsule in gonococcus was demonstrated. Interestingly, the polyP pseudo-capsule was found to be present in specific strains only. To investigate its putative role in host immune evasion mechanisms, such as resistance to serum bactericidal activity, antimicrobial peptides and phagocytosis, the enzymes involved in polyP metabolism were genetically deleted, generating mutants with altered polyP external content. The mutants with lower polyP content on their surface compared to the wild-type strains, became sensitive to complement-mediated killing in presence of normal human serum. Conversely, naturally serum sensitive strains that did not display a significant polyP pseudo-capsule became resistant to complement in the presence of exogenous polyP. The presence of polyP pseudo-capsule was also critical in the protection from antibacterial activity of cationic antimicrobial peptide, such as cathelicidin LL-37. Results showed that the minimum bactericidal concentration was lower in strains lacking polyP than in those harboring the pseudo-capsule. Data referring to phagocytic killing resistance, assessed by using neutrophil-like cells, showed a significant decrease in viability of mutants lacking polyP on their cell surface in comparison to the wild-type strain. The addition of exogenous polyP overturned the killing phenotype of sensitive strains suggesting that gonococcus could exploit environmental polyP to survive to complement-mediated, cathelicidin and intracellular killing. Taken together, data presented here indicate an essential role of the polyP pseudo-capsule in the gonococcal pathogenesis, opening new perspective on gonococcal biology and more effective treatments.

Analyses of human papillomavirus, Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, and co-infections in a gynecology outpatient clinic in Haikou area, China.

BACKGROUND: The purpose of this study was to study the infection rates of Chlamydia trachomatis (CT), Ureaplasma urealyticum (UU), Neisseria gonorrhoeae (NG), and co-infections with human papillomavirus (HPV) in a hospital gynecology outpatient clinic in the Haikou region in 2021. METHODS: From January to December 2021, the Women and Children Medical Center of Hainan Province collected 2389 samples of cervical exfoliated cells and vaginal swab specimens from gynecologic outpatients. The samples were then analyzed descriptively for data, and the detection rate of each pathogen was tallied. All vaginal swabs were obtained for CT, UU, and NG DNA testing, and cervical exfoliated cells for HPV genotyping. Analyses were performed on the detection rate of each group. RESULTS: In 2389 samples, the frequencies of pathogen identification among the 2389 samples were as follows: UU (58.43%); HPV (17.29%); CT (7.99%); and NG (0.38%). HPV, CT, UU, and NG were detected in 33.33%, 22.55%, 77.45%, and 2.94% of individuals between 15 and 20 years of age, respectively. The detection rates of CT, UU, and NG were substantially greater in the HPV-positive group than the the HPV-negative group (P < 0.05). CONCLUSION: Among gynecologic outpatients at a hospital in the Haikou area, the probability of mixed infections with genital tract pathogens in HPV-positive patients was higher compared to HPV-negative patients. Reproductive tract infections are becoming more prevalent in younger people, hence adolescent sexual health education needs improvement.

An optimized Factor H-Fc fusion protein against multidrug-resistant Neisseria gonorrhoeae.

Novel therapeutics against the global threat of multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococci evade killing by complement by binding factor H (FH), a key inhibitor of the alternative pathway. FH comprises 20 short consensus repeat (SCR) domains organized as a single chain. Gonococci bind FH through domains 6 and 7, and C-terminal domains 18 through 20. Previously, we showed that a chimeric protein comprising (from the N- to C-terminus) FH domains 18-20 (containing a point mutation in domain 19 to prevent lysis of host cells) fused to human IgG1 Fc (called FH*/Fc1) killed gonococci in a complement-dependent manner and reduced the duration and bacterial burden in the mouse vaginal colonization model of gonorrhea. Considering the N. gonorrhoeae-binding FH domains 18-20 are C-terminal in native FH, we reasoned that positioning Fc N-terminal to FH* (Fc1/FH*) would improve binding and bactericidal activity. Although both molecules bound gonococci similarly, Fc1/FH* displayed a 5-fold lower IC50 (the concentration required for 50% killing in complement-dependent bactericidal assays) than FH*/Fc1. To further increase complement activation, we replaced human IgG1 Fc in Fc1/FH* with Fc from human IgG3, the most potent complement-activating IgG subclass, to obtain Fc3/FH*. Bactericidal activity was further increased ~2.3-fold in Fc3/FH* compared to Fc1/FH*. Fc3/FH* killed (defined by <50% survival) 45/45 (100%) diverse PorB1B-expessing gonococci, but only 2/15 PorB1A-expressing isolates, in a complement-dependent manner. Decreased Fc3/FH* binding accounted for the limited activity against PorB1A strains. Fc3/FH* was efficacious against all four tested PorB1B gonococcal strains in the mouse vaginal colonization model when administered at a dose of 5 µg intravaginally, daily. Furthermore, Fc3/FH* retained bactericidal activity when reconstituted following lyophilization or spray-drying, suggesting feasibility for formulation into intravaginal rings. In conclusion, Fc3/FH* represents a promising prophylactic immunotherapeutic against multidrug-resistant gonococci.

Application of Isothermal Signal Amplification Technique in the Etiological Diagnosis of Gonorrhea and Drug Resistance Gene Detection.

Background: Isothermal signal amplification technique is developed based on the rolling ring amplification mechanism of cyclic DNA molecules in nature. This technique plays an extremely beneficial role in gonorrhea pathogen identification and drug resistance gene detection. Aims: This study analyzes the isothermal signal amplification techniques in the etiological diagnosis of gonorrhea and drug resistance gene detection. Materials and Methods: Urethral, cervical secretion, or prostatic fluid samples from 322 cases of gonorrhea collected from January 2018 to December 2021 at the STD clinic of our hospital dermatology department were selected for direct smear examination and gonococcal culture examination; DNA was extracted from urethral, cervical secretion, or prostatic fluid samples and then used for pathogen identification by SAT assay and rolling loop nucleic acid amplification technique, smear examination and pathogen culture examination methods, SAT assay, and isothermal signal amplification technique for comparative sensitivity and specificity analysis. Results: The highest rate of gonorrhea positivity was for the urine rolling loop nucleic acid amplification technique, followed by the swab rolling loop nucleic acid amplification technique, and the lowest rate of gonorrhea positivity was for the urine SAT test. The difference in the positivity rate between the two urine testing methods was statistically significant (P < 0.05). The highest sensitivity of the urine rolling loop nucleic acid amplification technique method for the detection of gonorrhea pathogens and the lowest sensitivity of the urine SAT method were statistically significant (P < 0.01). The differences in sensitivity and specificity between the swab rolling loop nucleic acid amplification technique and the swab SAT method were not statistically significant (P > 0.05). ROC curves were plotted based on sensitivity and specificity, with swab SAT assay (AUC = 0.998) > rolling loop nucleic acid amplification technique (AUC = 0.981). Comparing the negative rates of urine and swab rolling loop nucleic acid amplification technique and urine SAT assay, the differences were not statistically significant (P > 0.05). Conclusion: The isothermal signal amplification technique improves the shortcomings of gonorrhea pathogen identification means and drug resistance gene detection methods, with good detection sensitivity and specificity, simple operation, low price, and easy promotion, which has obvious advantages in clinical applications and epidemiological studies.

The Neisseria gonorrhoeae type IV pilus promotes resistance to hydrogen peroxide- and LL-37-mediated killing by modulating the availability of intracellular, labile iron.

The Neisseria gonorrhoeae Type IV pilus is a multifunctional, dynamic fiber involved in host cell attachment, DNA transformation, and twitching motility. We previously reported that the N. gonorrhoeae pilus is also required for resistance against hydrogen peroxide-, antimicrobial peptide LL-37-, and non-oxidative, neutrophil-mediated killing. We tested whether the hydrogen peroxide, LL-37, and neutrophil hypersensitivity phenotypes in non-piliated N. gonorrhoeae could be due to elevated iron levels. Iron chelation in the growth medium rescued a nonpiliated pilE mutant from both hydrogen peroxide- and antimicrobial peptide LL-37-mediated killing, suggesting these phenotypes are related to iron availability. We used the antibiotic streptonigrin, which depends on free cytoplasmic iron and oxidation to kill bacteria, to determine whether piliation affected intracellular iron levels. Several non-piliated, loss-of-function mutants were more sensitive to streptonigrin killing than the piliated parental strain. Consistent with the idea that higher available iron levels in the under- and non-piliated strains were responsible for the higher streptonigrin sensitivity, iron limitation by desferal chelation restored resistance to streptonigrin in these strains and the addition of iron restored the sensitivity to streptonigrin killing. The antioxidants tiron and dimethylthiourea rescued the pilE mutant from streptonigrin-mediated killing, suggesting that the elevated labile iron pool in non-piliated bacteria leads to streptonigrin-dependent reactive oxygen species production. These antioxidants did not affect LL-37-mediated killing. We confirmed that the pilE mutant is not more sensitive to other antibiotics showing that the streptonigrin phenotypes are not due to general bacterial envelope disruption. The total iron content of the cell was unaltered by piliation when measured using ICP-MS suggesting that only the labile iron pool is affected by piliation. These results support the hypothesis that piliation state affects N. gonorrhoeae iron homeostasis and influences sensitivity to various host-derived antimicrobial agents.

Variable Expression of Opa Proteins by Neisseria gonorrhoeae Influences Bacterial Association and Phagocytic Killing by Human Neutrophils.

Neisseria gonorrhoeae infection is characterized by local and abundant recruitment of neutrophils. Despite neutrophils' antimicrobial activities, viable N. gonorrhoeae is recovered from infected individuals, leading to the question of how N. gonorrhoeae survives neutrophil attack. One feature impacting N. gonorrhoeae-neutrophil interactions is the phase-variable opacity-associated (Opa) proteins. Most Opa proteins engage human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to facilitate bacterial binding and invasion. Neutrophils express two transmembrane CEACAMs, CEACAM1 and the granulocyte-specific CEACAM3. While N. gonorrhoeae isolated from infected individuals is frequently Opa+, expression of OpaD from strain FA1090, which interacts with CEACAMs 1 and 3, is associated with reduced N. gonorrhoeae survival after exposure to human neutrophils. In this study, we hypothesized that the receptor-binding capability of individual Opa proteins impacts bacterial survival in the presence of neutrophils. To test this hypothesis, we introduced opa genes that are constitutively expressed into a derivative of strain FA1090 with all 11 opa genes deleted. The engineered genes encode Opa proteins that bind CEACAM1 and -3, CEACAM1 but not CEACAM3, or neither CEACAM1 nor -3. N. gonorrhoeae expressing CEACAM3-binding Opa proteins survived significantly less well than bacteria expressing other Opa proteins when exposed to primary human neutrophils. The CEACAM3-binding N. gonorrhoeae had significantly greater association with and internalization by neutrophils. However, once internalized, bacteria were similarly killed inside neutrophils, regardless of Opa expression. Furthermore, Opa expression did not significantly impact neutrophil granule mobilization. Our findings indicate that the extent to which Opa proteins mediate nonopsonic binding is the predominant determinant of bacterial survival from neutrophils. IMPORTANCE Neisseria gonorrhoeae, the cause of gonorrhea, is an urgent-threat pathogen due to increasing numbers of infections and increased antibiotic resistance. Many surface components of N. gonorrhoeae are phase variable, including the Opa protein family of adhesins and invasins. While Opa protein expression is selected for in vivo, bacteria expressing some Opa proteins are readily killed by neutrophils, which are recruited to sites of infection. The reason for this discrepancy has remained unresolved. Our work shows that Opa-dependent differences in bacterial survival after exposure to primary human neutrophils correlates with Opa-dependent bacterial binding and phagocytosis. These findings underscore how the ability of N. gonorrhoeae to change Opa expression through phase variation contributes to bacterial resistance to neutrophil clearance.

The diagnostic accuracy of pooled testing from multiple individuals for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae: a systematic review.

OBJECTIVES: Molecular testing for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) is costly. Therefore, we appraised the evidence regarding pooling samples from multiple individuals to test for CT/NG. METHODS: In this systematic review, we searched 5 databases (2000-2021). Studies were included if they contained primary data describing pooled testing. We calculated the pooled sensitivities and specificities for CT and NG using a bivariate mixed-effects logistic regression model. RESULTS: We included 22 studies: most were conducted in high-income countries (81.8%, 18 of 22), among women (73.3%, 17 of 22), and pooled urine samples (63.6%, 14 of 22). Eighteen studies provided 25 estimates for the meta-analysis of diagnostic accuracy, with data from 6,913 pooled specimens. The pooled sensitivity for CT was 98.4% (95% confidence intervals [CI]: 96.8-99.2%, I2=77.5, p<0.001), and pooled specificity was 99.9% (95% CI: 99.6-100.0%, I2=62.6, p<0.001). Only 2 studies reported pooled testing for NG, and both reported similarly high sensitivity and specificity as for CT. Sixteen studies provided data on the cost of pooling, reporting cost-savings ranging from 39%-90%. CONCLUSIONS: Pooled testing from multiple individuals for CT is highly sensitive and specific compared with individual testing. This approach has the potential to reduce the cost of screening in populations for which single anatomic site screening is recommended.

High proportions of rectal and pharyngeal chlamydia and gonorrhoea cases among cisgender men are missed using current CDC screening recommendations.

OBJECTIVES: Pharyngeal and rectal Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections are often undiagnosed due to their asymptomatic nature. This study aims to determine (1) the prevalence of CT/NG infections by anatomical site among cisgender men; (2) the proportion of missed CT/NG rectal/pharyngeal infections if urogenital testing alone was performed or screening depended on self-reported behaviour alone; and (3) the predictive probability of self-reported behaviours for rectal CT/NG. METHODS: This cross-sectional study used electronic health records collected at a sexual health clinic in Los Angeles from 18 November 2018 until 28 February 2020. The included patients were ≥18 years of age cisgender men who received CT/NG testing at least once during the study period. We calculated the proportion of missed pharyngeal/rectal CT/NG infections if only urogenital testing had been done and if testing was based only on self-reported anal sex. Separately, we ran logistic regressions for predictive probability of self-reported anal sex on CT/NG rectal infections. RESULTS: Overall, there were 13 476 unique patients with 26 579 visits. The prevalence of any extragenital CT/NG infection was 37.28%. Over 80% rectal/pharyngeal CT cases and over 65% rectal/pharyngeal NG cases would be missed if urogenital testing alone was performed. Likewise, over 35% rectal CT/NG cases would be missed had testing relied on self-reported sexual behaviours alone. CONCLUSIONS: The proportion of missed rectal and pharyngeal CT/NG infections is high. Our data from a sexual health clinic lend support to three-site opt-out testing for cisgender men attending a sexual health/Lesbian, Gay, Bisexual, Transgender, Queer/Questioning (LGBTQ+) specialty clinic regardless of their sexual orientation or reported sexual behaviours.

Self-Inhibitory Peptides Targeting the Neisseria gonorrhoeae MtrCDE Efflux Pump Increase Antibiotic Susceptibility.

Neisseria gonorrhoeae is an increasing public health threat due to its rapidly rising incidence and antibiotic resistance. There are an estimated 106 million cases per year worldwide, there is no vaccine available to prevent infection, and N. gonorrhoeae strains that are resistant to all antibiotics routinely used to treat the infection have emerged. In many strains, antibiotic resistance is mediated by overexpression of the MtrCDE efflux pump, which enables the bacteria to transport toxic antibiotics out of the cell. Genetic mutations that inactivate MtrCDE have previously been shown to render resistant strains susceptible to certain antibiotics. Here, we show that peptides rationally designed to target and disrupt the activity of each of the three protein components of MtrCDE were able to increase the susceptibility of N. gonorrhoeae strains to antibiotics in a dose-dependent manner and with no toxicity to human cells. Cotreatment of bacteria with subinhibitory concentrations of the peptide led to 2- to 64-fold increases in susceptibility to erythromycin, azithromycin, ciprofloxacin, and/or ceftriaxone in N. gonorrhoeae strains FA1090, WHO K, WHO P, and WHO X. The cotreatment experiments with peptides P-MtrC1 and P-MtrE1 resulted in increased susceptibilities of WHO P and WHO X to azithromycin, ciprofloxacin, and ceftriaxone that were of the same magnitude seen in MtrCDE mutants. P-MtrE1 was able to change the azithromycin resistance profile of WHO P from resistant to susceptible. Data presented here demonstrate that these peptides may be developed for use as a dual treatment with existing antibiotics to treat multidrug-resistant gonococcal infections.

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin.

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Extragenital Screening of Chlamydia trachomatis and Neisseria gonorrhoeae Among Women in the College Health Setting.

BACKGROUND: Despite high frequencies of oral and receptive anal intercourse among young women, the Centers for Disease Control and Prevention does not recommend routine oropharyngeal or anorectal screening for CT and GC. Risk-based extragenital screening of women has not been adopted at the majority of college health centers, and existing research has not focused on the female or college population. METHODS: We examined health records of women at a college health center in a large urban university for 3 years to evaluate the effectiveness of CT and GC screening. We also evaluated the proportion of CT and GC infections that would have been missed if risk-based extragenital screening was not performed. Decisions to screen at extragenital sites were based on patient-reported risk behavior. RESULTS: For 8027 unique chlamydia screens and 7907 unique gonorrhea screens, approximately 20% of the visits used extragenital screening in response to self-reported risk behaviors. More than 44% of patients were non-Hispanic White, and approximately 48% fell within the 20- to 24-year age group. The case positivity rates for CT were 2.85% with urogenital-only screening and 1.30% with risk-prompted extragenital screening (1.1% throat, 4.3% rectal). The case positivity rates for GC were 0.11% with urogenital-only screening and 0.37% with risk-prompted extragenital screening (0.37% throat, 0% rectal). If the college health center had relied solely on urogenital screening rather than adding risk-based extragenital screening, 4.41% of CT infections would have been missed and 28.57% of GC infections would have been missed. CONCLUSIONS: Nearly 1 of 22 CT infections and nearly 1 of 3 GC infections would have been missed without extragenital screening in this analysis of college women. Inclusion of risk-prompted extragenital screening in asymptomatic STD screening protocols can help clinicians diagnose CT and GC infections that would be have been missed with urogenital-only screening. Although rectal GC infections among women seem to be less common, oropharyngeal testing, in particular, for GC is suggested for women based on sexual risk. However, clinicians might only identify these risks if they ask patients directly about these potential exposures. Because guidelines exist only for men, future studies should focus on extragenital screening in college women to build the evidence that this particular population of patients may benefit from this practice, given the high risk of STDs in young adults.

[MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA].

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Šfrom the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

The Lst Sialyltransferase of Neisseria gonorrhoeae Can Transfer Keto-Deoxyoctanoate as the Terminal Sugar of Lipooligosaccharide: a Glyco-Achilles Heel That Provides a New Strategy for Vaccines to Prevent Gonorrhea.

The lipooligosaccharide (LOS) of Neisseria gonorrhoeae plays key roles in pathogenesis and is composed of multiple possible glycoforms. These glycoforms are generated by the process of phase variation and by differences in the glycosyltransferase gene content of particular strains. LOS glycoforms of N. gonorrhoeae can be terminated with an N-acetylneuraminic acid (Neu5Ac), which imparts resistance to the bactericidal activity of serum. However, N. gonorrhoeae cannot synthesize the CMP-Neu5Ac required for LOS biosynthesis and must acquire it from the host. In contrast, Neisseria meningitidis can synthesize endogenous CMP-Neu5Ac, the donor molecule for Neu5Ac, which is a component of some meningococcal capsule structures. Both species have an almost identical LOS sialyltransferase, Lst, that transfers Neu5Ac from CMP-Neu5Ac to the terminus of LOS. Lst is homologous to the LsgB sialyltransferase of nontypeable Haemophilus influenzae (NTHi). Studies in NTHi have demonstrated that LsgB can transfer keto-deoxyoctanoate (KDO) from CMP-KDO to the terminus of LOS in place of Neu5Ac. Here, we show that Lst can also transfer KDO to LOS in place of Neu5Ac in both N. gonorrhoeae and N. meningitidis Consistent with access to the pool of CMP-KDO in the cytoplasm, we present data indicating that Lst is localized in the cytoplasm. Lst has previously been reported to be localized on the outer membrane. We also demonstrate that KDO is expressed as a terminal LOS structure in vivo in samples from infected women and further show that the anti-KDO monoclonal antibody 6E4 can mediate opsonophagocytic killing of N. gonorrhoeae Taken together, these studies indicate that KDO expressed on gonococcal LOS represents a new antigen for the development of vaccines against gonorrhea.IMPORTANCE The emergence of multidrug-resistant N. gonorrhoeae strains that are resistant to available antimicrobials is a current health emergency, and no vaccine is available to prevent gonococcal infection. Lipooligosaccharide (LOS) is one of the major virulence factors of N. gonorrhoeae The sialic acid N-acetylneuraminic acid (Neu5Ac) is present as the terminal glycan on LOS in N. gonorrhoeae In this study, we made an unexpected discovery that KDO can be incorporated as the terminal glycan on LOS of N. gonorrhoeae by the alpha-2,3-sialyltransferase Lst. We showed that N. gonorrhoeae express KDO on LOS in vivo and that the KDO-specific monoclonal antibody 6E4 can direct opsonophagocytic killing of N. gonorrhoeae These data support further development of KDO-LOS structures as vaccine antigens for the prevention of infection by N. gonorrhoeae.

Performance of human papillomavirus DNA detection in residual specimens taken for Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid amplification testing in men who have sex with men.

OBJECTIVES: Rectal swab specimens, either alone or pooled with first-void urine (FVU) and pharyngeal swab specimens, are used to test for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection in men who have sex with men (MSM). Following introduction of human papillomavirus (HPV) vaccination for MSM attending UK sexual health services (SHSs), HPV testing of residual CT/NG test specimens has been proposed to monitor HPV prevalence in this population. Performance of HPV detection in such specimens has not been evaluated previously. METHODS: MSM attending a UK SHS provided three specimens: (1) rectal swab for CT/NG, (2) pooled rectal/pharyngeal/FVU specimen for CT/NG and (3) dedicated anal swab for HPV. Specimen 3 and residual material from specimens 1 and 2 were tested for type-specific HPV DNA. HPV detection was by an in-house multiplex PCR and luminex-based genotyping assay. RESULTS: A total of 129 MSM were recruited with a mean age of 38.1 years; 24% were HIV-positive. Of the 129 MSM, 92 (71%) had any type-specific HPV DNA in ≥1 specimen; 80 (62%) had high risk (HR) HPV. Of 123 participants with sufficient residual pooled and dedicated specimens, 70 (56.9%) had detectable HPV on both, and 40 (32.5%) were negative on both; overall concordance was 89% (95% CI 83% to 94%), and kappa statistic was 0.78 (95% CI 0.66 to 0.89). Pooled samples had a 4.1% (95% CI -1.9% to 10.0%) higher test positivity rate than dedicated samples.Of 125 participants with sufficient residual rectal and specimens, 74 (59.2%) had detectable HPV on both, and 36 (28.8%) were negative on both; overall concordance was 88% (95% CI 81% to 93%), and kappa statistic was 0.74 (95% CI 0.61 to 0.86). Residual rectal samples had 5.6% (95%CI -0.6% to 11.8%) higher test positivity than dedicated samples. CONCLUSIONS: We observed high concordance between the dedicated and residual STI test specimens. Our data support the strategy of testing residual specimens for HPV prevalence monitoring in MSM to evaluate the impact of the targeted vaccination programme.

Evidence supporting the standardisation of extragenital gonorrhoea and chlamydia screenings for women.

OBJECTIVE: Current guidelines for women do not include extragenital screening for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) and do not mention anal sex behaviour. The objective of this cross-sectional study was to determine the number of potentially missed CT and NG cases by relying on urogenital screening and self-reported anal sex behaviour among women. METHODS: Demographic and clinical data of 4658 women attending a community health centre in Los Angeles, California, USA from 2015 to 2018 were examined. CT and NG were detected using nucleic acid amplification test (APTIMA Combo 2, Hologic Gen-Probe, San Diego, California). Demographic and behavioural factors were also examined to assess potentially missed NG/CT cases. Multivariable regression analyses were used to determine whether reported anal sex behaviour predicts NG/CT rectal infection. RESULTS: A total of 193 NG cases and 552 CT cases were identified; however, 53.9% of NG cases and 25.5% of CT cases were identified exclusively through extragenital screening. Of all positive cases of rectal CT, 87.0% did not report anal sex without a condom and 91.3% did not report any anal sex with their last sexual partner. Of all positive cases of rectal NG, 78.9% did not report anal sex without a condom and 76.3% did not report any anal sex with their last sexual partner. Anal sex with last partner was not predictive of NG/CT rectal infection. CONCLUSIONS: Relying solely on urogenital screening and reported behaviour misses NG/CT cases. Extragenital NG/CT screening should be conducted in all women regardless of reported anal sex behaviour.

Pilot implementation of a home-care programme with chlamydia, gonorrhoea, hepatitis B, and syphilis self-sampling in HIV-positive men who have sex with men.

BACKGROUND: Not all men who have sex with men (MSM) at risk for sexually transmitted infections (STIs) and human immunodeficiency virus (HIV) infection currently receive sexual healthcare. To increase the coverage of high-quality HIV/STI care for MSM, we developed a home-care programme, as extended STI clinic care. This programme included home sampling for testing, combined with treatment and sexual health counselling. Here, we pilot implemented the programme in a hospital setting (HIV-positive MSM) to determine the factors for the successful implementation of STI home sampling strategies. METHODS: Healthcare providers from the HIV hospital treatment centre (Maastricht) were invited to offer free STI sampling kits (syphilis, hepatitis B, [extra]genital chlamydia and gonorrhoea laboratory testing) to their HIV-positive MSM patients (March to May 2018). To evaluate implementation of the program, quantitative and qualitative data were collected to assess adoption (HIV care providers offered sampling kits to MSM), participation (MSM accepted the sampling kits) and sampling-kit return, STI diagnoses, and implementation experiences. RESULTS: Adoption was 85.3% (110/129), participation was 58.2% (64/110), and sampling-kit return was 43.8% (28/64). Of the tested MSM, 64.3% (18/28) did not recently (< 3 months) undergo a STI test; during the programme, 17.9% (5/28) were diagnosed with an STI. Of tested MSM, 64.3% (18/28) was vaccinated against hepatitis B. MSM reported that the sampling kits were easily and conveniently used. Care providers (hospital and STI clinic) considered the programme acceptable and feasible, with some logistical challenges. All (100%) self-taken chlamydia and gonorrhoea samples were adequate for testing, and 82.1% (23/28) of MSM provided sufficient self-taken blood samples for syphilis screening. However, full syphilis diagnostic work-up required for MSM with a history of syphilis (18/28) was not possible in 44.4% (8/18) of MSM because of insufficient blood sampled. CONCLUSION: The home sampling programme increased STI test uptake and was acceptable and feasible for MSM and their care providers. Return of sampling kits should be further improved. The home-care programme is a promising extension of regular STI care to deliver comprehensive STI care to the home setting for MSM. Yet, in an HIV-positive population, syphilis diagnosis may be challenging when using self-taken blood samples.

Sudden emergence of a Neisseria gonorrhoeae clade with reduced susceptibility to extended-spectrum cephalosporins, Norway.

Neisseria gonorrhoeae multilocus sequence type (ST)-7827 emerged in a dramatic fashion in Norway in the period 2016-2018. Here, we aim to shed light on the provenance and expansion of this ST. ST-7827 was found to be polyphyletic, but the majority of members belonged to a monophyletic clade we termed PopPUNK cluster 7827 (PC-7827). In Norway, both PC-7827 and ST-7827 isolates were almost exclusively isolated from men. Phylogeographical analyses demonstrated an Asian origin of the genogroup, with multiple inferred exports to Europe and the USA. The genogroup was uniformly resistant to fluoroquinolones, and associated with reduced susceptibility to both azithromycin and the extended-spectrum cephalosporins (ESCs) cefixime and ceftriaxone. From a genetic background including the penA allele 13.001, associated with reduced ESC susceptibility, we identified repeated events of acquisition of porB alleles associated with further reduction in ceftriaxone susceptibility. Transmission of the strain was significantly reduced in Norway in 2019, but our results indicate the existence of a recently established global reservoir. The worrisome drug-resistance profile and rapid emergence of PC-7827 calls for close monitoring of the situation.

Is it time to use nucleic acid amplification tests for identification of persons with sexually transmitted infections?: evidence from seroprevalence and behavioral epidemiology risk surveys in men with chlamydia and gonorrhea.

Chlamydia and gonorrhea are common sexually transmitted infections (STIs) that can cause multiple problems, and can be easily treated, but frequently present without symptoms. Because of this, commonly used syndromic diagnosis misses a majority of infected persons. Previously, diagnostic tests were expensive and invasive, but newer nucleic-acid amplification tests (NAATs) are available that use urine to non-invasively test for these infections. These analyses used data from seroprevalence studies conducted in five militaries. Data included self-reported current symptoms of STIs as well as chlamydia and gonorrhea NAAT results. A total of 4923 men were screened for chlamydia and gonorrhea from these 5 militaries during April 2016 to October 2017. The combined prevalence of chlamydia and gonorrhea in these five militaries ranged from 2.3% in Burundi to 11.9% in Belize. These infections were not successfully identified by symptomology; for example, only 2% of cases in Belize reported symptoms. In three of the five countries there was no statistical association between symptoms and positive NAAT results. The majority of individuals with these infections (81% to 98%) would be undiagnosed and untreated using only symptomology. Therefore, using symptoms alone to diagnose cases of chlamydia and gonorrhea is not an effective way to control these infections. We propose that automated, cartridge-based NAATs, be considered for routine use in diagnosing those at risk for STIs.

Comprehensive Molecular Screening in a Cohort of Young Men Who Have Sex With Men and Transgender Women: Effect of Additive Rectal Specimen Source Collection and Analyte Testing.

BACKGROUND: This study's purposes were to characterize detection rates of several sexually transmitted infection (STI) agents and describe the effect additional specimen source and analyte screening has on STI detection within a cohort of young men who have sex with men and transgender women. METHODS: Within a 16-month interval, 1966 encounters involved dual urine and rectal swab submissions assessed by commercial transcription-mediated amplification-based assays for Chlamydia trachomatis and Neisseria gonorrhoeae and by off-label transcription-mediated amplification-based Trichomonas vaginalis and Mycoplasma genitalium testing. Identification of STI carriers used algorithms involving Food and Drug Administration-cleared screening methods, laboratory-modified testing for extraurogenital C. trachomatis and N. gonorrhoeae, and laboratory-developed tests for T. vaginalis and M. genitalium. RESULTS: Food and Drug Administration-indicated urine C. trachomatis and N. gonorrhoeae screening revealed 39 encounters (2.0%) yielding one or both agents. Via C. trachomatis and N. gonorrhoeae screening that included rectal swab analysis, 264 encounters (13.4%) yielded evidence of either (140 C. trachomatis, 88 N. gonorrhoeae) or both (36 participants) infections. Detection rates for C. trachomatis and N. gonorrhoeae were 1.4% and 0.6% for urine screening and 8.2% and 6.2% for rectal screening, respectively. Off-label screening identified 413 additional encounters with STI (5 T. vaginalis, 396 M. genitalium, 12 with both). Of these identifications, 82.1% were generated from analysis of rectal swabs (4 participants with T. vaginalis, 323 participants with M. genitalium, 12 with both). Overall detection rates of T. vaginalis (0.2% urine, 1.3% rectal) and M. genitalium (9.1% urine, 21.5% rectal) were variable. CONCLUSIONS: Additive analyte testing, including extraurogenital collections, contributes to comprehensive STI screening within a high-risk demographic.