Neisseria Gonorrhoeae Research Articles

First-line treatment with ceftriaxone for Neisseria gonorrhoeae infection less likely to be prescribed to patients with a penicillin allergy label: a retrospective audit of medical records.

Background Gonorrhoea notifications have increased substantially in Australia over the past decade. Neisseria gonorrhoeae is already highly resistant to several antibiotics and so, alternatives to first-line treatment are generally strongly discouraged. The penicillin allergy label (AL) on patient medical records has previously been shown to influence prescribing practices, to the detriment of best-practice management and antimicrobial stewardship. This study aimed to understand how the penicillin AL influences antibiotic selection for gonorrhoea treatment at Canberra Sexual Health Centre. Methods A retrospective chart audit of gonorrhoea cases treated at Canberra Sexual Health Centre between January 2020 and October 2023 (n =619 patients, n =728 cases). Antibiotic selection was assessed according to penicillin AL status. Ceftriaxone selection was assessed according to penicillin allergy severity reported in the medical records and as determined using a validated antibiotic allergy assessment tool. Results Cases with a penicillin AL were more likely to receive antibiotics other than ceftriaxone (n =7/41, 17.1%) than cases without the label (n =8/687, 1.2%, P n =28/41, 68.3%) to apply the assessment tool. Those reported as low-severity in the records were more likely to receive ceftriaxone (n =21/22, 95.5%) than those reported as moderate-high (n =7/11, 63.6%) or unreported (n =6/8, 0.75%). Conclusions Treatment of gonorrhoea in outpatient settings requires an understanding of penicillin allergy, and the ability to quickly and accurately identify penicillin-AL patients who can safely tolerate ceftriaxone. Institutionally endorsed penicillin allergy de-labelling protocols and access to easy-to-navigate prescribing advice within national sexually transmitted infection management guidelines would support this.

Gynecologic infection rates after ablation treatment for cervical intraepithelial neoplasia grade 2 and higher (CIN2+): Secondary analysis of a non-inferiority randomized trial.

Although concerns have been raised regarding potential infection and morbidity in women undergoing ablation treatment for cervical precancer in low- and middle-income countries (LMIC), there is extremely limited data to substantiate this claim. This is a secondary analysis of a randomized non-inferiority trial (id: NCT03084081) that compares the efficacy and safety of three ablation treatments for biopsy-confirmed cervical intraepithelial neoplasia grade 2 or higher (CIN2+): CO2 gas-based cryotherapy, non-gas cryotherapy, and thermal ablation (TA). Here, we present findings regarding the incidence of sexually transmitted infections (STI) and vaginitis post-treatment. Samples were collected at enrollment and again at 6 weeks post-treatment and assessed for STIs (Chlamydia trachomatis (CT), Neisseria gonorrhea (NG), and Trichomonas vaginalis (TV)) and vaginitis (Bacterial vaginosis (BV) and/or Candida albicans (Candida)). This analysis reflects 864 women with baseline and 6-week follow-up data. None of the ablative treatments put women at increased risk for STIs (CT, NG, TV) or vaginitis (BV, Candida). While most women adhered to post-treatment recommendations (97%) and no difference by treatment arm was observed, the incidence of STIs at follow-up in women that did not adhere with a given recommendation was higher compared to their adherent counterparts. The incidence of gynecologic infection did not increase with any of the ablation treatments from baseline to the six-week follow-up. Non-gas cryotherapy and TA emerge as safe alternatives to gas-based cryotherapy with respect to gynecologic infection rates.

Dual species transcriptomics reveals conserved metabolic and immunologic processes in interactions between human neutrophils and Neisseria gonorrhoeae.

Neisseria gonorrhoeae (the gonococcus, Gc) causes the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe lifelong sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs, neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing was used to define Gc and PMN transcriptional profiles alone and after infection. Core host and bacterial responses were assessed for two strains of Gc and three human donors' PMNs. Comparative analysis of Gc transcripts revealed overlap between Gc responses to PMNs, iron, and hydrogen peroxide; 98 transcripts were differentially expressed across both Gc strains in response to PMN co-culture, including iron-responsive and oxidative stress response genes. We experimentally determined that the iron-dependent TbpB is suppressed by PMN co-culture, and iron-limited Gc have a survival advantage when cultured with PMNs. Analysis of PMN transcripts modulated by Gc infection revealed differential expression of genes driving cell adhesion, migration, inflammatory responses, and inflammation resolution pathways. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and prostaglandin PGE2 were induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of two isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease.

In silico gepotidacin target mining among 33 213 global Neisseria gonorrhoeae genomes from 1928 to 2023 combined with gepotidacin MIC testing of 22 gonococcal isolates with different GyrA and ParC substitutions.

The novel dual-target triazaacenaphthylene, gepotidacin, recently showed promising results in its Phase III randomized controlled trial for the treatment of gonorrhoea. We investigated alterations in the gepotidacin GyrA and ParC targets in gonococci by in silico mining of publicly available global genomes (n = 33 213) and determined gepotidacin MICs in isolates with GyrA A92 alterations combined with other GyrA and/or ParC alterations. We examined gonococcal gyrA and parC alleles available at the European Nucleotide Archive. MICs were determined using the agar dilution method (gepotidacin) or Etest (four antimicrobials). Models of DNA gyrase and topoisomerase IV were obtained from AlphaFold and used to model gepotidacin in the binding site. GyrA A92 alterations were identified in 0.24% of genomes: GyrA A92P/S/V + S91F + D95Y/A/N (0.208%), A92P + S91F (0.024%) and A92P (0.003%), but no A92T (previously associated with gepotidacin resistance) was found. ParC D86 alterations were found in 10.6% of genomes: ParC D86N/G (10.5%), D86N + S87I (0.051%), D86N + S88P (0.012%) and D86G + E91G (0.003%). One isolate had GyrA A92P + ParC D86N alterations, but remained susceptible to gepotidacin (MIC = 0.125 mg/L). No GyrA plus ParC alterations resulted in a gepotidacin MIC > 4 mg/L. Modelling of gepotidacin binding to GyrA A92/A92T/A92P suggested that gepotidacin resistance due to GyrA A92T might be linked to the formation of a new polar contact with DNA. In silico mining of 33 213 global gonococcal genomes (isolates from 1928 to 2023) showed that A92 is highly conserved in GyrA, while alterations in D86 of ParC are common. No GyrA plus ParC alterations caused gepotidacin resistance. MIC determination and genomic surveillance of potential antimicrobial resistance determinants are imperative.

A novel murine model mimicking male genital Neisseria species infection using Neisseria musculi†.

With ~78 million cases yearly, the sexually transmitted bacterium Neisseria gonorrhoeae is an urgent threat to global public health due to continued emergence of antimicrobial resistance. In the male reproductive tract, untreated infections may cause permanent damage, poor sperm quality, and subsequently subfertility. Currently, few animal models exist for N. gonorrhoeae infection, which has strict human tropism, and available models have limited translatability to human disease. The absence of appropriate models inhibits the development of vital new diagnostics and treatments. However, the discovery of Neisseria musculi, a mouse oral cavity bacterium, offers much promise. This bacterium has already been used to develop an oral Neisseria infection model, but the feasibility of establishing urogenital gonococcal models is unexplored. We inoculated mice via the intrapenile route with N. musculi. We assessed bacterial burden throughout the male reproductive tract, the systemic and tissue-specific immune response 2-weeks postinfection, and the effect of infection on sperm health. Neisseria musculi was found in penis (2/5) and vas deferens (3/5) tissues. Infection altered immune cell counts: CD19+ (spleen, lymph node, penis), F4/80+ (spleen, lymph node, epididymus), and Gr1+ (penis) compared with noninfected mice. This culminated in sperm from infected mice having poor viability, motility, and morphology. We hypothesize that in the absence of testis infection, infection and inflammation in other reproductive is sufficient to damage sperm quality. Many results herein are consistent with outcomes of gonorrhoea infection, indicating the potential of this model as a tool for enhancing the understanding of Neisseria infections of the human male reproductive tract.

Are Chlamydia Trachomatis and Neisseria Gonorrhoeae Screenings in Pregnant Women Being Properly Performed? A Single-Center Retrospective Observational Study in Italy.

A new Italian intersociety position statement on the prevention of ophthalmia neonatorum was published in 2023. In this document, attention was paid to the indications for the screening of gonococcal and chlamydial infections during pregnancy according to the international and national guidelines for the prevention of sexually transmitted infections (STIs). We conducted an observational retrospective study to assess whether the current guidelines for the prevention of STIs are being followed correctly. From February to August 2022, 2507 women nearing childbirth were enrolled. Among them, 42.4% received a swab for Chlamydia and only 0.5% for gonococcus. Concerning the geographical area of origin, most of the screened women came from Western Europe. None of the women who received gonococcal swabs and only 105 women out of 1062 screened for Chlamydia were under 25 years of age. Overall, only seven swabs were positive for Chlamydia, while none were positive for gonococcus. Concerning the age, geographical area of origin, and medical history of the women with a positive screening for Chlamydia, all were over 25 years old, six were from Western Europe, one was from South America, and none had other STIs. Although monocentric in nature, this study shows that the guidelines are not being followed correctly.

Revolutionizing chronic endometritis diagnosis: real-time polymerase chain reaction unveils microbial pathogens in Indian women with abnormal bleeding and reproductive challenges

BackgroundThis study aimed to assess the utility of real-time-polymerase chain reaction (PCR) for diagnosing chronic endometritis (CE) by targeting 11 prevalent pathogens and to compare the outcomes with conventional culture-based diagnosis. Study DesignA retrospective analysis was conducted on 500 patients with clinical conditions such as abnormal bleeding, in vitro fertilization failure, recurrent implantation failure, recurrent miscarriage, and recurrent pregnancy loss. The prevalence of 11 key pathogens associated with CE was evaluated in endometrial biopsy samples. ResultsIn our study, PCR identified 318 cases (63.6%) positive for at least one of the 11 investigated pathogens, while culture-based methods detected 115 cases (23%). Predominant pathogens detected by PCR included Enterococcus faecalis (E. faecalis) (19%), Escherichia coli (E. coli) (6.8%), Staphylococcus aureus (S. aureus) (9%), Mycoplasma hominis (5%), Mycoplasma genitalium (6.2%), Streptococcus agalactiae (S. agalactiae) (4.2%), Ureaplasma urealyticum (4%), nontuberculous Mycobacterium (5.2%), Mycobacterium tuberculosis (1.2%), Neisseria gonorrhoeae (0.6%), and Chlamydia trachomatis (2.4%). Standard culture methods identified E. faecalis (10.8%), S. aureus (6.2%), E. coli (3.8%), and S. agalactiae (2.2%). ConclusionThe DICE panel proves itself as a swift, precise, and cost-effective diagnostic tool for detecting both culturable and nonculturable endometrial pathogens in CE. Demonstrating superiority, the Molecular method outshines microbial culture, ensuring accurate and sensitive detection of CE-associated pathogens, harmonizing seamlessly with histology and hysteroscopy findings.

Gonococcal Mimitope Vaccine Candidate Forms a Beta-Hairpin Turn and Binds Hydrophobically to a Therapeutic Monoclonal Antibody.

The spread of multidrug-resistant strains of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, represents a global health emergency. Therefore, the development of a safe and effective vaccine against gonorrhea is urgently needed. In previous studies, murine monoclonal antibody (mAb) 2C7 was raised against gonococcal lipooligosaccharide (LOS). mAb 2C7 elicits complement-dependent bactericidal activity against gonococci, and its glycan epitope is expressed by almost every clinical isolate. Furthermore, we identified a peptide, cyclic peptide 2 (CP2) that mimicked the 2C7 LOS epitope, elicited bactericidal antibodies in mice, and actively protected in a mouse vaginal colonization model. In this study, we performed structural analyses of mAb 2C7 and its complex with the CP2 peptide by X-ray crystallography, NMR spectroscopy, and molecular dynamics (MD) simulations. The crystal structure of Fab 2C7 bound to CP2 showed that the peptide adopted a beta-hairpin conformation and bound the Fab primarily through hydrophobic interactions. We employed NMR spectroscopy and MD simulations to map the 2C7 epitope and identify the bioactive conformation of CP2. We also used small-angle X-ray scattering (SAXS) and native mass spectrometry to obtain further information about the shape and assembly state of the complex. Collectively, our new structural information suggests strategies for humanizing mAb 2C7 as a therapeutic against gonococcal infection and for optimizing peptide CP2 as a vaccine antigen.

Automating the Generation of Notifiable Bacterial Disease Reports: Proof-of-Concept Study and Implementation in Six Hospitals in Thailand.

Information on notifiable bacterial diseases (NBD) in low- and middle-income countries (LMICs) is frequently incomplete. We developed the AutoMated tool for the Antimicrobial resistance Surveillance System plus (AMASSplus), which can support hospitals to analyze their microbiology and hospital data files automatically (in CSV or Excel format) and promptly generate antimicrobial resistance surveillance and NBD reports (in PDF and CSV formats). The NBD reports included the total number of cases and deaths after Brucella spp., Burkholderia pseudomallei, Corynebacterium diphtheriae, Neisseria gonorrhoeae, Neisseria meningitidis, nontyphoidal Salmonella spp., Salmonella enterica serovar Paratyphi, Salmonella enterica serovar Typhi, Shigella spp., Streptococcus suis, and Vibrio spp. infections. We tested the tool in six hospitals in Thailand in 2022. The total number of deaths identified by the AMASSplus was higher than those reported to the national notifiable disease surveillance system (NNDSS); particularly for B. pseudomallei infection (134 versus 2 deaths). This tool could support the NNDSS in LMICs.

Bacterial Profile of Ocular Infections: A Mini Review

Ocular infections are a growing global health concern, affecting millions worldwide. This review explores the bacterial profile of various ocular infections to aid in developing effective treatments and preventive strategies. The eye's complex anatomy, including the cornea, pupil, iris, lens, and retina, makes it susceptible to infections that can lead to vision loss. Bacteria are a leading cause of these infections, with common culprits including Escherichia coli, Streptococcus, Neisseria gonorrhoeae, and Staphylococcus aureus. Environmental factors, allergies, and poor hygiene can exacerbate the risk. Treatment options range from antibiotics and antivirals to eye drops and surgery. Understanding the bacterial profile of ocular infections is crucial for targeted interventions and improved public health outcomes.

Global trends of antimicrobial resistance rates in Neisseria gonorrhoeae: a systematic review and meta-analysis.

Antimicrobial resistance (AMR) of Neisseria gonorrhoeae (NG) is a significant public health concern. The objective of our study was to assess global AMR rates and test them both temporally and geographically. We conducted a systematic search of relevant reports from international databases up to 2021. The R statistical package was used for all statistical analyses. A total of 225 articles were analyzed, and 432,880 NG isolates were examined. The weighted pooled resistance (WPR) rate of different antibiotics was as follows: ciprofloxacin, 51.6%; tetracycline, 45.4%; trimethoprim/sulfamethoxazole, 42.4%; chloramphenicol, 4.1%; kanamycin, 2.1%; gentamicin, 0.6%; and spectinomycin, 0.3%. The resistance to spectinomycin, gentamicin, and kanamycin decreased over time. Significant differences in antibiotic resistance rates were found between the countries. Our findings reveal a continuous increase in resistance to some antibiotics (tetracycline and ciprofloxacin) historically used for gonorrhea, even after discontinuation. However, encouraging trends of decreasing resistance to spectinomycin, gentamicin, and kanamycin were observed. Continued global monitoring of AMR profiles in NG isolates is essential for informing appropriate treatment strategies and mitigating the threat of untreatable gonorrhea.

Impact of point-of-care gonorrhea and chlamydia testing in the emergency department on reducing overtreatment rates

BackgroundChlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections continue to increase in the United States. Advancement in technology with point-of-care (POC) testing can improve the overall treatment of sexually transmitted infections (STI) in the emergency department (ED) by shortening the time to test result and administration of accurate treatment. The purpose of this study was to assess if the POC test reduced the rate of overtreatment for CT and/or NG compared to the standard-of-care (SOC) test. MethodsThis retrospective cohort study included adult patients tested for CT and NG at two urban EDs between August 2020 and October 2022. This cohort excluded hospital admissions, elopement, pregnancy, rectal and oral samples, victims of sexual assault, and diagnoses for which antimicrobial treatment overlapped that of CT/NG. The primary outcome assessed overtreatment, defined as receiving treatment in the ED or a prescription prior to discharge for patients who tested negative for CT and/or NG. Secondary outcomes included undertreatment rates, overtreatment rates in select populations, test turnaround time, and ED length of stay (LOS). ResultsOf 327 patients screened, 97 patients were included in the SOC group and 100 in POC. Overtreatment for CT was provided in zero POC patients and 29 (29.9%) SOC patients (p < 0.001). NG was overtreated in 1 (1%) POC and 23 (23.7%) SOC (p < 0.001). POC was associated with undertreatment of CT and/or NG in two patients, compared to four patients tested with SOC. Overall, treatment was deemed inappropriate for 5 (5%) of those tested with POC, compared to 35 (36%) tested with SOC (p < 0.001). There was no difference in ED LOS (2.7 vs 3.01 h, p = 0.41). ConclusionsPOC testing facilitated the return of results prior to patients being discharged from the ED. Compared to standard testing, POC improved appropriateness of CT and NG treatment by reducing the rates of overtreatment.

Susceptibility of Neisseria gonorrhoeae against a dual treatment antibiotics regimen in primary health centres in Surabaya, Indonesia.

There were 82.4 million new gonorrhoea cases worldwide in 2020. Dual treatment with ceftriaxone or cefixime and azithromycin or doxycycline is currently recommended for gonorrhoea in Indonesia. However, reduced susceptibility and resistance to cephalosporins and azithromycin are increasing. We evaluated the susceptibility pattern of Neisseria gonorrhoeae to cefixime, ceftriaxone, azithromycin and doxycycline. N. gonorrhoeae isolates were obtained from 19 male participants with clinically and laboratory-confirmed gonorrhoea. Antibiotic susceptibility testing was conducted by disc diffusion and interpreted according to Clinical and Laboratory Standards Institute and Centers for Disease Control and Prevention criteria. Reduced susceptibility or resistance was observed against doxycycline in 19 isolates (100%), cefixime in six (31.6%), ceftriaxone in three (15.8%) and azithromycin in zero (0%) isolates. A dual treatment regimen with ceftriaxone and azithromycin can still be recommended as first-line therapy for gonorrhoea in Indonesia. Antibiotic susceptibility surveillance of N. gonorrhoeae should be routinely conducted.

World Health Organization Enhanced Gonococcal Antimicrobial Surveillance Programme, Cambodia, 2023.

To determine antimicrobial susceptibility of Neisseria gonorrhoeae, we analyzed phenotypes and genomes of 72 isolates collected in Cambodia in 2023. Of those, 9/72 (12.5%) were extensively drug resistant, a 3-fold increase from 2022. Genomic analysis confirmed expansion of newly emerging resistant clones and ongoing resistance emergence across new phylogenetic backbones.

Erratum for Balthazar et al., "A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase".

Erratum for Balthazar et al., "A laboratory-based predictive pathway for the development of Neisseria gonorrhoeae high-level resistance to corallopyronin A, an inhibitor of bacterial RNA polymerase".

A Study of Sexually Transmitted Infections Among Patients Attending Suraksha Clinic &amp; Targeted Intervention Clinics In Nainital District: A Cross-Sectional Survey

Background: Sexually transmitted infections (STIs) pose a global health threat, particularly among vulnerable populations. Aligned with the National AIDS Control Program, this study focuses on estimating the prevalence of STIs in India, targeting Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, and Trichomonas vaginalis. The aim is to identify gaps, understand factors influencing STI screening, and propose improvements. Objectives: Uncover barriers in STI screening, diagnosis, and management at Suraksha and targeted intervention clinics. Identify socio-demographic factors associated with screening barriers. Methodology: Sample unit: Patients at Suraksha and TI clinics. Sample size: 577 participants, including community-surveyed and health camp attendees. Sampling design: Complete enumeration. Cross-sectional survey in specified Haldwani and Nainital clinics. Results: Age group 20-40 exhibits higher symptomatic individuals, underscoring the need for screening in young and middle-aged adults. Only 55% of positive cases sought treatment, citing stigma, mild symptoms, and unawareness. Health workers, especially spouses, play a crucial role in testing and counseling. Delayed presentation at health facilities due to long waiting times, mild symptoms, lack of awareness, and distance. Conclusions: While the program succeeds in various aspects, infrastructure improvement, heightened awareness, and addressing stigma are crucial. Recommendations include more staff, wider service publicity, and better availability of essential resources.

Modelling molecular and culture-based surveillance of tetracycline resistance in Neisseria gonorrhoeae

Modelling molecular and culture-based surveillance of tetracycline resistance in Neisseria gonorrhoeae

The use of an imperfect vaccination and awareness campaign in the control of antibiotic resistant gonorrhoea infection: A mathematical modelling perspective

The multi drug-resistant Neisseria gonorrhoeae has been classified by the World Health Organisation (WHO) as a high-priority global public health problem. This underlines the need for better understanding of the transmission dynamics and proposing an optimal intervention strategy to control the disease. In this article, a deterministic mathematical model for the transmission dynamics of gonorrhoea as an antibiotic resistant disease in a population with an imperfect vaccination is proposed and analysed. The model incorporates the classes of vaccinated individuals and individuals equipped with self protection interventions to reduce antibiotic resistant cases. The threshold parameter R0, the basic reproduction number, for the analysis of the model is calculated. In the given setting, the model exhibits a backward bifurcation for R0<1. However, if the efficacy of the vaccine is 100% without a waning effect, the model is shown to be without a backward bifurcation and the disease-free equilibrium is globally asymptotically stable whenever R0<1. The global sensitivity analysis of the model to variations in parameter values is also performed to determine the most influential parameters on the disease transmission. Moreover, the optimal control analysis of the full model is presented and the optimal intervention strategies are proposed. The proposed intervention strategies are shown to be able to control the disease within a relatively shorter period of time. Finally, numerical experiments are presented to support the theoretical analysis of the model.

The Prevalence of Sexually Transmitted Infections among Male Patients at a Dermato-Venereology Outpatient Clinic in Gdańsk, Poland: Findings from a Single-Center Study.

Background: Sexually transmitted infections (STIs) are a significant public health concern worldwide, yet data on their prevalence and epidemiology, particularly in Central and Eastern Europe, remain scarce. This study aimed to assess the prevalence, anatomical localization, symptomatic/asymptomatic course, and co-infection patterns of STIs among men. Methods: This retrospective study analyzed data collected between May 2021 and July 2023, including sociodemographic, sexual behavior, and clinical data from 139 male participants. Molecular polymerase chain reaction (PCR) tests were conducted for Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium, and Trichomonas vaginalis. Results: Of the participants, 36% tested positive for at least one STI, with the urethra being the most common site of infection. NG and CT were the most prevalent infections. The majority of infections were asymptomatic, highlighting the importance of comprehensive screening, especially in high-risk populations like men who have sex with men (MSM). Conclusions: This study emphasizes the need for targeted screening strategies, particularly for extragenital STIs, and underscores the role of MSM in STI epidemiology. The findings highlight the importance of routine screening, even for asymptomatic individuals, to effectively control STI spread. Future research should validate and expand upon these findings to enhance STI prevention and management efforts.

The Prevalence of Antibiotic Tolerance in Neisseria gonorrhoeae Varies by Anatomical Site.

Tolerance enables bacteria to survive intermittent antibiotic exposure without an increase in antimicrobial susceptibility. In this study, we investigated the presence of tolerance to three antimicrobials, ceftriaxone, azithromycin and ciprofloxacin, in clinical isolates and the WHO (World Health Organization) reference panel of Neisseria gonorrhoeae. We used the modified tolerance disk (TD test) to assess for tolerance to ceftriaxone, azithromycin and ciprofloxacin in 14 WHO reference strains and 62 N. gonorrhoeae clinical isolates-evenly divided between anorectal and urogenital infections. The isolates underwent a three-step incubation process wherein the isolates were exposed to an antibiotic disk for 20 h of incubation (Step I), followed by the replacement of the antibiotic disk with a nutrient disk for overnight incubation (Step II) and additional overnight incubation with extra nutrients (Step III). A total of 4 of the 62 clinical anorectal isolates and none of the urogenital isolates exhibited tolerance to azithromycin (p = 0.033). Tolerance to ceftriaxone and ciprofloxacin was observed in eight and four isolates, respectively, with no difference between infection sites. Tolerance was also detected in 8 (K, M, N, O, P, U, V, W) out of the 14 WHO reference strains, with varying patterns of tolerance to ceftriaxone (n = 8), ciprofloxacin (n = 2) and azithromycin (n = 1). This study identified ceftriaxone, azithromycin and ciprofloxacin tolerance in clinical and WHO reference N. gonorrhoeae isolates. Azithromycin tolerance was more common in anorectal than urogenital infections.