Gonococci – Pathogens of Growing Importance. Part 1. Current Data on Diagnostics, Genotyping and Therapy.

Microscopically, N. gonorrhoeae is a Gram-negative diplococcus with a diameter of 0.6 to 1 μm, arranged in characteristic pairs resembling coffee beans. It does not have a capsule and does not produce spores.

Direct Gram-stained microscopic preparation should always be performed for purulent or mucopurulent urethral discharge in men, a symptom of gonococcal urethritis. Observing numerous polymorphonuclear leucocytes (PMNs) and Gram-negative gonococci within or closely associated with some PNMs in the microscopic image is diagnostically significant and allows us to identify gonococcal infection in clinically symptomatic men. In this case, if the preparation is appropriately performed and evaluated by an experienced microbiologist, the microscopic examination has high sensitivity (90–95%) and specificity (95–100%) (Janda and Gaydos 2007). However, direct microscopic examination has shown low sensitivity (< 55%) in diagnosing asymptomatic men. It is also the case for the diagnosis of cervicitis (sensitivity < 50%) and rectal inflammation (sensitivity < 40%). Hence, direct examination of Gram-stained preparation of smears from the vagina, cervical canal, pharynx, or rectum has no diagnostic significance (Bignell et al. 2013). Methylene blue (M.B.) stain may also establish the intracellular location of gonococci in leucocytes (PMNs) of male urethral exudate accompanying gonococcal urethritis (Workowski 2021).

N. gonorrhoeae is an aerobic bacterium with high nutritional requirements, is oxidase- and catalase-positive and oxidizes only glucose to acid. It requires an energy source of glucose, pyruvate or lactate and the presence of cysteine to grow on culture media. Because of changes in metabolic pathways, some isolates show special growth requirements for amino acids, purines, and pyrimidines (Ng and Martin 2005; Quillin et al. 2018).

The culture of gonococci requires special nutrientrich growth media and selective media that inhibit the growth of other microorganisms on human mucous membranes. The pathogen grows at a temperature of 35–37°C in an atmosphere with up to 5–10% CO₂ and increased humidity. Gonococci outside the host are sensitive to environmental factors. They die within hours at temperatures above 40°C. The bacteria are sensitive to drying, light and low concentrations of disinfectants. Collected clinical materials should be inoculated into culture media as soon as possible. Culture media with added animal protein are used, primarily chocolate agar, modified Thayer-Martin (MTM) medium, Roiron medium, Martin-Lewis (ML) medium, GC-Lect agar and New York City (NYC) agar. MTM, ML, and GC-Lect media based on chocolate agar are supplemented with growth factors (e.g., IsoVitalex, PolyVitex, and Kellog supplement). NYC medium is a transparent medium based on peptone, corn starch with yeast extract, horse plasma and lyophilized horse erythrocytes. Roiron medium contains, among others, enzymatic casein hydrolysate, horse plasma and glucose. Antibiotics and antimycotics are added to selective media to inhibit the growth of commensal microorganisms present in clinical materials. Vancomycin and colimycin inhibit Gram-positive and Gram-negative microorganisms, respectively, trimethoprim Proteus species, while nystatin, amphotericin B or anisomycin have antifungal effects (Janda and Gaydos 2007).

For urogenital tract specimens, cultures for N. gonorrhoeae are usually conducted in parallel on Columbia agar with the addition of 5% sheep blood, chocolate agar without any selection factors, and selective medium with the selection factors mentioned above. An example of a selective medium is chocolate agar with polyvitex with the addition of vancomycin, colimycin, amphotericin and trimethoprim (Chocolate + PVS + VCAT). Basal media without the addition of antibiotics are necessary to ensure the growth of those few N. gonorrhoeae strains that show sensitivity to antibiotics present in selective media. On chocolate agar, gonococci grow after a 24- to 48-hour culture as small, round, smooth colonies of greyish-beige colour or clear and shiny, 0.5 to 2 mm in diameter. Their morphology depends on the number of in vitro passages. Fine, elevated colonies are associated with the production of pili.

Further morphotypes appear during passages on solid media, variants of larger diameter, flat colonies, often with irregular edges. Under the influence of phase changes of Opa surface proteins, dull and iridescent variants appear. Due to the different types of colonies, cultures on chocolate agar can give the impression of a mixed culture (Janda and Gaydos 2007).

After a 24–48 hour incubation, the characteristic small grey-beige or transparent colonies of N. gonorrhoeae grown on chocolate agar or selective medium require isolation to identify the species level and determine drug susceptibility. Identification by classical microbiological methods is based on the evaluation of colony morphology on chocolate medium, finding of Gram-negative diplococci in a Gram-stained slide, confirmation of the ability to produce oxidase, catalase, and the results of biochemical tests, considering carbohydrate degradation and enzymatic activity. The bacterium oxidizes glucose and does not metabolize maltose, sucrose, fructose or lactose. It shows proli-iminopeptidase (PIP) activity, while it does not have β-galactosidase nor γ-glutamylaminopeptidase activity, unlike N. lactamica and N. meningitidis. The specific enzymatic activity is used to identify N. gonorrhoeae in manual enzyme chromogenic assays such as API NH – bioMerieux, Gonochek II – TCS Biosciences, Neisseria PET – BioConnections (Alexander et al. 2005; Meyer and Buder 2020). The automated system VITEK 2 (bioMerieux) also uses biochemical traits for species identification of N. gonorrhoeae. A modern identification method by which gonococci can be reliably identified is MALDI-TOF (Matrix-Assisted Laser Desorption/ Ionization – Time Of Flight) mass spectrometry (Ilina et al. 2009; Cassagne et al. 2011; Carannante et al. 2015; van Belkum et al. 2013; Morrel et al. 2018), which analyses the protein profile, mainly of the microbe’s ribosomal proteins. 16S rRNA gene sequencing can also be used for species identification of N. gonorrhoeae. A fragment of the rplF gene (413 pz), encoding the L6 50S ribosomal protein, is a suitable genetic target for differentiating this species (Bennett et al. 2014).

According to the Centers for Disease Control and Prevention (CDC), NAAT methods are currently recommended for diagnosing and screening gonococcal infections among men and women (Table I). They have high sensitivity and specificity and the added advantage of simultaneously detecting other sexually transmitted diseases (Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis). Examples of NAATs that detect N. gonorrhoeae approved by the Food and Drug Administration (FDA) for clinical use are shown in Table I (CDC 2014; Low and Unemo 2016). Recommendations for transporting protected swabs for gonorrhoea diagnosis by molecular methods are less restrictive compared to culture methods. Any delay in inoculation of materials into appropriate microbiological media can adversely affect the result of N. gonorrhoeae culture.

In contrast, this is not the case with NAAT methods. Longer sample storage time does not impair the method sensitivity. The very high sensitivity of molecular methods, associated with the ability to multiplicate even single copies of target DNA or RNA, allows diagnostic use of samples with low bacterial loads, such as the initial urine stream or vaginal swabs. On the other hand, due to the high sensitivity, there is the possibility of false-positive results, such as in cases of crossreactions with genetic material from other species of the genus Neisseria or patients just after treatment, due to the persistence of the pathogen’s DNA in the body. With molecular techniques, it is essential to strictly control the course of laboratory procedures to ensure the reliability of test results.

Most importantly, all safeguards should be used to prevent contamination of the laboratory environment with pathogen amplicons, the presence of which can be a source of false positives. Control of the efficiency of isolation, amplification and detection process of the pathogen’s DNA is ensured by using so-called positive controls, and each time, the inclusion of negative control, i.e. sterile water, verifies the possibility of contamination of, for example, reagents or the environment. To date, the development and good standardization of molecular methods have improved the diagnosis of gonococcal infections but have not enabled routine determination of drug susceptibility.

Antimicrobial resistance (AMR) of N. gonorrhoeae has a multifactorial basis, both chromosomal and plasmid-mediated (Unemo et al. 2016; Unemo and Shafer 2014). The drug susceptibility profiles of gonococci and plasmid and chromosomal resistance prevalence vary by geographic region and change over time. The increasing antimicrobial resistance of the pathogen is a global problem and requires constant monitoring (Tapsall et al. 2009; Town et al. 2020). With the introduction of sulfonamides and then penicillin for the treatment of gonorrhoea in the 1930s, N. gonorrhoeae has acquired resistance to successive groups of antibiotics formerly used in therapeutic regimens: sulfonamides, penicillin, tetracyclines, fluoroquinolones (Ohnishi et al. 2011; Unemo and Shafer 2014; Grad et al. 2016). Third-generation cephalosporins, along with azithromycin or tetracycline, appeared in treatment recommendations, but the use of these antibiotics may soon prove less effective or even ineffective (Grad et al. 2016; Martin et al. 2016; Martin et al. 2019). Strains with a wild-type phenotype regarding drug susceptibility, which do not show resistance or reduced sensitivity to antibiotics, have become increasingly rare. Multidrug-resistant strains emerge worldwide, such as those with Multi Drug Resistance (MDR) or Extensively Drug-Resistant (XDR) phenotypes. N. gonorrhoeae strains with the MDR phenotype are defined as resistant or with reduced sensitivity to one of the antibiotics recommended for current empiric therapy (i.e., cefixime, ceftriaxone or azithromycin) with resistance to two other therapeutic groups (e.g., fluoroquinolones or tetracyclines). In contrast, strains with extensive drug resistance, XDR N. gonorrhoeae, are understood to be resistant or with reduced sensitivity to both recommended drugs, with concurrent resistance to two other antibiotic groups (Tapsall et al. 2009). In 2011, the first case of treatment failure of gonorrhoeal pharyngitis caused by a strain with the XDR phenotype was described in Japan (Ohnishi et al. 2011). Europe’s first ceftriaxone-resistant gonococcal strains were isolated in France in 2010 (Unemo et al. 2012) and Spain in 2011 (Carnicer-Pont et al. 2012). They showed phenotypic and genotypic similarity (NG-MAST 1407, MLST ST1901). In 2018, XDR strains emerged in the U.K. and Australia, resistant to ceftriaxone and with high azithromycin resistance (> 256 mg/l) (Eyre et al. 2017; Whiley et al. 2018; Jennison et al. 2019). Due to the inreasing resistance of gonococci, the WHO presented the “Global Action Plan to Control the Spread and Prevent Antibiotic Resistance of N. gonorrhoeae “ (WHO 2012), and in 2017, N. gonorrhoeae was included in the list of bacterial pathogens for which there is a particular need for new drugs (WHO 2021). The drug susceptibility of gonococci is monitored mainly through widely conducted national and international surveillance programs: WHO-Global GASP (Gonococcal Antimicrobial Surveillance Programme) (Unemo et al. 2019), Euro GASP (Cole et al. 2013), US-GISP (Gonococcal Isolate Surveillance Project) (Grad et al. 2016), Canadian GASP (Martin et al. 2019), Australian AGSP (Australian Gonococcal Surveillance Programme) (Lahra et al. 2015), UK GRASP (Gonococcal Resistance to Antimicrobials Surveillance Programme) (Unemo et al. 2020).

Over the past 30 years, several methods have been developed to analyze the genetic diversity of N. gonorrhoeae strains. They aim to identify polymorphisms at a single locus, several loci, or even the entire genome. The variability of gonococcal strains was assessed based on patterns of electrophoretically separated strands of amplified DNA, which allowed the demonstration of, among others, different plasmid profiles (PPA – Plasmid Profile Analysis), restriction fragment length polymorphism (RFLP/PFGE pulse electrophoresis method), ribotyping and Opa protein typing (Unemo and Dillon 2011). The use of sequencing techniques allowed the differentiation of gonococci initially for the porB gene (Bash et al. 2005; Liao et al. 2009), and then, after expanding the scope, gave the possibility to type several loci. Nowadays the most popular molecular typing schemes are MLST (Maiden et al. 1998), NG-MAST (Martin et al. 2004), NG-STAR (Demczuk et al. 2017). The whole genome sequencing method (WGS) is now playing an increasingly important role in epidemiological and phylogenetic studies, as well as in monitoring antibiotic resistance of gonococci. This method certainly facilitates surveillance of circulating N. gonorrhoeae strains in an epidemiological context (Eyre et al. 2018; Harris et al. 2018; Sánchez-Busó et al. 2019; Sánchez-Busó et al. 2022). The Multi-Locus Sequence Typing (MLST) method is based on the sequencing of 7 housekeeping genes: abcZ (putative ABC transporter), adenylate kinase (adk), aroE (shikimate dehydrogenase) , fumC (fumarate hydratase), gdh (glucose-6-phosphate dehydrogenase), pdhC (pyruvate dehydrogenase subunit), pgm (phosphoglucomutase) (Maiden 2008). Sequence type relatedness analysis allowed us to define MLST CC clonal complexes grouping related clonal lines. With the same scheme being used to type other species in the human genus Neisseriaceae, the pathogenic N. meningitidis and the commensal N. lactamica, the MLST database is a valuable source of data for genetic analyses within the genus and broad studies in an evolutionary context over the long term (Maiden 2008; Harrison et al. 2020). As of June 2022, the Pub-MLST database contained 16700 ST types for the genus Neisseria. The PubMLST website is maintained by the Department of Zoology at Oxford University (Jolley et al. 2018). N.gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) method is based on the sequencing of two highly variable loci within the porB (490 bp) and tbpB (390 bp) genes, which encode two N. gonorrhoeae membrane proteins: porin B (PorB) and transferrin binding protein (tbpB), respectively. The combination of both loci allows the determination of the Sequence Type (ST). NG-MAST STs are grouped into geno-groups for phylogenetic and epidemiological analysis (Chisholm et al. 2013). The NG-MAST method is widely used worldwide. The website https://pubmlst.org contains the current database for typing with this method. As of May 2023, 12735 porB and 3147 tbpB alleles and 21490 ST profiles of N. gonorrhoeae have been published on the site (Jolley et al. 2018). N. gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) is the typing scheme that considers seven chromosomal determinants of N. gonorrhoeae resistance to β-lactam antibiotics, macrolides, fluoroquinolones and tetracyclines. The method requires sequencing of the following loci: penA, mtrR, porB, ponA, gyrA, parC and 23S rRNA and is consistent with classical Sanger sequencing and WGS (Demczuk et al. 2017). The unique sequence of each gene is assigned an allele number, while the combination of alleles of seven genes allows to determine the sequence type of NG-STAR. Sequences can be analyzed and compared through an online application in a publicly available database (https://ngstar.canada.ca/) maintained by the Public Health Agency of Canada and the National Microbiology Laboratory. As of May 2023, 5259 ST types of N. gonorrhoeae have been published on the NG-STAR database website. NG-STAR typing allows analysis of strain relatedness, tracking of clonal lineage development and monitoring of antibiotic resistance (Golparian et al. 2021).

Today’s challenge is to develop a quicker, more straightforward, and more sensitive diagnostic method to detect N. gonorrhoeae infection (Bignell et al. 2013; Li et al. 2019). Both standard methods for identifying N. gonorrhoeae – NAAT or culturing of the pathogen prove expensive and require specialized laboratory equipment, making its use in low- and middle-income countries much limited or impossible.

In 2004, the WHO drew attention to the urgent need for rapid diagnostic tests for bacterial sexually transmitted diseases. The ASSURED criteria were established to determine the characteristics that such a diagnostic test should have. First and foremost, it is to be affordable, sensitive and specific, avoiding false results. In addition, it is to be user-friendly, i.e., simple to perform, with a minimum number of steps and requiring no advanced training. Moreover, the test must be fast and durable to survive various transportation conditions. It should also require no additional equipment (Land et al. 2019).

Rapid gonococcal diagnostic tests are emerging on the market. Such tests allow bypassing the multi-step and lengthy identification of the pathogen. Most often, they consist of a carrier such as a cassette, frequently containing all the reagents necessary for analysis, in which the collected sample is placed directly and inserted into the appropriate analyzer. These tests allow rapid identification of the pathogen for up to 30 minutes. However, they have limitations. First, not every test will prove suitable for every type of sample.

One rapid diagnostic test is Xpert CT/NG, which uses the GeneXpert platform (Cepheid). It is a realtime in vitro PCR performed directly from the collected sample. The FDA approves the test for use in urine, vaginal, cervical, rectal, and pharynx samples. It shows a sensitivity and specificity between 86–99% (Herbst De Cortina et al. 2016; Doernberg et al. 2019). Another rapid test is binx health io CT/NG (binx health, INC), a PCR-based qualitative test consisting of a stationary device and easy-to-use coffers. The waiting time for the result is about 30 minutes. As of 2019, the FDA has authorized the use of this test in vaginal swab samples. In vaginal samples, its high sensitivity and specificity of 96–97.7% was stated (Van Der Pol and Gaydos 2021; Gaydos et al. 2013). The Truelab Preal Time micro PCR system (Molbio Diagnostics Pvt Ltd) is a rapid semi-quantitative PCR-based test. The entire process involves sample collection and an automated extraction system. Then, the nucleic acid is transferred to an analyzer chip using a fluorophore-capturing optical sensor for determination. Analysis results are available after about an hour. The test is not FDA-certified (Nair et al. 2016). The STI Array test (Randox Biosciences) requires the Vivalytic Analyzer (Bosch Healthcare Solutions), a device that enables quantitative PCR reaction, to perform the assay. It comes with a coffer containing all the necessary reagents for the analysis (Adamson et al. 2020). A different type of assay is loop-mediated isothermal amplification (LAMP). It is a reliable, low-cost, sensitive, rapid technique for amplifying nucleic acids. It has been widely used to identify pathogens, including SARS-CoV-2, Mycobacterium tuberculosis and Brucella (Shete et al. 2019). DNA polymerase in the LAMP method can effectively amplify target genes at 58–69°C (Notomi et al. 2000). The N. gonorrhoeae - LAMP-PNB assay combines isothermal amplification with a polymer nanoparticle-based biosensor. The entire identification process takes about 60 minutes (Wong et al. 2018; Chen et al. 2021). The documented development of rapid diagnostic methods for N. gonorrhoeae infection is insufficient. There is still a need for specific, sensitive assays that are simple and inexpensive.

According to current European recommendations for treating uncomplicated gonorrhoea in adults, ceftriaxone is administered intramuscularly in a single dose of 1 g (Unemo et al. 2021). In CDC recommendations for anogenital and pharyngeal infections, the dose of ceftriaxone depends on the patient’s body weight (500 mg for individuals < 150 kg and 1 g for individuals > 150 kg). If chlamydial infection has not been excluded, chlamydia should be treated with doxycycline 100 mg orally twice daily for seven days, except for pregnant patients for whom a single dose of azithromycin 1 g is recommended. An alternative treatment for uncomplicated gonococcal infections of the urethra or anus is intramuscular gentamicin in a single dose of 240 mg with azithromycin (2 g) or a single oral dose of cefixime (800 mg). In cases of gonococcal throat infections with an allergy to ceftriaxone, CDC recommends consultation with an infectious disease specialist to choose alternative therapy. The treatment regimen is the same for HIV-positive patients. Abstaining from sexual activity is necessary during treatment and for seven days afterwards. According to recommendations, all patients diagnosed with gonorrhoea should also be tested for other STIs, including chlamydia, syphilis, and HIV. Recent sex partners of diagnosed persons should be referred for testing and presumptive treatment (Workowski et al. 2021).