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The present article describes the laboratory diagnosis of Neisseria gonorrhoeae by culturing of the organism from different types of clinical specimens followed by confirmatory tests. The success of culture methods requires good quality collection and transport of clinical specimens. The present guide describes the media requirements and cultural c...
Contexts in source publication
Context 1
... current preferred laboratory method for the diagnosis of N gonorrhoeae infections is the isolation and identification of the agent (Figure 1). Culturing isolates is important for antimi- crobial susceptibility testing, surveillance purposes, detecting treatment failure and characterizing outbreaks. ...
Context 2
... cultures that are frequently used as controls should be stored as multiple aliquots to avoid freezing and thawing. Presumptive identification of N gonorrhoeae: The presump- tive identification of N gonorrhoeae rests on the isolation of an oxidase-positive, Gram-negative diplococcus recovered from urogenital sites that grows on selective media (5) (Figure 1). The Gram stain of a smear of urethral exudates or endocervical secretions shows typical Gram-negative intracellular diplococci. ...
Citations
... Gonorrhea, which is caused by Ng is highly prevalent in less-developed countries and lower-middle-income countries [12,15]. The prevalence of which varies by anatomic sites (whether urethral, rectal, or oropharyngeal) [16] and the methods of detection, e.g., Gram's stain, standard culture, and molecular test [17]. Chlamydia, an equally concerning sexually transmitted infection caused by Ct [18], has been on the rise since 1995, and it is now the most pervasive STI [10,19,20], especially among untreated asymptomatic patients [21]. ...
Background
Men who have sex with men (MSM) are disproportionately affected by sexually transmitted infections (STI) including Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct). The lack of robust data on STIs among African MSM has limited the development of evidence-based screening strategies. This study aimed at documenting the pooled prevalence of Ng/Ct among MSM in sub-Saharan Africa (SSA).
Methods
This systematic review was performed according to the Preferred Reporting Items for Systematic Review and Meta-analyses (PRISMA) 2020 guidelines. Relevant articles from the following databases were searched: PubMed, Scopus, ISI Web of Science, and the Directory of Open Access Journals (DOAJ). Eligible studies reported on the prevalence of Ng/Ct among the MSM population in SSA. Publication bias was assessed using the Hoy tool, Doi plot, and LFK ratio. Due to heterogeneity among studies, subgroup analyses were performed using the MetaXL add-on tool for Microsoft Excel.
Results
Of 525 articles screened, 20 were selected for inclusion. Six were cross-sectional, four had a prospective cohort study design, and one was an epidemiological study. The pooled prevalence of Ng/Ct in MSM was 27% (95% CI, 19–39%), with an I² of 98% signifying heterogeneity among the studies. Subgroup analysis by country revealed South Africa had the highest prevalence (38%).
Discussion
Interpretation
The high prevalence of Ng/Ct infection among MSM in SSA is of concern.
Limitations
Due to limited data available on Ng/Ct prevalence, the true prevalence of SSA and its associated risk factors is uncertain.
Conclusion
As the first study to systematically review the available literature on STI prevalence among the MSM population in SSA, it showed the burden of Ng/Ct is higher than in other regions, warranting the strengthening of health systems to improve education, testing, and treatment in MSM population.
Systematic review registration
PROSPERO CRD42022327095.
... It requires an energy source of glucose, pyruvate or lactate, and cysteine to grow on culture media. Due to changes in metabolic pathways, some isolates show special growth requirements for amino acids, purines, and pyrimidines (Ng et al. 2005;Quillin et al. 2018). The structure of N. gonorrhoeae cell wall is typical of Gram-negative bacteria. ...
... An important virulence factor is the glycolipid outer membrane antigen LOS, composed of endotoxinactive lipid A and a core oligosaccharide. It stimulates the release of pro-inflammatory cytokines, which promotes neutrophil recruitment to the infection site (Ng et al. 2005; Quillin and Seiferth 2018). The structure of LOS lacks the repeated polysaccharide chains characteristic of the analogous cell wall antigen of Enterobacterales, lipopolysaccharide. ...
Neisseria gonorrhoeae (gonococcus) is a human pathogen, the aetiological agent of gonorrhoea, which is the second most common bacterial sexually transmitted disease (STD) in the world. The structure of N. gonorrhoeae cell wall is typical of Gram-negative bacteria, poses variable antigens porin B (PorB), and opacity-associated proteins (Opa proteins), lipooligosaccharide (LOS) and type IV pili (TFP) playing an essential role in pathogenesis. In addition to adhesins, gonococcus presents other virulence factors such as reducing modifiable protein (Rmp), iron transporters, membrane pumps, and IgA peptidase. The pathogen produces outer membrane vesicles (OMVs), releases peptidoglycan (PG) fragments and is well adapted to develop infection in diverse niches of the female and male reproductive tracts. The characteristic genotypic trait of N. gonorrhoeae is the state of natural competence, which allows DNA uptake from the environment. The antigenic and phase variability is essential to gonococcal defence against the human immune system. Because of the increasing antimicrobial resistance (AMR) of N. gonorrhoeae and the high incidence rate of gonococcal infections, developing an antigonococcal vaccine has become an urgent need. Vaccine development difficulties are mainly due to the gonococcal ability of immune evasion, the lack of an animal model, and the limited understanding of protective immune response mechanisms.
... The relatively few causative bacterial agents associated with STIs makes targeted NAAT-based diagnostics an effective solution for infection identification. Furthermore, the characterisation of common AMR causing genes found in C. trachomatis and N. gonorrhoeae, also lend themselves well to NAAT-based AMR detection 24,60-62 and the preferred laboratory method for these two strains has shifted from culture 63 to NAAT 64 increasing sensitivity and specificity, and faster turnaround time 63 . WGS offers an alternative that would be able to strain ID and screen for AMR at the same time (and rapidly), but the high incidence would be too expensive in comparison to NAAT. ...
Antimicrobial resistance is a global threat to public health. Without proactive intervention, common infections may become untreatable, restricting the types of clinical intervention that can be undertaken and reversing improvements in mortality rates. Effective antimicrobial stewardship represents one approach to restrict the spread of antimicrobial resistance but relies on rapid and accurate diagnostics that minimise the unnecessary use of antibiotics. This is increasingly a key unmet clinical need. In this paper, we describe existing techniques for the detection of antimicrobial resistance, while examining their drawbacks and limitations. We also discuss emerging diagnostic technologies in the field, and the need for standardisation to allow for swifter and more widespread clinical adoption.
... Polymerase Chain Reaction (PCR) based methods are sensitive but require expensive thermocycling equipment, electricity, and cold chains for reagents [9][10][11][12][13]. The high sensitivity of nucleic acid amplification technologies (NATs) such as 16S rRNA PCR with a detection limit of 10 fg/μl in N. gonorrhoeae-positive clinical samples is attributed to their ability to produce a positive test result from a few copies of DNA or RNA. ...
... Large-scale clinical use of the loop-mediated isothermal amplification (LAMP) test, especially in low-and middle-income countries, has not been achieved. This is due to affordability and challenges with field deployment, including the need for cold chain storage of temperature-sensitive reagents [10,16,17]. ...
Background
Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose.
Methods
We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/μL to 1 × 10⁻³ pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel.
Results
Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL.
Conclusion
Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.
... Neisseria gonorrhoeae is a gram-negative, host-adapted infectious agent responsible for gonorrhea. Gonorrhea is characterized by cervicitis, urethritis, pharyngitis, and proctitis (Ng and Martin 2005). According to the WHO's alert, gonorrhea has annual incidence rate of more than 78 million cases globally due to lack of effective treatments (Quillin and Seifert 2018).The need for treatment is also alarmed by quick arrival of antimicrobial resistance in N. gonorrhoeae to approved antibiotics (Russell et al. 2020). ...
Neisseria gonorrhoeae, a World Health Organization (WHO) declared superbug and the second-most frequent cause of bacterial sexually transmitted infections worldwide is responsible for gonorrhea. Hypothetical proteins are gene products that are predicted to be encoded by a particular gene based on the DNA sequence, but their specific functions and characteristics have not been experimentally determined or verified. In the context of this research, annotating hypothetical proteins is crucial for identifying their potential as therapeutic targets. Without proper annotation, these proteins would remain vague, hindering efforts to understand their roles in disease. The methodology used aims to bridge this gap by employing algorithm-based tools and software to annotate hypothetical proteins and assess their suitability as therapeutic targets based on factors such as essentiality, virulence, subcellular localization, and druggability. Out of 716 N. gonorrhoeae hypothetical proteins reported in UniProt, assessment of crucial pathogenic factors, including essentiality, virulence, subcellular localization, and druggability, effectively filtered and prioritized the hypothetical proteins for further therapeutic exploration and lead to 5 proteins being chosen as targets. The molecular docking studies conducted identified 10 hits targeting the five targets. Conclusively, this study aided in identification of targets and hit compounds for therapeutic targeting of gonorrhea disease.
Graphical abstract
... Gonorrhea can be asymptomatic or symptomatic. Individuals afflicted with gonorrhoea may not suffer any visible symptoms in some circumstances, which is why the condition can often go misdiagnosed and spread unwittingly Ng and Martin, 2005;Kirkcaldy et al., 2019). In men, it can appear as urethritis, with symptoms including urethral stricture, epididymitis, and prostatitis. ...
... For this, efficient specimen transport and submission, from the clinic to the laboratory, is significant (Gumede et al., 2017). Several factors affect the recovery and final isolation of the organism, such as the method of collection, time of transport, and transport conditions, including temperature, CO 2 content, overgrowth of contaminants, or dilution of the organism in surrounding collection medium (Ng and Martin, 2005). Recovery rates can be improved by following appropriate guidelines for isolation, including the use of Dacron swabs for specimen collection, and immediate plating onto a suitable medium with minimal delay. ...
... The inoculated CA plates should be held at room temperature for no longer than 5 hours in a CO 2 -enriched atmosphere while being transported to a local laboratory. If long-distance mailing is required, the specimens should be inoculated onto media contained in a CO 2 -generating system, incubated for 18-24 hours before shipping, and should have visible growth (Ng and Martin, 2005). Neisseria gonorrhoeae cultures have been reported to survive up to 4 days on Thayer Martin media upon mailing (Cross et al., 1970;Jephcott, 1997). ...
Abstract Background: Neisseria gonorrhoeae (Gonococcus-GC) is a fastidious, autolytic Gram-negative diplococcus with stringent growth requirements, and cannot be cultivated in a routine microbiology laboratory, without well-equipped incubators, reagents, and special media. Hence, many clinics and laboratories prefer to ship the specimens or isolates to a dedicated referral laboratory for confirmation of isolates and antimicrobial susceptibility testing. Thus, transportation conditions for gonococcal isolates, become crucial for its recovery and successful isolation in the laboratory. This retrospective study was conducted at a national referral laboratory for gonococcus, in India, over 3 years. Aim: In this study, an attempt was made to determine the factors affecting the revival of isolates of gonococci, that were despatched, from across India, to the referral laboratory for confirmation of species and antimicrobial susceptibility patterns. Method: Over 3 years, the culture plates, test tubes, or vials used for transporting gonococcal isolates, and their modes of transport to the referral laboratory, were studied in detail. The isolates were revived (whenever possible), subcultured, and identified by standard methods in the referral laboratory. Results: A total of 77 samples were processed for revival and 83.12% of isolates were recovered, with failure of recovery in 16.88% of specimens. Conclusion: Several factors play a role in the successful revival of N. gonorrhoeae from culture isolates transported across the Indian subcontinent. These include purity of growth, culture media used for transport, sending of isolates in duplicates, temperature, time, distance, and season of transport. All these factors must be kept in mind when transporting gonococcal isolates, for successful revival. Finally, the skills of the laboratory technician are of immense importance too. Keywords: Gonococcus, Neisseria gonorrhoeae, Speciation, Transport media, Transit time.
... A gram-negative bacteria Neisseria gonorrhoeae is an obligate humanoid pathogen that causes the sexually transmitted disease gonorrhea to include cervicitis in women and urethritis, pharyngitis, and proctitis in both sexes (Ng and Martin 2005). Extensive use of antibiotics causes Neisseria gonorrhoeae resistance to thirdgeneration cephalosporins and fluoroquinolone. ...
A significant threat to global public health is antimicrobial resistance (AMR). The estimated cause of at least 700,000 deaths each year worldwide is medication-resistant bacterial infections (including tuberculosis). Estimates suggest that around ten million deaths are expected annually by 2050, due to drug-resistant bacteria. The World Health Organization (WHO) surveillance report concedes resistance in common human pathogens like Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, and Staphylococcus aureus is one of the biggest threats to humankind. Antibiotic resistance is becoming globalized due to the significant high evolutionary pressure. Thus, developing new therapeutic agents against new targets or antibacterials with different approaches to target pathogenic bacteria has become imperative. This review listed out various antibiotics act with different mechanisms, and bacteria also acquire different mechanisms to dodge these antibiotics. The mechanism includes removing antibiotics out of the cell by increased efflux, not allowing the antibiotic to enter by decreased uptake, decreasing the antibiotic binding by modifying the target, degrading the antibiotics by enzyme, etc. So, the novel antibacterial agent uses four criteria: absence of known cross-resistance, new class, new target, and a new mode of action developed to treat AMR.KeywordsAntibioticAntimicrobial resistanceFluoroquinolesβ-lactamPathogensTopoisomerases
... Polymerase Chain Reaction (PCR) are sensitive but need expensive equipment, electricity, and cold chains for reagents [10][9] [11][12][13]. The high sensitivity of nucleic acid amplification technologies (NATs) such as the 16S rRNA PCR with a detection limit of 10 fg/μl in N. gonorrhoeae positive clinical samples is attributed to their ability to produce a positive test result from few copies of DNA or RNA. ...
... Use of Loop-Mediated Isothermal Amplification (LAMP) test especially in low and middle-income countries has not been achieved. This is due to affordability and challenges with field deployment including need for cold chain storage of temperature sensitive reagents [10] [16] [17]. ...
Purpose
Curable sexually transmitted infections (STIs) such as Neisseria gonorrhoeae ( N. gonorrhoeae ) is a major cause of poor pregnancy outcome. The infection is often asymptomatic in pregnant women and a syndrome-based approach of testing leads to missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard Nucleic Acid Amplification Tests (NAATs) require advanced infrastructure settings whilst point of care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose.
Materials and methods
Here, we have identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100pg/μL to 1×10 ⁻³ pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products resolved on 1% agarose gel.
Results
Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity compared to the 16S rRNA PCR assay with analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated with clinical urethral swab samples. We further advanced this technique by developing an iso-thermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining the iso-thermal IMRS with a low-cost Lateral Flow Assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL.
Conclusion
Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.
... Among all these tests, nucleic acid amplification test (NAAT) is the gold standard and recommended. The culture of the infected person swab is also recommended to detect the drug resistance (Ng and Martin 2005). The major drawback of hybridization and amplification methods is the time consumption and required specialized facility that delayed the diagnosis. ...
As the world fights the COVID-19 pandemic, point-of-care detection and diagnostic systems have gained immense importance. Microfluidics has revolutionized the domain of point-of-care (PoC) devices meant for the on-site detection of diseases. Microfluidic platforms provide an integrated, miniaturized, and cost-effective analytical alternative to conventional point-of-care devices with a massive potential for commercialization. These platforms also offer the additional advantage of low sample volume and lesser time for detection. The amalgamation of nanobiotechnology with microfluidics has given rise to highly selective and sensitive stand-alone devices that detect early disease onset and progression biomarkers. Early detection helps to decide the therapeutic strategy for the patient in as little time as possible. These devices are compact, portable, and convenient, hence ideal for PoC applications. The incorporation of nanoscale sensing elements, including nanoprobes, graphene, and magnetic and noble metal nanoparticles (Gold, Silver, and Platinum), further enhances the sensitivity of nanobiosensor-based immunoassays. The future of medical diagnostics heavily relies on these novel sensing platforms, thus helping in proper planning and management of the treatment of any disease. In this chapter, to better understand, we illustrate the fabrication, characterization, and applications of these intelligent point-of-care biosensing platforms. A brief account of challenges and future scope associated with applying such point-of-care nanobiosensors has also been discussed.
