Laboratory Diagnosis of Gonorrhea

The name gonorrhea, which means “flow of seed,” was initially used in the second century when the urethral discharge was mistaken for sperm. For years, the illnesses syphilis and gonorrhea were confused since they were frequently seen in the same person. In 1530, gonorrhea was regarded to be an early indication of syphilis. Gonorrhea was also known as “the clap,” a term derived from the French word clapoir, which means “brothel.”

Gonorrhea is a common sexually transmitted infection caused by the bacteria Neisseria gonorrhoeae. Direct contact with fluids from the mouth, vagina, penis, or perianal area may transmit the disease. The organism reproduces in warm, wet parts of the body such as the urethra of both males and females, the fallopian tubes, the uterus, and the cervix. Females get symptoms 2 to 5 days after infection. Males may not show symptoms for up to a month after infection. Female symptoms include vaginal discharge, discomfort and frequency of urine, sore throat, stomach pain, fever, and painful sexual intercourse.

Males have discomfort and frequency during urinating, a penile discharge, a red or inflamed urethra, and testicular soreness. The features of the urethral discharge can range from murky to clear, making it an inconsistent predictor of gonococcal urethritis in men.

Pathophysiology

Growth and metabolism: N. gonorrhoeae is a fastidious bacterium that is susceptible to many environmental parameters, including oxygen, non-physiological temperatures, desiccation, and the accumulation of toxic substances (such as many fatty acids); as a result, the bacterium does not survive long outside of the human host and is difficult to culture. Many strains have insufficient amino acid synthesis capabilities, possibly as amino acids and other critical nutrients are easily acquired from the human host. Iron (which is required for bacterial development) is obtained from the host by binding iron-containing host proteins such as transferrin, lactoferrin, and hemoglobin at the bacterial cell surface and depleting these components of iron, which is subsequently transported to the bacterial cytoplasm. Because of the wide variety of oxygen levels in different habitats of the men’s and women urogenital tracts, N. gonorrhoeae may meet aerobic, microaerobic, and anaerobic environments within the host, and the bacteria may flourish in all of these situations.

Bacterial Genetics: Well almost all gonococcal strains possess a cryptic plasmid (with no defined functions); most constitute a plasmid gene that encodes a penicillinase (mostly TEM-1 or TEM-135 – lactamase), resulting in high-level penicillin resistance, and conjugative plasmids, which occasionally carry tetM, causing high-level tetracycline resistance, though these plasmids are not as prevalent Several penicillinase-encoding plasmids of various sizes have been identified in N. gonorrhoeae and called by their epidemiological origin, such as the widely distributed and most frequent African, Asian, and Rio/Toronto plasmids.

Colonization determinants: Many colonization factors are shared by N. gonorrhoeae and other human-restricted Neisseria species that rarely cause illness. The type IV pilus, the opacity protein family (Opa proteins), the porin PorB, efflux pumps, and metal transport mechanisms are all necessary to build a host niche. N. gonorrhoeae is likely to compete for colonization with the resident microbiota, but little is known about how various resident commensal species may restrict or collaborate with N. gonorrhoeae during colonization.

Host damage: Because it is highly adapted to its human host and is rarely fatal, N. gonorrhoeae is not a particularly disruptive pathogen. It does not create exotoxins capable of destroying host cells, but does discharge peptidoglycan components, outer membrane vesicles (OMVs), and LOS that are toxic to human cells and may selectively suppress ciliated cells on fallopian tube tissue. Furthermore, when PMNLs are attracted to areas of infection, anti-microbial chemicals from the PMNLs are produced, which may cause tissue damage. All of these variables lead to the fallopian tube tissue damage and scarring that is typical of PID. These elements may also induce harm at other sites of infection, especially during DGIs, which can result in fever, dermatitis, infectious arthritis, and (less commonly) septicemia, as well as endocarditis and meningitis.

Clinical Presentation and Laboratory Diagnosis

The incubation time for urogenital gonorrhea varies from ~2 days to 8 days. The clinical signs of gonorrhea are heterogeneous and vary substantially in men and women. Laboratory diagnosis of gonorrhea is established by direct identification of the organism in urogenital, anorectal, pharyngeal, or conjunctival swab specimens or first-catch urine.

Microscopy

Methylene blue staining and Gram’s staining are adopted for direct microscopy. The slide is covered with a 1 % aqueous methylene blue. The preparation is washed with water and dried after a brief exposure (15 s). When bacteria are stained with methylene blue, they all become blue. It should only be used in conjunction with normal clinical signs to diagnose simple male urethritis.

Gram’s staining is essential in female’s gonorrhea and all other indications of disease. Commercially available staining packages are also available. Gram stain is ineffective for diagnosing Neisseria gonorrhoeae from pharyngeal or rectal specimens (since other Neisseria species with similar appearance are common in the oral and nasopharyngeal cavities).

Gonococcus gram Stain

Culture

Antimicrobial resistance in Neisseria gonorrhoeae is a serious issue all over the globe, and reliable findings are required for an effective treatment. Bacterial culture is very particular and sensitive. Under ideal circumstances, sensitivity in urogenital specimens may reach 85–95 %, and specificity can approach 100 % when species identification is conducted.

Gonococci are fastidious pathogens. They do not tolerate dehydration and should be inoculated into culture media shortly after swab collection (nutritious selective culture medium and non-selective culture medium). Culture plates must be incubated at 35–37°C, high humidity (70–80%), pH 6.75–7.5, and in a 4–6% CO2-enriched environment. After 18–24 (~48) h, tiny, glossy gray colonies form, allowing for colony growth fluctuations. Following cultivation, the identity of Neisseria gonorrhoeae is determined by combining different detection techniques.

A microscopic Gram’s staining preparation and positive cytochrome oxidase reaction are used to achieve a preliminary identification. Biochemical studies, immunological testing, spectrometric tests, or molecular tests are used to confirm the identification and discriminate between other Neisseria species, such as Neisseria meningitidis and apathogenic Neisseria spp., particularly in extragenital areas.

Antimicrobial Susceptibility Testing

The WHO-recommended gold standard technique for antimicrobial susceptibility testing in Neisseria gonorrhoeae is agar dilution. An antibiotic agent is integrated into nutrient medium in a defined series of strengths. The corresponding inhibition zone is read off as the antibiotic concentration increases. The MIC value in g/ml (mg/l) is the lowest concentration that inhibits growth. However, this procedure is sophisticated and best suited to a large variety of tests. As a result, the standardized and quality-assured MIC gradient stripe test technique (Etest), which correlates with the agar dilution method, is presently the preferred method.

Nucleic Acid Amplification Technologies (NAATs)

NAATs are currently a major diagnostic test since specimen collection is noninvasive (urine or self-collected swabs, particularly vaginal swabs); live organisms are not required for detection. This facilitates less restrictive transportation and storage procedures.

NAATs are also the most efficient tests for detecting extragenital Neisseria gonorrhoeae infection and are so recommended for laboratory detection of rectal or pharyngeal infection. Further have a glance at FDA approved NAAT tests.

Rapid/POCT tests

Despite the fact that NAATs are considered the standard tests for detecting Neisseria gonorrhoeae, their application in low- and middle-income countries is severely constrained due to their relatively expensive prices. Given the availability of a microscope, microscopy may be regarded a Neisseria gonorrhoeae RDT that can be conducted at the point of care. Microscopic examination, on the other hand, necessitates expert investigators and lacks sensitivity in asymptomatic infections as well as anorectal and pharyngeal specimens. However, some lateral flow immunoassays are also available.

References:

  1. Bailey Scott’s Diagnostic Microbiology 14th Edition
  2. Textbook of Diagnostic Microbiology by Connie R Mahon 6th Edition
  3. Meyer, T., & Buder, S. (2020). The Laboratory Diagnosis of Neisseria gonorrhoeae: Current Testing and Future Demands. Pathogens (Basel, Switzerland)9(2), 91. https://doi.org/10.3390/pathogens9020091
  4. Unemo, M., Seifert, H.S., Hook, E.W. et al. Gonorrhoea. Nat Rev Dis Primers 5, 79 (2019). https://doi.org/10.1038/s41572-019-0128-6

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