Jonah Patel
Neisseria gonorrhoeae, commonly known as gonorrhea, is a sexually transmitted disease caused by a bacterium. It can be transmitted during vaginal, anal, or oral sex, as well as from mother to child during childbirth. The current preferred laboratory method for diagnosing N. gonorrhoeae infections is the isolation and identification of the agent through culturing isolates. This involves inoculating primary specimens onto selective and non-selective agar to differentiate N. gonorrhoeae from other species. Presumptive identification of N. gonorrhoeae typically involves the use of biochemical, serological, colorimetric, and nucleic acid testing methods.
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What You'll Learn
Gram stain of urethral exudates or endocervical secretions
Gram staining is a common technique used to identify bacteria in urethral discharge, or endocervical secretions. The Gram stain procedure involves applying a sample of the discharge to a microscope slide, which is then treated with a series of stains to identify the bacteria. The test is often carried out in a healthcare provider's office and can be used to identify the presence of bacteria in cases of suspected sexually transmitted infections.
The Gram stain of urethral exudates or endocervical secretions is a test used to identify bacteria in fluid from the urethra, which is the tube that drains urine from the bladder. Urethral discharge may be collected on a cotton swab and applied to a microscope slide. The sample is then treated with a series of stains, which allow for the identification of the bacteria under a microscope. The colour, size, and shape of the cells are all factors that help to identify the type of bacteria causing the infection.
The Gram stain procedure is a method of staining microorganisms on a slide using crystal violet. When viewed under a microscope, the bacteria will be either Gram-positive or Gram-negative, depending on how they react to the stain. If the bacteria remain purple, they are Gram-positive, but if they turn pink, they are Gram-negative.
In the case of Neisseria gonorrhoeae, the Gram stain of urethral exudates or endocervical secretions shows typical Gram-negative intracellular diplococci. When these Gram-negative diplococci are found outside the neutrophils, cultures of specimens from males will usually be positive for N. gonorrhoeae. However, in specimens of cervical secretions, a predominance of extracellular organisms indicates that many are nonpathogenic Neisseria species rather than N. gonorrhoeae.
The presumptive identification of N. gonorrhoeae involves the isolation of an oxidase-positive, Gram-negative diplococcus recovered from urogenital specimens. The oxidase test uses a commercially available reagent, or one prepared in-house, which turns dark purple within 10 seconds to indicate a positive sample. Other rapid tests used in the presumptive identification of N. gonorrhoeae include the catalase test and superoxol test.
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Oxidase test
The oxidase test is used in the presumptive identification of N gonorrhoeae, which causes gonorrhea. The oxidase test uses the tetramethyl derivative of the oxidase reagent (1% aqueous solution of N, N, N, N-tetramethyl-1, 4-phenylenediamine). This reagent is commercially available (BACTIDROP Oxidase, Remel Inc, USA) or can be prepared in-house.
To perform the test, a drop of reagent is applied to filter paper or the tip of a cotton swab. Culture is then applied to the filter paper or swab tip using a platinum or plastic loop, wooden applicator stick, or toothpick. A dark purple colour change within 10 seconds indicates a positive sample. N gonorrhoeae will produce a positive reaction with bubbling within 1-2 seconds. Weak bubbling or bubbling after 3 seconds indicates a negative reaction.
The oxidase test is one of several methods available to confirm the identification of N gonorrhoeae. Other methods include biochemical testing, serological testing, colourimetric testing, and nucleic acid methods. N gonorrhoeae can be differentiated from other species based on its ability to grow on selective and non-selective media, produce acid from glucose, maltose, lactose, sucrose, and fructose, reduce nitrate, produce polysaccharide from sucrose, and exhibit DNase production.
The primary specimens for the oxidase test should be inoculated onto non-selective chocolate agar and selective agar containing antimicrobial agents that inhibit the growth of commensal bacteria and fungi. The antibacterial agents in modified Thayer-Martin, Martin Lewis, and New York City medium are vancomycin, colistin, trimethoprim lactate, and the antifungal agents nystatin and anisomycin or amphotericin B.
Culture is the preferred method for the diagnosis of N gonorrhoeae infections, as it allows for the isolation and identification of the agent. However, culturing for Neisseria is problematic due to the difficulty of maintaining the bacteria's viability during transport and storage. Other drawbacks include the time needed to obtain preliminary results (24-72 hours) and the possibility of false-positive results in medico-legal cases. In such cases, culture is required due to the potential for false positives with nonculture methods.
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Culture methods
Culture Specimens
Culture specimens for *N. gonorrhoeae* are typically collected from symptomatic males and females through urethral and endocervical swabs, respectively. These specimens are then transported to a laboratory for testing, ideally within 72 hours of collection. In the laboratory, the specimens are cultured on selective media, such as GC agar base or Columbia agar supplemented with lysed horse blood, to promote the growth of *N. gonorrhoeae*.
Culture for Presumptive Identification
The presumptive identification of *N. gonorrhoeae* relies on specific characteristics of the bacterium. *N. gonorrhoeae* is an oxidase-positive, Gram-negative diplococcus. Microscopic examination of Gram-stained smears can reveal the presence of these distinct kidney-shaped diplococci, providing an initial presumptive diagnosis. However, further confirmation is often required.
Culture Confirmation Techniques
To confirm the presence of *N. gonorrhoeae*, additional biochemical, immunological, or nucleic acid tests are employed. Biochemical tests, such as the rapid carbohydrate test, assess the ability of the bacterium to produce acid from carbohydrates. Immunological tests, such as serological testing, detect specific antigens or antibodies associated with *N. gonorrhoeae*. Nucleic acid tests, including NAATs (nucleic acid amplification tests), amplify specific nucleic acid sequences, allowing for the detection of small amounts of the pathogen's genetic material. These tests provide definitive identification of *N. gonorrhoeae*, ensuring accurate diagnosis and guiding appropriate treatment regimens.
Culture Storage
Proper storage of *N. gonorrhoeae* cultures is essential for long-term reference and research purposes. Cultures can be stored in a -80°C freezer, in liquid nitrogen (-196°C), or through lyophilization. Lyophilization involves suspending the cells in skim milk and distributing them into vials, which are then stored at 4°C or room temperature. To maintain viability, reference cultures should be subcultured every five years.
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Biochemical testing
The ability of N. gonorrhoeae to grow on selective and non-selective media is a critical aspect of biochemical testing. This involves inoculating primary specimens onto non-selective chocolate agar and selective agar containing antimicrobial agents. The selective agar inhibits the growth of commensal bacteria and fungi, allowing for the specific identification of N. gonorrhoeae.
Additionally, N. gonorrhoeae can be distinguished by its ability to produce acid from carbohydrates like glucose, maltose, lactose, sucrose, and fructose. This is traditionally determined using the conventional cystine trypticase agar medium (CTA) with 1% sugar. However, the CTA method is not sensitive enough to detect acid production from oxidative species, and newer rapid tests have been developed.
Another important aspect of biochemical testing is the oxidase test, which uses the tetramethyl derivative of the oxidase reagent (a 1% aqueous solution of N, N, N, N-tetramethyl-1, 4-phenylenediamine). A drop of this reagent is applied to a filter paper or swab, and then the culture is applied. A dark purple colour change within 10 seconds indicates a positive sample of N. gonorrhoeae.
Other rapid tests used in presumptive identification include the catalase test (3% hydrogen peroxide) and superoxol (30% hydrogen peroxide). These tests provide a rapid method for identifying N. gonorrhoeae in the laboratory.
While biochemical testing plays a crucial role in the presumptive identification of N. gonorrhoeae, it is often used in conjunction with other testing methods to confirm the presence of this pathogen.
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Serological testing
There are several serological tests available for the diagnosis of N. gonorrhoeae infections, including the fluorescent antibody test and the complement-fixation test. These tests detect the presence of specific antibodies or antigens associated with the N. gonorrhoeae infection.
One example of a fluorescent antibody test is the indirect fluorescent-antibody technique, which involves the use of an antigen derived from N. gonorrhoeae to detect the presence of specific antibodies in the patient's serum. This test has been used to study uncomplicated gonorrhea and can help characterize the strain of N. gonorrhoeae causing the infection.
Serological tests for N. gonorrhoeae can also be used in combination with other techniques, such as nucleic acid detection methods, to increase the accuracy of diagnosis. However, it is important to note that serological testing alone may not be sufficient for confirming an N. gonorrhoeae infection due to its limited sensitivity and specificity.
The collection of specimens for serological testing of N. gonorrhoeae typically involves the use of Dacron or rayon swabs, as calcium alginate may be toxic to gonococci. Fatty acids can also inhibit the growth of gonococci, so cotton swabs that do not meet acceptable manufacturer specifications should be avoided. Discharge from the meatus is preferred for the detection of N. gonorrhoeae, but an intraurethral swab can be used if there is no meatal exudate present.
Overall, serological testing plays an important role in the presumptive identification of N. gonorrhoeae, but it should be used in conjunction with other diagnostic techniques to ensure accurate and reliable results.
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Frequently asked questions
N. gonorrhoeae is a common sexually transmitted disease caused by the bacterium Neisseria gonorrhoeae, which can be transmitted during vaginal, anal, or oral sex and at birth to a newborn.
N. gonorrhoeae is identified by the isolation and identification of the agent. Culturing isolates is important for antimicrobial susceptibility testing, surveillance purposes, detecting treatment failure, and characterizing outbreaks.
Several methods are available to confirm the identification of N. gonorrhoeae, including biochemical testing, serological testing, colorimetric testing, and nucleic acid methods.
Specimens that are positive for N. gonorrhoeae are reported to the Medical Officer of Health as per the relevant Health Protection and Promotion Act. Culture is the specimen of choice for all potentially medico-legal specimens.
N. gonorrhoeae can be effectively treated by antibiotics. Clinicians should first assess a patient’s allergy history, including type of reaction, associated medications, and previous prescription records.
