Neisseria gonorrhoeae- Laboratory Diagnosis, Treatment, Prevention
The laboratory diagnosis of Neisseria gonorrhoeae (Ng) infection is important for the effective management and prevention of gonorrhea, a sexually transmitted infection that can cause serious complications such as pelvic inflammatory disease, infertility, ectopic pregnancy, and disseminated gonococcal infection. The ideal laboratory test to detect Ng should be sensitive, specific, easy to use, rapid, affordable and provide information about susceptibility to antimicrobial drugs. However, such a test is not available and presumably will not be in the near future. Thus, diagnosis of gonococcal infections presently includes application of different techniques to address these requirements.
The specimen choice and collection method depends on the testing technique used in a laboratory and the age, sex and sexual orientation of the patient. The following are some common types of specimens and how to collect them:
- Urethral: Express urethral exudates when patients have discharge. If there is no discharge, compress the meatus vertically to open the distal urethra and insert a thin, water-moistened swab (calcium alginate or Dacron) with flexible wire slowly (3 cm to 4 cm in males or 1 cm to 2 cm in females), rotate slowly and withdraw gently.
- Urine: Ask patients to collect only the first 10 mL to 15 mL of urine. Patients should not have voided for at least 2 h before specimen collection to increase the chance of detecting the organism.
- Cervical: Insert a speculum into the vagina to view the cervix. Insert a swab 1 cm to 3 cm into the endo-cervical canal and rotate for 10 s to 30 s to allow absorption of exudates.
- Vaginal: Collect pooled vaginal secretions, if present. Vaginal wash specimens are most preferred and acceptable to pre-pubertal girls. If not possible, rub a sterile cotton swab against the posterior vaginal wall and allow the swab to absorb the specimen.
- Rectal: Specimens may be obtained blindly or, preferably, through an anoscope. Insert a swab 2 cm to 3 cm into the anal canal. Avoiding fecal material, rotate to sample crypts just inside the anal ring; allow the swab to absorb specimen for 10s.
- Oropharyngeal: Rub sterile swabs over the posterior pharynx and tonsillar crypts, or obtain nasopharyngeal aspirate from infants.
- Conjunctiva: Any exudate or pus present in the eye should be carefully removed with a sterile swab. A second swab moistened with saline should be used to rub the affected conjunctiva. This swab should be broken off into a vial of transport medium.
- Sterile body fluids: Clean skin puncture site with iodine (1% to 2%, or 10% solution of povidone-iodine ). If tincture of iodine is used, remove with 70% ethanol to avoid burn. Perform percutaneous aspiration for pleural, pericardial, peritoneal or synovial fluids. Use non-heparinized collection if possible.
To minimize the inhibitory effects of unknown substances in the specimen, the swabs should be inoculated directly onto growth medium or placed in swab transport medium immediately after sampling. The transport medium should be suitable for the preservation of N. gonorrhoeae and should not contain any antibiotics or antiseptics that may interfere with the growth of the organism. Some examples of transport media are Stuart`s, Amies and modified Thayer-Martin media.
If the inoculated media are being transported to a local laboratory, the plates should be held at room temperature for no more than 5 h in a CO2-enriched atmosphere using a candle jar or a commercial CO2-generating system. Alternatively, the plates can be incubated at 35°C to 37°C in a CO2 incubator until they reach the laboratory.
If long-distance shipping is required, the specimens should be inoculated onto media contained in a CO2-generating system, incubated for 18 h to 24 h and have visible growth on the plate before shipping. The plates should be packed in insulated containers with ice packs or dry ice to maintain a low temperature during transit. The plates should also be labeled with the date and time of collection, the specimen type and the patient identification.
The specimens should be processed as soon as possible after arrival at the laboratory. If there is any delay in processing, the plates should be stored at 2°C to 8°C for no longer than 24 h. The specimens should not be frozen or exposed to high temperatures or direct sunlight.
A direct smear for Gram staining may be performed as soon as the swab specimen is collected from the urethra, cervix, vagina or rectum. This method can provide rapid results, but it has low sensitivity and specificity in some cases.
The Gram stain of a smear of urethral exudates or endocervical secretions shows typical Gram-negative, non-motile, intracellular diplococci. The diplococci have kidney/coffee bean shape and are arranged in pairs with the flattening of the adjacent sides. The diameter ranges from 0.6-1 μm.
The Gram stain of a smear of vaginal or rectal specimens may show extracellular diplococci, but they may be confused with other Gram-negative bacteria that are normally present in these sites. Therefore, microscopy alone is not sufficient to confirm the diagnosis of gonorrhea and should be followed by culture or nucleic acid amplification tests.
The current preferred laboratory method for the diagnosis of N. gonorrhoeae infections is the isolation and identification of the agent. The primary specimens should be inoculated onto nonselective chocolate agar and selective agar containing antimicrobial agents that inhibit the growth of commensal bacteria and fungi.
Modified Thayer Martin Medium (Chocolate agar containing antibiotics (vancomycin, colistin, trimethoprim, and nystatin)) is most often used. Modified Newyork City Medium (MNC) is also used for the culture of N. gonorrhoeae. The inoculated plates should be incubated at 35°C to 37°C in a moist atmosphere enriched with CO2 (3% to 7%). An 18 h to 24 h culture should be used as the inoculum for additional tests.
Note: Plates should not be incubated for longer than 48 h because most old cultures would not survive storage conditions. Autolysis may occur during prolonged incubation, and growth from agar plates becomes difficult to suspend in solutions. Isolates should be sub-cultured at least once on nonselective medium after initial isolation before being used in a diagnostic test that requires pure culture or heavy inoculum.
The presumptive identification of N. gonorrhoeae rests on the isolation of an oxidase-positive, catalase-positive, Gram-negative diplococcus recovered from urogenital sites that grow on selective media. This means that the organism produces the enzymes oxidase and catalase, has a spherical shape in pairs, and has a thin cell wall that stains pink with Gram stain. The selective media contain antibiotics that inhibit the growth of other bacteria and fungi that may contaminate the specimen.
According to the CDC recommendations, the presumptive identification of N. gonorrhoeae can be made if ONE of the following criteria is met:
- Direct Gram stain from symptomatic males only: The presence of typical Gram-negative intracellular diplococci in urethral exudates or secretions indicates a high probability of N. gonorrhoeae infection. This criterion is not applicable to females or asymptomatic males because of the low specificity and sensitivity of Gram stain in these populations.
- Culture: The growth of oxidase-positive, Gram-negative diplococci on selective media (e.g., modified Thayer-Martin or New York City medium) from any anatomic site (e.g., urethra, cervix, rectum, pharynx, or conjunctiva) suggests N. gonorrhoeae infection. However, some nonpathogenic Neisseria species may also grow on these media and require further confirmation.
- Nucleic acid amplification tests (NAATs): The detection of N. gonorrhoeae-specific nucleic acid sequences by NAATs from any anatomic site indicates N. gonorrhoeae infection. NAATs are highly sensitive and specific for N. gonorrhoeae and can be performed on various specimen types, including urine, swabs, and self-collected vaginal swabs. However, NAATs may not be able to distinguish between viable and nonviable organisms, and may cross-react with some nonpathogenic Neisseria species.
Presumptive identification of N. gonorrhoeae should be followed by confirmatory tests to ensure accurate diagnosis and appropriate treatment. Confirmatory tests include biochemical tests, chromogenic enzyme substrate tests, immunoassays and nucleic acid methods.
Confirmatory tests are used to verify the presumptive identification of Neisseria gonorrhoeae based on the isolation of an oxidase-positive, catalase-positive, Gram-negative diplococcus recovered from urogenital sites that grow on selective media.
Confirmatory tests include biochemical tests, chromogenic enzyme substrate tests, immunoassays and nucleic acid methods.
Biochemical tests involve testing the ability of the isolate to ferment glucose and other sugars, produce deoxyribonuclease (DNase), beta-galactosidase (ONPG) and glutamyl-aminopeptidase (GAP). N. gonorrhoeae is positive for oxidase, catalase and glucose fermentation, but negative for maltose, sucrose and lactose fermentation, DNase, ONPG and GAP.
Chromogenic enzyme substrate tests use specific substrates that release a colored compound when hydrolyzed by an enzyme produced by N. gonorrhoeae. The most commonly used substrate is hydroxyprolylaminopeptidase (HPA), which is positive for N. gonorrhoeae.
Immunoassays use antibodies that bind to specific antigens on the surface of N. gonorrhoeae. These antibodies can be labeled with fluorescent dyes, enzymes or radioisotopes for detection. Immunoassays can be performed on direct specimens or cultured isolates.
Nucleic acid methods use probes or primers that hybridize or amplify specific sequences of DNA or RNA from N. gonorrhoeae. These methods can be performed on direct specimens or cultured isolates and can provide high sensitivity and specificity. Some nucleic acid methods can also detect mutations associated with antimicrobial resistance.
The choice of confirmatory test depends on the availability of resources, the type of specimen and the clinical setting.
Monitoring of antimicrobial susceptibilities of N. gonorrhoeae is important to investigate treatment failure and to evaluate the efficacy of currently recommended therapies. Antibiotic resistance in N. gonorrhoeae has severely compromised the successful treatment of gonorrhea. Penicillins, tetracyclines, and newer macrolides have limited utility, and spectinomycin (and in many parts of the world, quinolones) have been withdrawn because of resistance. Of the usually recommended treatments, only the third-generation cephalosporins, and most notably ceftriaxone have retained their efficacy, but decreased susceptibility to these antibiotics has also appeared.
The ideal laboratory test to detect N. gonorrhoeae (Ng) should be sensitive, specific, easy to use, rapid, and affordable and should provide information about susceptibility to antimicrobial drugs. Currently, such a test is not available and presumably will not be in the near future. Therefore, different methods are used for antimicrobial susceptibility testing (AST) of N. gonorrhoeae, depending on the availability of resources, expertise, and isolates.
The most commonly used methods for AST of N. gonorrhoeae are:
- Disk diffusion: This method involves placing antibiotic-impregnated disks on an agar plate inoculated with a standardized suspension of bacteria and measuring the zone of inhibition around each disk after incubation. The zone size is compared with interpretive criteria to determine if the isolate is susceptible, intermediate or resistant to the antibiotic. This method is simple, inexpensive and widely used, but it has some limitations such as subjectivity in reading the results, lack of standardization of inoculum density and disk potency, and inability to determine the minimum inhibitory concentration (MIC) of the antibiotic.
- Agar dilution: This method involves preparing a series of agar plates containing different concentrations of antibiotics and inoculating them with a standardized suspension of bacteria. After incubation, the MIC is defined as the lowest concentration of antibiotic that inhibits visible growth of the isolate. This method is considered the gold standard for AST of N. gonorrhoeae, as it is accurate, reproducible and allows testing multiple antibiotics simultaneously. However, it is also labor-intensive, time-consuming and requires specialized equipment and expertise.
- Gradient strip testing: This method involves placing a plastic strip impregnated with a gradient of antibiotic concentrations on an agar plate inoculated with a standardized suspension of bacteria. After incubation, the MIC is read from the point where the edge of the inhibition ellipse intersects with the strip. This method is rapid, easy to perform and interpret, and provides quantitative results. However, it is also expensive and requires specific quality control procedures.
- Resazurin microdilution assay: This is a novel method that involves adding resazurin dye to microtiter plates containing serial dilutions of antibiotics and inoculating them with a standardized suspension of bacteria. After incubation, the MIC is determined by observing the color change of resazurin from blue to pink in wells with bacterial growth. This method is fast (results can be obtained within 6 h), inexpensive and objective. However, it has not been extensively validated and standardized for AST of N. gonorrhoeae.
Regardless of the method used for AST of N. gonorrhoeae, it is essential to follow standardized protocols and quality control procedures to ensure reliable and comparable results. Moreover, it is important to report and interpret the results according to the most updated clinical breakpoints and epidemiological cut-off values established by relevant authorities such as the Clinical and Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) or the World Health Organization (WHO).
Treatment of gonorrhea involves antibiotics to kill the bacteria and clear the infection. However, antibiotic resistance in Neisseria gonorrhoeae has severely compromised the successful treatment of gonorrhea. Therefore, it is important to follow the current treatment guidelines and monitor the antimicrobial susceptibility of the isolates.
According to the CDC, the recommended treatment for uncomplicated gonorrhea is a single dose of 500 mg of intramuscular ceftriaxone. This is combined with oral azithromycin (1 g) to provide additional coverage for possible co-infection with Chlamydia trachomatis and to prevent the emergence of ceftriaxone resistance.
Alternative regimens are available when ceftriaxone cannot be used to treat urogenital or rectal gonorrhea. These include oral cefixime (400 mg) plus azithromycin (1 g), or oral gemifloxacin (320 mg) plus azithromycin (2 g), or intramuscular gentamicin (240 mg) plus oral azithromycin (2 g). However, these alternatives are less effective and more toxic than ceftriaxone.
For pharyngeal gonorrhea, the only recommended treatment is ceftriaxone (500 mg) plus azithromycin (1 g). This is because oral antibiotics have poor efficacy against pharyngeal infection.
Sexual partners of people with gonorrhea should also be notified, tested and treated. The CDC advises that people with gonorrhea abstain from sexual intercourse for at least seven days after completion of treatment.
If symptoms persist after receiving treatment, a reevaluation should be done to rule out treatment failure or reinfection. A test-of-cure is not needed for genital and rectal infections; however, it is needed 7-14 days after treatment for pharyngeal infections. Because reinfection is common, people with gonorrhea should be retested three months after treatment of the initial infection, regardless of whether they believe that their sex partners were successfully treated.
Treatment of gonorrhea is also important to prevent complications such as pelvic inflammatory disease, infertility, ectopic pregnancy, epididymitis, disseminated gonococcal infection and neonatal conjunctivitis.
To summarize, treatment of gonorrhea involves:
- A single dose of intramuscular ceftriaxone plus oral azithromycin as the first-line therapy
- Alternative regimens when ceftriaxone is not available or contraindicated
- Treatment of sexual partners and abstaining from sex for seven days after treatment
- Reevaluation and retesting if symptoms persist or recur
- Prevention of complications by timely diagnosis and treatment
The best way to prevent gonorrhea infection is to avoid sexual contact with anyone who has the disease or who may be at risk of getting it. However, if you are sexually active, you can reduce your chances of getting gonorrhea by following these tips:
- Use latex condoms or dental dams correctly and consistently every time you have vaginal, anal, or oral sex. Condoms and dental dams can help prevent the transmission of gonorrhea and other sexually transmitted diseases (STDs) by creating a barrier between the mucous membranes of the genitals, rectum, and mouth.
- Limit your number of sexual partners and avoid having sex with people who have multiple partners or who do not know their STD status. Having fewer partners reduces your exposure to gonorrhea and other STDs.
- Get tested regularly for gonorrhea and other STDs if you are sexually active, especially if you have new or multiple partners, or if you have symptoms of an infection. Early detection and treatment can prevent complications and transmission of gonorrhea to others.
- If you are diagnosed with gonorrhea, inform your sexual partners as soon as possible so they can get tested and treated too. Do not have sex until you and your partners have completed the treatment and are cured of the infection.
- If you are pregnant or planning to become pregnant, talk to your healthcare provider about getting tested for gonorrhea and other STDs. Gonorrhea can cause serious health problems for your baby during pregnancy and delivery, such as premature birth, eye infection, blindness, joint infection, and blood infection. Treatment during pregnancy can prevent these complications and protect your baby from infection.
There is currently no vaccine available to prevent gonorrhea infection. Researchers are working on developing a vaccine that can stimulate the immune system to fight against the bacteria that cause gonorrhea. Until then, practicing safe sex and getting regular testing and treatment are the most effective ways to prevent gonorrhea infection and its consequences.
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